Roflumilast N-oxide Research Articles

Prediction of drug-drug interactions between roflumilast and CYP3A4/1A2 perpetrators using a physiologically-based pharmacokinetic (PBPK) approach.

This study aimed to develop a physiologically-based pharmacokinetic (PBPK) model to predict changes in the pharmacokinetics (PK) and pharmacodynamics (PD, PDE4 inhibition) of roflumilast (ROF) and ROF N-oxide when co-administered with eight CYP3A4/1A2 perpetrators. The population PBPK model of ROF and ROF N-oxide has been successfully developed and validated based on the four clinical PK studies and five clinical drug-drug interactions (DDIs) studies. In PK simulations, every ratio of prediction to observation for PK parameters fell within the range 0.7 to 1.5. In DDI simulations, except for tow peak concentration ratios (Cmax) of ROF with rifampicin (prediction: 0.63 vs. observation: 0.19) and with cimetidine (prediction: 1.07 vs. observation: 1.85), the remaining predicted ratios closely matched the observed ratios. Additionally, the PBPK model suggested that co-administration with the three perpetrators (cimetidine, enoxacin, and fluconazole) may use with caution, with CYP3A4 strong inhibitor (ketoconazole and itraconazole) or with dual CYP3A41A2 inhibitor (fluvoxamine) may reduce to half-dosage or use with caution, while co-administration with CYP3A4 strong or moderate inducer (rifampicin, efavirenz) should avoid. Overall, the present PBPK model can provide recommendations for adjusting dosing regimens in the presence of DDIs.

A Single-center, Open-label, Parallel Control Study Comparing the Pharmacokinetics and Safety of a Single Oral Dose of Roflumilast and Its Active Metabolite Roflumilast N-oxide in Healthy Chinese and Caucasian Volunteers.

Roflumilast is a phosphodiesterase-4 inhibitor which treats chronic obstructive pulmonary disease (COPD). Roflumilast N-oxide is the major metabolite of roflumilast with a similar mechanism of action to roflumilast. Although racial differences in roflumilast drug disposition have been observed, the necessity of dose adjustment is subject to debate. This study compares the pharmacokinetics of a single 500μg dose of roflumilast in healthy Chinese and Caucasian subjects under uniform conditions. Chinese subjects were found to have longer t1/2 and higher AUC0-t and Cmax than Caucasian subjects. The point estimates on the geometric mean of AUC0-t in Chinese subjects were 22% higher for roflumilast and 46% higher for roflumilast N-oxide. Point estimates on the geometric mean of Cmax were 9% and 24% higher for roflumilast and roflumilast N-oxide, respectively. Total phosphodiesterase-4 (PDE4) inhibitory (tPDE4i) activity, a theoretical parameter that describes the combined contribution to PDE4 inhibitory activity of roflumilast and roflumilast N-oxide, was 44% higher in Chinese subjects than in Caucasian subjects. With about a 10-fold higher plasma AUC compared to the parent roflumilast and a much longer observed half-life, roflumilast N-oxide has been estimated to contribute about 90% of tPDE4i, with 10% attributed to the parent compound roflumilast. Following body weight normalization, these figures were lower but remained significant. Safety analysis showed signs of reduced tolerance or different pharmacodynamic response to roflumilast in Chinese recipients than in Caucasians. Our results suggest that Chinese patients should receive a dose of roflumilast lower than 500μg daily during future clinical trials.

Pharmacokinetics of Roflumilast Cream in Chronic Plaque Psoriasis: Data from Phase I to Phase III Studies.

