vardenafil-dihydrochloride and Cystic-Fibrosis

Chronic inflammation that progressively disrupts the lung tissue is a hallmark of cystic fibrosis (CF). In mice, vardenafil, an inhibitor of phosphodiesterase type 5 (PDE5), restores transepithelial ion transport and corrects mislocalization of the most common CF mutation, F508del-CFTR. It also reduces lung pro-inflammatory responses in mice and in patients with CF. To test the hypothesis that macrophages are target effector cells of the immunomo-dulatory effect of vardenafil, we isolated lung macrophages from mice homozygous for the F508del mutation or invalidated for the

Topics: Animals; Cell Polarity; Cells, Cultured; Cyclic Nucleotide Phosphodiesterases, Type 5; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Drug Evaluation, Preclinical; Inflammation Mediators; Macrophages; Macrophages, Alveolar; Mice, Inbred CFTR; Molecular Targeted Therapy; Phosphodiesterase 5 Inhibitors; Tumor Necrosis Factor-alpha; Vardenafil Dihydrochloride

Morbi-mortality in cystic fibrosis (CF) is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.

Topics: Animals; Bleomycin; Cell Line; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Female; Fibroblasts; Gene Expression; Imidazoles; Immunohistochemistry; Inflammation Mediators; Lipopolysaccharides; Lung; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Inbred CFTR; Phenotype; Piperazines; Pseudomonas aeruginosa; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; Skin; Sulfones; Triazines; Vardenafil Dihydrochloride; Vasodilator Agents

Although lung disease is the major cause of mortality in cystic fibrosis (CF), gastrointestinal (GI) manifestations are the first hallmarks in 15-20% of affected newborns presenting with meconium ileus, and remain major causes of morbidity throughout life. We have previously shown that cGMP-dependent phosphodiesterase type 5 (PDE5) inhibitors rescue defective CF Transmembrane conductance Regulator (CFTR)-dependent chloride transport across the mouse CF nasal mucosa. Using F508del-CF mice, we examined the transrectal potential difference 1 hour after intraperitoneal injection of the PDE5 inhibitor vardenafil or saline to assess the amiloride-sensitive sodium transport and the chloride gradient and forskolin-dependent chloride transport across the GI tract. In the same conditions, we performed immunohistostaining studies in distal colon to investigate CFTR expression and localization. F508del-CF mice displayed increased sodium transport and reduced chloride transport compared to their wild-type littermates. Vardenafil, applied at a human therapeutic dose (0.14 mg/kg) used to treat erectile dysfunction, increased chloride transport in F508del-CF mice. No effect on sodium transport was detected. In crypt colonocytes of wild-type mice, the immunofluorescence CFTR signal was mostly detected in the apical cell compartment. In F508del-CF mice, a 25% reduced signal was observed, located mostly in the subapical region. Vardenafil increased the peak of intensity of the fluorescence CFTR signal in F508del-CF mice and displaced it towards the apical cell compartment. Our findings point out the intestinal mucosa as a valuable tissue to study CFTR transport function and localization and to evaluate efficacy of therapeutic strategies in CF. From our data we conclude that vardenafil mediates potentiation of the CFTR chloride channel and corrects mislocalization of the mutant protein. The study provides compelling support for targeting the cGMP signaling pathway in CF pharmacotherapy.

Topics: Animals; Cations, Monovalent; Cell Polarity; Chlorides; Colon; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Expression; Humans; Imidazoles; Intestinal Mucosa; Ion Transport; Male; Membrane Potentials; Mice; Mice, Inbred CFTR; Phosphodiesterase 5 Inhibitors; Piperazines; Sodium; Sulfones; Triazines; Vardenafil Dihydrochloride

We tested the hypothesis that vardenafil, a common drug used for improving erectile dysfunction and able to partially normalize transepithelial chloride transport in cystic fibrosis (CF), modulates CF lung inflammation.. Inflammatory markers in lungs of F508del-CF and wild-type mice were monitored in response to lipopolysaccharide from Pseudomonas aeruginosa (LPS). The effect of pretreatment with vardenafil (0.14 mg/kg) was evaluated.. A latent inflammatory status, characterized by neutrophil infiltrate, mouse macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-α, was found in baseline conditions in F508del-CF mice. Inflammatory markers were increased after LPS with higher responses in CF. Vardenafil globally attenuated inflammatory responses in both genotypes however reduction of macrophage infiltration, macrophage chemoattractant chemokine and interleukin-1β was observed in the CF group only.. Vardenafil reduces lung inflammation with a more pronounced effect in F508del-CF mice, particularly on macrophage cell markers.

