Sildenafil and analogues as potential therapy for cystic fibrosis
(2011)
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- (en) Cystic fibrosis (CF) is a common lethal genetic disease affecting mainly Caucasian populations and caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The encoded protein, also named CFTR, is a cAMP regulated anion channel expressed in airways, intestinal, pancreatic, and other cells. It regulates luminal fluid volume and composition in the respiratory and gastrointestinal tracts. The most common CFTR mutation results in deletion of a single phenylalanine residue at position 508 (F508del) and causes defective synthesis and folding of the mutant protein that fails to escape the endoplasmic reticulum and reach the apical membrane of many epithelial cell types. CF epithelia are characterized by defective electrolyte transport, namely reduced chloride conductance and increased sodium conductance. These abnormalities can be assessed in vivo by measuring transepithelial nasal potential difference (PD), which has been increasingly used as an index of therapeutic efficacy in novel fundamental therapies either in patients or in animal models. Airway dehydrated hyperviscous mucus in patients with CF disrupts normal mucociliary clearance of inhaled pathogens and leads to recurring airway infection. Intestinal epithelia are also abnormal, and intestinal obstruction (meconium ileus) is an early manifestation in newborns with CF. Despite the clear link between abnormal ion transport and CF, the pathogenesis of lung disease in CF is complex and still debated. The initiating event in CF-airway disease pathogenesis seems to be reduced airway surface liquid volume that develops, in large part, as a consequence of abnormal ion transport. A compound capable of rescuing the F508del protein function and of normalizing ion transport abnormalities could be considered, in contrast to symptomatic or alleviating treatments currently available, as a disease-modifying therapeutic agent and would be beneficial for most patients with CF. The generation of animal models that mimic the human phenotype has been an important progress in CF research. To date, 13 mouse models have been created and 3 F508del strains are available. The prevalence of the F508del mutation along with its severe phenotype makes the F508del-CF mouse a useful model to test novel drugs that target the mutant CFTR processing and turnover. Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of the second messengers, cAMP and/or cGMP, by controlling their rates of degradation. Eleven families of PDEs with varying selectivities for cAMP and/or cGMP have been identified in mammalian tissues. Within these families, over 50 isoforms are expressed, either as products of different genes or as products of the same gene through alternative splicing. PDE5 is highly specific for cGMP and is involved in the regulation of the intracellular concentration of cGMP in various tissues. It was recently reported that sildenafil, a PDE5 inhibitor, increases the expression of F508del-CFTR protein in nasal epithelial cells harvested from patients with CF. The effect, observed in the presence of 150 μM sildenafil, a dose roughly 300 times larger than that usually used in male erectile dysfunction, was associated with an increase in the activity of chloride transport. This project aimed at evaluating the potential of clinical doses of PDE5 inhibitors (sildenafil, vardenafil and tadalafil) for the treatment of CF. The F508del-CFTR mouse model was chosen because: - F508del is the most common mutation in CF. - Epithelia of this model display defective electrolyte transport (Cl- and Na+) - P. aeruginosa lipopolysaccharide (LPS) exposure mimics several aspects of CF airway epithelial inflammation such as increased of pro-inflammatory cytokine (IL-8, IL-6, TNF-α,…) and neutrophil infiltrate cells. The first aim of this work was to evaluate in vivo the ability of clinical doses of sildenafil, vardenafil and tadalafil, to correct ion transport in CF. We measured the transepithelial PD across the nasal mucosa as readout for sodium and chloride transport. The first step was to test the effect of a single intraperitoneal injection of sildenafil or vardenafil in F508del, cftr knockout and normal homozygous mice. In the second step, we developed a restraint-free mouse chamber for inhalation studies; PDE5 inhibitors were nebulized for 15 minutes at concentrations adjusted from recommended therapeutic oral doses for male erectile dysfunction. We have shown that PDE5 inhibitors, applied in vivo at clinical doses can restore chloride secretion in airway epithelium of F508del-CFTR mutant mice. We also demonstrated that inhalation of PDE5 inhibitors restores CFTR-dependent chloride transport function across the respiratory epithelium. The inhaled route is preferred for the delivery of small doses of PDE5 inhibitors directly to the airway epithelium, leading to rapid onset of action and low incidence of side effects. The mechanism of action through which PDE5 inhibitors activate F508del-CFTR chloride channel function remains to be elucidated. The second aim of this work was to evaluate the therapeutic efficacy of vardenafil for modulating the inflammatory response in CF airways. The effect of vardenafil pretreatment, given as a single intraperitoneal injection, was evaluated, after induction of inflammation by intratracheal instillation of lipopolysaccharide (LPS) from P. aeruginosa, on cellular and molecular markers of lung inflammation in F508del-CF and control mice. We have demonstrated for the first time the benefits of vardenafil in attenuating LPS induced airway inflammation in F508del-CF mice in vivo. An additional potential interest of PDE5 inhibitors in CF might be raised by assuming that PDE5 inhibitors have a possible anti-inflammatory action. In conclusion, our results provide evidence that sildenafil, vardenafil and tadalafil, administered in vivo at clinical doses, activate CFTR-dependent chloride transport in CF respiratory epithelium. In addition, the drugs induce a possible anti-inflammatory action in F508del-CF mice.
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Lubamba, B. (2011). Sildenafil and analogues as potential therapy for cystic fibrosis. https://hdl.handle.net/2078.5/151154