Most patients with chronic plaque psoriasis receive topical treatment; however, available options lack a balance of efficacy with long-term safety and tolerability. Roflumilast cream 0.3% is a highly potent phosphodiesterase 4 (PDE4) inhibitor approved by the US FDA for treatment of psoriasis. The aim of this study was to define the pharmacokinetic (PK) profile of roflumilast delivered topically from a phase I maximal usage study and data from phase II and phase III studies. PK data for roflumilast and the active metabolite, roflumilast N-oxide, were determined from a phase I PK and safety maximal usage study of roflumilast cream 0.3% applied once daily for 14 days in patients with plaque psoriasis affecting body surface area (BSA) ≥20% (N=26). Serial plasma samples were obtained on Days 1 and 15 to determine maximum plasma concentration (Cmax) and area under the concentration-time curve (AUC). Plasma concentrations were also assessed at Weeks 3, 4, and 5 for terminal half-life (t½). Concentrations of roflumilast and roflumilast N-oxide in skin were assessed at Day 28 for 14 patients with psoriasis in a phase I/IIa study of once-daily roflumilast cream 0.5% and 0.15% for 28 days. Systemic exposure (Ctrough and AUC) of roflumilast and roflumilast N-oxide in two phase III trials (DERMIS-1, n=245; DERMIS-2, n=250) of roflumilast cream 0.3% for 8 weeks was assessed at Weeks 4 and 8. Bioavailability of roflumilast cream 0.3% after topical administration was 1.5%. Unlike after oral dosing, the plasma concentration-time curve was flat, with a peak-to-trough ratio of 1.2. Roflumilast N-oxide concentrations were eightfold higher than roflumilast concentrations. The t½ in adult patients was 4.0 days for roflumilast and 4.6 days for roflumilast N-oxide following the last dose administered. Steady state was reached by Day 15. Concentrations of roflumilast in skin were, on average, 126- and 61.8-fold higher than corresponding mean plasma Ctrough following administration of roflumilast cream 0.15% and 0.5% daily for 28 days. Roflumilast N-oxide was quantifiable in only one skin sample (N=27). Following 8 weeks of treatment in DERMIS-1, mean plasma Ctrough of roflumilast was 1.78 ng/mL, and 9.86 ng/mL for roflumilast N-oxide. In DERMIS-2, mean plasma Ctrough was 1.72 ng/mL and 10.2 ng/mL, respectively. In the maximal usage study (mean BSA: 27.5%), eight patients (30.8%) experienced adverse events (AEs) and all were mild or moderate, with no reports of diarrhea, headache, insomnia, or application-site pain; no patients discontinued treatment due to an AE. Topical administration of roflumilast cream 0.3% results in concentrations in skin 126- and 61.8-fold higher relative to plasma, which are much higher than expected to be achievable with oral dosing. PDE4 inhibition in the skin is likely due to roflumilast as compared with its active metabolite, as there is no significant conversion to roflumilast N-oxide in the skin. Consistent with reservoir formation and retention of drug in the stratum corneum, roflumilast is slowly released from the skin (t½ 4 days) and peak-to-trough ratiois 1.2. NCT04279119, NCT03392168, NCT04211363, NCT04211389.

Clinical Relevance of the Anti-inflammatory Effects of Roflumilast on Human Bronchus: Potentiation by a Long-Acting Beta-2-Agonist.

Background: Roflumilast is an option for treating patients with severe COPD and frequent exacerbations despite optimal therapy with inhaled drugs. The present study focused on whether the phosphodiesterase (PDE) 4 inhibitor roflumilast and its active metabolite roflumilast N-oxide affect the release of tumor necrosis factor (TNF)-α and chemokines by lipopolysaccharide (LPS)-stimulated human bronchial explants. We also investigated the interactions between roflumilast, roflumilast N-oxide and the β2-agonist formoterol with regard to cytokine release by the bronchial preparations. Methods: Bronchial explants from resected lungs were incubated with roflumilast, roflumilast N-oxide and/or formoterol and then stimulated with LPS. An ELISA was used to measure levels of TNF-α and chemokines in the culture supernatants. Results: At a clinically relevant concentration (1 nM), roflumilast N-oxide and roflumilast consistently reduced the release of TNF-α, CCL2, CCL3, CCL4, CCL5 and CXCL9 (but not CXCL1, CXCL5, CXCL8 and IL-6) from human bronchial explants. Formoterol alone decreased the release of TNF-α, CCL2, and CCL3. The combination of formoterol with roflumilast (1 nM) was more potent than roflumilast alone for inhibiting the LPS-induced release of TNF-α, CCL2, CCL3, CCL4, and CXCL9 by the bronchial explants. Conclusions: At a clinically relevant concentration, roflumilast N-oxide and its parent compound, roflumilast, reduced the LPS-induced production of TNF-α and chemokines involved in monocyte and T-cell recruitment but did not alter the release of chemokines involved in neutrophil recruitment. The combination of formoterol with roflumilast enhanced the individual drugs’ anti-inflammatory effects.

Clinical relevance of the relaxant effects of roflumilast on human bronchus: potentiation by a long-acting beta-2-agonist.