Topics: Animals; Animals, Outbred Strains; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Female; Imidazoles; Immunomodulation; Lipopolysaccharides; Macrophages; Male; Mice; Mice, 129 Strain; Mice, Inbred CFTR; Phosphodiesterase 5 Inhibitors; Piperazines; Pneumonia; Pseudomonas Infections; Sulfones; Triazines; Vardenafil Dihydrochloride

Sildenafil and vardenafil, two selective inhibitors of phosphodiesterase type 5 (PDE5) are able, when applied by intraperitoneal injection, to activate chloride transport in cystic fibrosis (CF) mice homozygous for the F508del mutation. Oral treatment with the drugs may be associated with adverse haemodynamic effects. We hypothesised that inhaled PDE5 inhibitors are able to restore ion transport in F508del CF airway epithelium. We developed a restraint-free mouse chamber for inhalation studies. PDE5 inhibitors were nebulised for 15 min at concentrations adjusted from recommended therapeutic oral doses for male erectile dysfunction. We measured in vivo nasal transepithelial potential difference 1 h after a single inhalation of sildenafil, vardenafil or tadalafil in F508del CF and normal homozygous mice. After nebulisation with the drugs in F508del mice, chloride transport, evaluated by perfusing the nasal mucosa with chloride-free buffer containing amiloride followed by forskolin, was normalised; the forskolin response was increased, with the largest values being observed with tadalafil and intermediate values with vardenafil. No detectable effect was observed on sodium conductance. Our results confirm the role of PDE5 inhibitors in restoring chloride transport function of F508del CF transmembrane conductance regulator protein and highlight the potential of inhaled sildenafil, vardenafil and tadalafil as a therapy for CF.

Topics: Administration, Inhalation; Animals; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Homozygote; Humans; Imidazoles; Mice; Mice, Transgenic; Nebulizers and Vaporizers; Phosphodiesterase 5 Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Purines; Sildenafil Citrate; Sulfones; Time Factors; Triazines; Vardenafil Dihydrochloride

Topics: Administration, Oral; Animals; Cyclic Nucleotide Phosphodiesterases, Type 5; Cystic Fibrosis; Disease Models, Animal; Epithelial Sodium Channels; Genetic Therapy; Humans; Imidazoles; Infant, Newborn; Mice; Mutation; Neonatal Screening; Oocytes; Piperazines; Sulfones; Triazines; Vardenafil Dihydrochloride; Xenopus

Sildenafil has been implicated in the activation of cystic fibrosis transmembrane conductance regulator (CFTR) protein. The effect was observed in vitro and in the presence of doses roughly 300 times larger than those commonly used for treating erectile dysfunction.. To evaluate in vivo the therapeutic efficacy of clinical doses of sildenafil and vardenafil, two clinically approved phosphodiesterase 5 inhibitors, for activating ion transport in cystic fibrosis.. We used transepithelial potential difference in vivo across the nasal mucosa as a measure of sodium and chloride transport. The effect of a single intraperitoneal injection of sildenafil (0.7 mg/kg) or vardenafil (0.14 mg/kg) was investigated in F508del, cftr knockout and normal homozygous mice.. In F508del mice, but not in cftr knockout mice, the chloride conductance, evaluated by perfusing the nasal mucosa with a chloride-free solution in the presence of amiloride and with forskolin, was corrected 1 hour after sildenafil administration. A more prolonged effect, persisting for at least 24 hours, was observed with vardenafil. The forskolin response was increased after sildenafil and vardenafil in both normal and F508del mutant animals. In F508del mice, the chloride conductance in the presence of 200 microM 4-4'-diisothiocyanostilbene-2,2'-disulphonic acid, an inhibitor of alternative chloride channels, was much higher after sildenafil injection than after placebo treatment. No effect on the sodium conductance was detected in any group of animals.. Our results provide preclinical evidence that both drugs stimulate chloride transport activity of F508del-CFTR protein.

Topics: Animals; Chlorides; Colforsin; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Imidazoles; Injections, Intraperitoneal; Ion Transport; Membrane Potentials; Mice; Mice, Inbred CFTR; Nasal Mucosa; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Piperazines; Protein Modification, Translational; Purines; Sildenafil Citrate; Sulfones; Triazines; Vardenafil Dihydrochloride