Roflumilast is an oral, add-on option for treating patients with severe COPD and frequent exacerbations despite optimal therapy with inhaled drugs. The present study focused on whether this phosphodiesterase 4 inhibitor and its active metabolite roflumilast N-oxide affect the tone of human bronchial rings. We also investigated the interactions between roflumilast, roflumilast N-oxide and the long-acting β2 -agonist formoterol with regard to the relaxation of isolated human bronchial rings at basal tone or pre-contracted with histamine. Our results demonstrated for the first time that at a clinically relevant concentration (1nm), roflumilast N-oxide and roflumilast induce a weak relaxation of the isolated human bronchus either at resting tone (22% and 16%, respectively) or even weaker on pre-contracted bronchus with histamine (7% and 5%, respectively). In addition, the combination of formoterol with roflumilast or roflumilast N-oxide is more potent than each component alone for relaxing pre-contracted isolated bronchi - the apparent pD2 of formoterol was significantly reduced for the threshold concentration of 1nm of the phosphodiesterase 4 inhibitors by a factor of 2.4 for roflumilast N-oxide and 1.9 for roflumilast. The full inhibition of phosphodiesterase 4 activity is achieved at 100nm but this high concentration only caused partial relaxations of the human bronchi. At a clinically relevant concentration, these oral phosphodiesterase 4 inhibitors are not effective direct bronchodilators but could enhance the efficacy of inhaled long-acting β2-agonists.

The effect and associated mechanism of action of phosphodiesterase 4 (PDE4) inhibitor on CD4+ lymphocyte proliferation.

PDE4 inhibitors are involved in anti-inflammatory and immunomodulatory responses. Recently, they have been getting attention as a new class of drugs treating inflammatory airway diseases. The T lymphocyte is a major cell type present in the inflammatory infiltrate in the airway wall in patients with chronic obstructive pulmonary disease (COPD), and a previous study found that treatment with a PDE4 inhibitor significantly suppressed T cell proliferation. However, the mechanism of action of PDE4 inhibitors has not been elucidated. The present study aimed to investigate major signal transduction pathways of T lymphocyte and identify the phase, during which PDE4 inhibitors affect T cell proliferation. Isolated splenic CD4+ T cells were grown under stimulation with an anti-CD3/CD28 antibody, and/or treated with roflumilast-n-oxide (RNO). A western blot assay was performed using major antibodies including anti-p-38, anti-p-PI3K, anti-p-JNK, anti-p-ERK 1/2, anti-NFAT1 (NFATc2), and anti-NF-kB antibodies. Additional experiments conducted on the pathway showed significant change following RNO treatment, thus providing further evidence for signal transduction pathway concerning PDE4 inhibitors. T cell proliferation was suppressed by RNO treatment. In the pathways involved in T cell proliferation, only expression of anti-NFAT1 antibody was suppressed by RNO treatment. In additional experiments on the NFAT pathway, the very first phase (TCR signalling) remained unchanged on treatment with RNO, but RNO treatment increased IP3R expression and suppressed calcineurin activity. Calcineurin activity, reduced by RNO, increased on treatment with an IP3 receptor agonist. PED4 inhibitor, roflumilast is speculated to suppress T cell proliferation by interfering with IP3-IP3R binding to inhibit calcium emission, blocking pathway activation from this phase onward, eventually decreasing the level of a growth factor for T cell proliferation, IL-2.

The Possible Nephroprotective Effect of Roflumilast Ameliorating Glycerol -Induced Myoglobinuric Acute Renal Injury in Rabbits Model

urther leads to tissue necrosis and inflammation. While, roflumilast N-oxide is an active metabolite ofroflumilast that rising the cyclic adenosine-3?, 5?-monophosphate by inhibiting PDE4.Thirty rabbits were divided into three groups, control group was received normal saline intra-muscularly anddrived from water for eighteen hours before. Second group was received a single dose glycerol injected intramuscularly after drived from water for eighteen hours .Third group was received roflumilast intraperitonealdaily for three days after received a single dose of glycerol .All animals were sacrificed after 72 hour, and then blood collected by cardiac puncture for evaluation thechanges in the levels of renal function tests and histopathological study of rabbit’s kidneys tissues areevaluated evaluation and scoring the changes of tested agents. The numerical data are expressed as means ±standard error means and analyzed by using SPSS version 20.The results of the levels serum urea and creatinine found to be significantly reduced by roflumilast usingANOVA test. Besides, the histopathological changes showed ameliorative effects of roflumilast in comparedto glycerol group. From this study, one could be concluded the protective effect of roflumilast in case ofrenal failure induction by glycerol.

Impact of Phosphodiesterase 4 Inhibition on the Operational Efficacy, Response Maxima, and Kinetics of Indacaterol-Induced Gene Expression Changes in BEAS-2B Airway Epithelial Cells: A Global Transcriptomic Analysis.

The effects of phosphodiesterase (PDE) 4 inhibitors on gene expression changes in BEAS-2B human airway epithelial cells are reported and discussed in relation to the mechanism(s) of action of roflumilast in chronic obstructive pulmonary disease (COPD). Microarray-based gene expression profiling failed to identify mRNA transcripts that were differentially regulated by the PDE4 inhibitor 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) after 1, 2, 6, or 18 hours of exposure. However, real-time polymerase chain reaction analysis revealed that GSK 256066 was a weak stimulus, and the negative microarray results reflected low statistical power due to small sample sizes. Furthermore, GSK 256066, roflumilast, and its biologically active metabolite roflumilast N-oxide generally potentiated gene expression changes produced by the long-acting β 2-adrenoceptor agonists (LABAs) salmeterol, indacaterol, and formoterol. Many of these genes encode proteins with antiviral, anti-inflammatory, and antibacterial activities that could contribute to the clinical efficacy of roflumilast in COPD. RNA-sequencing experiments established that the sensitivity of genes to salmeterol varied by ∼7.5-fold. Consequently, the degree to which a PDE4 inhibitor potentiated the effect of a given concentration of LABA was gene-dependent. Operational model fitting of concentration-response curve data from cells subjected to fractional, β 2-adrenoceptor inactivation determined that PDE4 inhibition increased the potency and doubled the efficacy of LABAs. Thus, adding roflumilast to standard triple therapy, as COPD guidelines recommend, may have clinical relevance, especially in target tissues where LABAs behave as partial agonists. Collectively, these results suggest that the genomic impact of roflumilast, including its ability to augment LABA-induced gene expression changes, may contribute to its therapeutic activity in COPD.

The Effect of Phosphodiesterase 4 Inhibition on Gene Expression Changes Produced by Long Acting β2‐adrenoceptor Agonists in BEAS‐2B Airway Epithelial Cells

Phosphodiesterase 4 (PDE4) inhibitors are prescribed as add‐on therapy to a subset of COPD patients that suffer from exacerbations despite treatment with a combination of long‐acting β2‐adrenoceptor agonists (LABA), long‐acting muscarinic receptor antagonist and inhaled corticosteroids. The molecular mechanisms of their beneficial effects in COPD are ill‐defined. We have previously shown that LABAs, through their effect on the canonical Gs‐cAMP pathway, promote genomic effects in airway epithelial cells. Herein, we hypothesized that PDE4 inhibitors, by inhibiting cAMP degradation, promote similar genomic effects, and further potentiate the effects of LABAs in a human bronchial epithelial cell line. Additionally, we investigated whether this interaction between PDE4 inhibitors and LABAs occurred in an additive or a synergistic manner.Gene expression changes were determined by population‐based RNA‐sequencing in BEAS‐2B cells treated with the PDE4 inhibitor, roflumilast N‐oxide (RNO), alone and in combination with LABA, salmeterol. RNO showed a weak stimulation of gene expression by itself but showed a strong rank‐order correlation with salmeterol‐induced gene expression changes, suggesting a common mechanism of action. RNO augmented the expression of all salmeterol‐regulated genes, but the magnitude of augmentation varied between genes. This variation can be explained by an estimated 7.5‐fold difference in sensitivities of genes to salmeterol. Deming regression analysis of the gene expression changes produced by the combination of salmeterol and RNO versus the sum of their individual effects showed a deviation away from unity slope that may be explained by a synergistic effect of RNO at some DEGs. Consistent with these results, PDE4 inhibitors, GSK 256066, and roflumilast potentiated the effects of LABAs, indacaterol, and formoterol, on a panel of randomly‐selected genes as measured by RT‐PCR.Collectively, these data suggest that PDE4 inhibition may synergistically augment LABA‐induced gene expression changes in airway epithelial cells. This interaction between LABAs and PDE4 inhibitors may contribute to the therapeutic benefits of their combination in COPD.Support or Funding InformationCanadian Institutes for Health Research (PJT 152904)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Direct Inhibitory Effect of the PDE4 Inhibitor Roflumilast on Neutrophil Migration in Chronic Obstructive Pulmonary Disease.

Neutrophilic inflammation is characteristic of chronic obstructive pulmonary disease (COPD); yet, there are no effective antiinflammatory therapies. The PDE4 inhibitor roflumilast is approved for use in COPD and suppresses sputum neutrophilia. The mechanism underlying this observation is unclear; therefore, this study addressed whether roflumilast directly affected neutrophil migration. Blood-derived neutrophils were isolated from nonsmokers, smokers, and patients with COPD, and chemotaxis was measured using Boyden chambers. Intracellular calcium ion concentration was measured by fluorimetry, and shape change and CD11b expression were measured by flow cytometry. Neutrophils from patients with COPD showed enhanced chemotactic responses toward both CXCL1 and leukotriene B4 compared with control cells. Chemotaxis was inhibited by both the active metabolite roflumilast N-oxide and rolipram in a concentration-dependent manner with no difference in responsiveness between subjects. Roflumilast N-oxide and rolipram were less efficacious against CXCL1 and leukotriene B4-mediated intracellular calcium ion concentration, suggesting that inhibition was not via this pathway. Both PDE4 inhibitors attenuated chemoattractant-mediated shape change and CD11b upregulation, suggesting common mechanisms. The stable cAMP analog 8-bromoadenosine 3',5'-cAMP inhibited chemotaxis, as did the direct Epac1 (exchange protein directly activated by cAMP 1) activator 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cAMP but not the direct protein kinase A activator N6-benzoyladenosine-3',5'-cAMP. These data suggest that roflumilast inhibits neutrophil chemotaxis directly via a cAMP-mediated mechanism requiring activation of Epac1 and that Epac1 activators could reduce COPD neutrophilic inflammation.

Determination of Phosphodiesterase Inhibitors Tadalafil, Roflumilast and Roflumilast N-Oxide Using LC-MS in Guinea Pig Plasma.

Phosphodiesterases are known as a super-family of 11 isoenzymes, which can exert various functions based on their organ distribution. In this work, a rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed for quantification of tadalafil (phosphodiesterase five inhibitor), roflumilast (RF) (phosphodiesterase four inhibitor) and its active metabolite, RF N-oxide in guinea pig plasma. Chromatographic separation was carried out on UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) at a flow rate 0.5 mL/min, using 0.2% formic acid in acetonitrile and 0.2% formic acid in water as mobile phases within 4 min. Detection was performed using a triple quadrupole mass spectrometer employing electrospray ionization operated in positive mode using multiple reaction monitoring mode. The method utilized deuterium labeled internal standards, and was validated according to European Medicines Agency guidelines. It showed excellent linearity in the range of 0.5-500.0 ng/mL for all analytes with coefficient of determination >0.99. The intra- and inter-day precisions (relative standard deviation %) were within 6.7%, and the recoveries were greater than 73.4%. Using this method, plasma samples from experiments of phosphodiesterase four, and five inhibitors in a model of ovalbumin-induced allergic inflammation in guinea pigs were analyzed.

The phosphodiesterase-4 inhibitor roflumilast reverts proteolysis in skeletal muscle cells of patients with COPD cachexia.

Peripheral muscle weakness and mass loss are characteristic features in severe chronic obstructive pulmonary disease (COPD). We hypothesized that the phosphodiesterase (PDE)-4 inhibitor roflumilast-induced cAMP may ameliorate proteolysis and metabolism in skeletal muscles of COPD patients with severe muscle wasting. In myogenic precursor cells (isolated from muscle biopsies and cultured up to obtain differentiated myotubes) from 10 severe COPD patients and 10 healthy controls, which were treated with 1 μM roflumilast N-oxide (RNO) for three time cohorts (1, 6, and 24 h), genes of antioxidant defense and oxidative stress marker, myogenesis and muscle metabolism, proteolysis (tyrosine release assay) and ubiquitin-proteasome system markers, autophagy, and myosin isoforms were analyzed using RT-PCR and immunoblotting. In COPD patients at 6 h RNO treatment, myotube tyrosine release, total protein ubiquitination, and tripartite motif-containing protein 32 levels were significantly lower than healthy controls, whereas at 24 h RNO treatment, myotube myosin heavy chain ( MyHC) -I and MyHC-IIx expression levels were upregulated in both patients and controls. In the 6-h RNO cohort, in patients and controls, myotube expression of nuclear factor (erythroid-derived 2)-like 2 ( NRF2) and its downstream antioxidants sirtuin-1, FGF-inducible 14, and insulin-like growth factor-1 was upregulated, whereas that of myocyte-specific enhancer factor 2C, myogenic differentiation, myogenin, myostatin, atrogin-1, and muscle RING-finger protein-1 was downregulated. In myotubes of severe COPD patients with cachexia, roflumilast-induced cAMP signaling exerts beneficial effects by targeting muscle protein breakdown (tyrosine release), along with reduced expression of proteolytic markers of the ubiquitin-proteasome system and that of myostatin. In both patients and controls, roflumilast also favored antioxidant defense through upregulation of the NRF2 pathway and that of the histone deacetylase sirtuin-1, whereas it improved the expression of slow- and fast-twitch myosin isoforms. These findings show that muscle dysfunction and wasting may be targeted by roflumilast-induced cAMP signaling in COPD. These results have potential therapeutic implications, as this PDE-4 inhibitor is currently available for the treatment of systemic inflammation and exacerbations in patients with severe COPD. NEW & NOTEWORTHY In myotubes of cachectic chronic obstructive pulmonary disease (COPD) patients, cAMP signaling exerted beneficial effects by targeting muscle proteolysis and reducing gene expression of proteolytic markers of the ubiquitin-proteasome system and that of myostatin. In myotubes of patients and controls, roflumilast also favored antioxidant defense through upregulation of the nuclear factor (erythroid-derived 2)-like 2 pathway, of sirtuin-1, and of gene expression of slow- and fast-twitch isoforms. These findings have potential clinical implications for the treatment of muscle wasting in patients with COPD and cachexia.

Roflumilast N-Oxide in Combination with Formoterol Enhances the Antiinflammatory Effect of Dexamethasone in Airway Smooth Muscle Cells.

Roflumilast is an orally active phosphodiesterase 4 inhibitor approved for use in chronic obstructive pulmonary disease. Roflumilast N-oxide (RNO) is the active metabolite of roflumilast and has a demonstrated antiinflammatory impact in vivo and in vitro. To date, the effect of RNO on the synthetic function of airway smooth muscle (ASM) cells is unknown. We address this herein and investigate the effect of RNO on β2-adrenoceptor-mediated, cAMP-dependent responses in ASM cells in vitro, and whether RNO enhances steroid-induced repression of inflammation. RNO (0.001-1,000 nM) alone had no effect on AMP production from ASM cells, and significant potentiation of the long-acting β2-agonist formoterol-induced cAMP could only be achieved at the highest concentration of RNO tested (1,000 nM). At this concentration, RNO exerted a small, but not significantly different, potentiation of formoterol-induced expression of antiinflammatory mitogen-activated protein kinase phosphatase 1. Consequently, tumor necrosis factor-induced IL-8 secretion was unaffected by RNO in combination with formoterol. However, because there was the potential for phosphodiesterase 4 inhibitors and long-acting β2-agonists to interact with corticosteroids to achieve superior antiinflammatory efficacy, we examined whether RNO, alone or in combination with formoterol, enhanced the antiinflammatory effect of dexamethasone by measuring the impact on IL-8 secretion. Although RNO alone did not significantly enhance the cytokine repression achieved with steroids, RNO in combination with formoterol significantly enhanced the antiinflammatory effect of dexamethasone in ASM cells. This was linked to increased mitogen-activated protein kinase phosphatase 1 expression in ASM cells, suggesting that a molecular mechanism is responsible for augmented antiinflammatory actions of combination therapeutic approaches that include RNO.

Roflumilast and aquaporin-2 regulation in rat renal inner medullary collecting duct.

Roflumilast is a cyclic nucleotide phosphodiesterase inhibitor that is FDA‐approved for treatment of chronic obstructive pulmonary disease. With a view toward possible use for treatment of patients with X‐linked nephrogenic diabetes insipidus (NDI) due to hemizygous mutations in the V2 vasopressin receptor, this study sought to determine the effect of roflumilast on aquaporin‐2 (AQP2) phosphorylation, AQP2 trafficking, and water permeability in the rat inner medullary collecting duct (IMCD). In the presence of the vasopressin analog dDAVP (0.1 nmol/L), both roflumilast and its active metabolite roflumilast N‐oxide (RNO) significantly increased phosphorylation at S256, S264, and S269, and decreased phosphorylation at S261 (immunoblotting) in IMCD suspensions in a dose‐dependent manner (3–3000 nmol/L). Another commonly used phosphodiesterase inhibitor, IBMX, affected phosphorylation only at the highest concentration in this range. However, neither roflumilast nor RNO had an effect on AQP2 phosphorylation in the absence of vasopressin. Furthermore, roflumilast alone did not increase AQP2 trafficking to the plasma membrane (immunofluorescence) or increase water permeability in freshly microdissected perfused IMCD segments. We conclude that roflumilast can be used to enhance vasopressin's action on AQP2 activity in the renal collecting duct, but has no detectable effect in the absence of vasopressin. These findings suggest that roflumilast may not have a beneficial effect in X‐linked NDI, but could find useful application in acquired NDI.

PDE4 Inhibition as Potential Treatment of Epidermolysis Bullosa Acquisita

Pemphigoid diseases such as epidermolysis bullosa acquisita (EBA) may be difficult to treat. In pemphigoid diseases, mucocutaneous blistering is caused by autoantibodies to hemidesmosomal antigens; in EBA the autoantigen is type VII collagen. Despite growing insights into pemphigoid disease pathogenesis, corticosteroids are still a mainstay of treatment. In experimental EBA, myeloid cell activation is a key event leading to blistering. Activation of these cells depends on phosphodiesterase (PDE) 4. We therefore evaluated the potential for PDE4 inhibition in EBA: PDE4 was highly expressed in inflammatory cells and in the epidermis of patients compared with healthy skin samples. PDE4 inhibitors rolipram, roflumilast, and roflumilast N-oxide prevented the release of immune complex-induced reactive oxygen species from polymorphonuclear leukocytes and separation of the dermal-epidermal junction of skin incubated with antibodies to collagen type VII and polymorphonuclear leukocytes. The PDE4 inhibitors also impaired CD62L shedding and decreased CD11b expression on immune complex-stimulated polymorphonuclear leukocytes. For invivo validation, experimental EBA was induced in mice by transfer of anti-collagen type VII IgG or immunization with collagen type VII. Roflumilast dose-dependently reduced blistering in antibody transfer-induced EBA and also hindered disease progression in immunization-induced EBA. PDE4 inhibition emerges as a new treatment modality for EBA and possibly other neutrophil-driven pemphigoid diseases.

Pharmacokinetics of Roflumilast and Its Active Metabolite Roflumilast N-Oxide in Healthy Chinese Subjects After Single and Multiple Oral Doses

Roflumilast is a selective, oral phosphodiesterase 4 inhibitor approved for the treatment of severe chronic obstructive pulmonary disease. The aim of this study was to evaluate the pharmacokinetics of roflumilast and roflumilast N-oxide in healthy Chinese subjects, and the effects of gender and food on their respective pharmacokinetic profiles. 36 healthy Chinese subjects were recruited in a randomized, single-center, open-label, parallel group study and assigned to 0.25-, 0.375-, and 0.5-mg dose groups. The single-dose pharmacokinetic studies in fasting condition were carried out in all groups. Moreover, the food effect study and multiple-dose study were conducted in 0.375-mg dose group. Serial blood samples were collected over 168h after dosing, and plasma concentrations of roflumilast and roflumilast N-oxide were determined using a validated LC-MS/MS method. After oral administration of single doses of 0.25, 0.375 and 0.5mg of roflumilast under fasting condition, the mean AUC0-72h for roflumilast was 21.7±8.3, 29.8±8.3 and 54.2±21.3ng·h/mL, respectively. Meanwhile the mean AUC0-168h for roflumilast N-oxide was 290±103, 385±107 and 673±245ng·h/mL, respectively. In the steady state after the multi-dose administration, the exposure to roflumilast in the subjects increased 20-40%, and the exposure to roflumilast N-oxide increased about 169%, compared to the single-dose administration. No statistically significant effect of gender on the disposition of roflumilast and roflumilast N-oxide was observed. Food had no effect on systemic exposure to roflumilast and roflumilast N-oxide in the subjects, but delayed the T max of roflumilast by 0.9h and reduced the C max of roflumilast by approximately 20%. Based upon between-study comparison, peak and systemic exposure of roflumilast and roflumilast N-oxide were higher in Chinese than that in Caucasian subjects after oral administration of the same dose (i.e., 0.25 and 0.5mg). It implies that the therapeutic dose for Chinese patients may be different from that for Caucasians, warranting further investigation.

Simultaneous determination of roflumilast and its metabolite in human plasma by LC-MS/MS: Application for a pharmacokinetic study.

Roflumilast had shown good efficacy and safety in Caucasian COPD patients after oral administration of 0.5mg. The main active metabolite of it is roflumilast N-oxide. A reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitation method was developed for the simultaneous determination of them in human plasma with rather low limits of quantitation for roflumilast (0.02ng/mL) and roflumilast N-oxide (0.04ng/mL). Human plasma samples were prepared by solid phase extraction (SPE), which ensured high recovery and slight matrix effect for the both analytes. This method showed good linearity, accuracy, precision and stability in the range of 0.02-10ng/mL and 0.04-50ng/mL for roflumilast and roflumilast N-oxide respectively. The developed method was successfully applied for the pharmacokinetic research in Chinese healthy volunteers after oral administration of 0.25mg, 0.375mg and 0.5mg of roflumilast tablet.

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

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. 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. 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). 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. 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 β-adrenergic receptor agonist. This study reveals novel antithrombotic activities by which roflumilast may exert protective effects against cardiovascular comorbodities in COPD.

Roflumilast n-oxide associated with PGE2 prevents the neutrophil elastase-induced production of chemokines by epithelial cells

Neutrophil chemotaxis is involved in the lung inflammatory process in conditions such as chronic obstructive pulmonary disease (COPD). Neutrophil elastase (NE), one of the main proteases produced by neutrophils, has an important role in the inflammatory process via the release of chemokines from airway epithelial cells. It was recently shown that roflumilast N-oxide has therapeutic potential in COPD. The aim of the present study was to investigate roflumilast N-oxide's effect on NE-induced chemokine production and signaling pathways in A549 epithelial cells.A549 cells were incubated with NE for 30min, washed with PBS and then cultured for 2h (for measurement of mRNA expression) and 24h (for chemokine release) or for 5 to 30min (for protein phosphorylation assays). Prior to the addition of NE, cells were also pre-incubated with prostaglandin E2 (PGE2), alone and in combination with roflumilast N-oxide.Addition of NE was associated with elevated chemokine production by A549 cells and induction of the p38α pathway. In contrast when combined with PGE2, the roflumilast N-oxide had an additive effect on the inhibition of NE-induced chemokine release and p38α and other kinases activation.In conclusion, we demonstrated that NE is able to increase the release of chemokines from epithelial cells via the activation of p38α MAP-kinase and that roflumilast N-oxide when combined with PGE2 lowers NE-induced kinase activation and chemokine production.

Phosphodiesterase-4 inhibitors ameliorates cognitive deficits in deoxycorticosterone acetate induced hypertensive rats via cAMP/CREB signaling system

Phosphodiesterase-4 (PDE-4) inhibitors promote memory by blocking the degradation of cAMP. Existing evidence also shows that neuronal survival and plasticity are dependent on the phosphorylation of cAMP-response element-binding protein. In this regard, PDE-4 inhibitors have also been shown to reverse pharmacologically and genetically induced memory impairment in animal models. In the present study, the authors examined the effect of both rolipram and roflumilast (PDE-4 inhibitors) on the impairment of learning and memory observed in hypertensive rats. Deoxycorticosterone acetate (DOCA) salt hypertensive model was used to induce learning and memory deficits. The mRNA expression of different PDE-4 subtypes along with the protein levels of pCREB and BDNF in the hippocampus was quantified. Systolic blood pressure was significantly increased in DOCA salt hypertensive rats when compared to sham operated rats. This effect was reversed by clonidine, an α2 receptor agonist, while PDE-4 inhibitors did not. PDE-4 inhibitors significantly improved the time-induced memory deficits in object recognition task (ORT). In DOCA salt hypertensive rats, the gene expression of PDE-4B and PDE-4D was significantly increased. Furthermore, both pCREB and BDNF showed decreased levels of expression in hypertensive rats in comparison to sham operated rats. Repeated administration of PDE-4 inhibitors significantly decreased both PDE-4B and PDE-4D with an increase in the expression of pCREB and BDNF in hypersensitive rats. Also, rolipram, roflumilast and roflumilast N-oxide showed a linear increase in the plasma and brain concentrations after ORT. Our present findings suggested that PDE-4 inhibitors ameliorate hypertension-induced learning impairment via cAMP/CREB signaling that regulates BDNF expression downstream in the rat hippocampus.