Cystic fibrosis (CF), the most common autosomal recessive disorder at least in western countries, is caused by mutations of the cystic fibrosis transmembranous conductance regulator molecule (CFTR) and affects approximately 40 000 patients in Europe. Most, if not all, CF-patients develop a chronic pulmonary infection with Pseudomonas aeruginosa (P. aeruginosa). At present, it is unknown why CF-patients are highly sensitive to P. aeruginosa infections and, most importantly, no curative treatment for cystic fibrosis is available.

Our studies provided a novel pathophysiological concept for cystic fibrosis. The investigators demonstrated that ceramide plays a crucial role in the development of cystic fibrosis and the high sensitivity of Cftr-deficient mice to infection with P. aeruginosa (1,2). Using biochemical techniques, fluorescence microscopy, and mass spectrometry, the investigators found that ceramide accumulates in the lungs of various Cftr-deficient mouse strains before any infection occurs, in particular in the epithelial cells of large and small bronchi and in alveolar macrophages. The accumulation of ceramide in Cftr-deficient epithelial cells may be mediated by an increase in pH from 4.5 to 6.0 in secretory lysosomes and pre-lysosomes of Cftr-deficient cells. The change in pH results in a reduction of approximately 90% in the activity of acid ceramidase which consumes ceramide, and a reduction of only 35% in the activity of acid sphingomyelinase which releases ceramide. An imbalance in the activity of these two enzymes, by which a relative over-activity of acid sphingomyelinase produces ceramide, may then result in an accumulation of ceramide. Partial inhibition of acid sphingomyelinase, either genetically or pharmacologically, returns ceramide concentrations to near normal levels in the lungs of Cftr-deficient mice. Genetic inhibition of acid sphingomyelinase was achieved by crossing Cftr-deficient mice with acid sphingomyelinase-deficient mice to create mice deficient in Cftr and heterozygous for acid sphingomyelinase (Cftr-/-/Smpd1+/- mice). The activity of acid sphingomyelinase in the lungs of these mice was approximately 50% lower than in the lungs of wild-type mice. Pharmacological inhibition of acid sphingomyelinase was achieved by treating Cftr-deficient mice with the functional acid sphingomyelinase inhibitor amitriptyline (1,2). Increased concentrations of ceramide in the bronchial epithelial cells of Cftr-deficient mice triggered death of these cells, the deposition of DNA in bronchi, chronic pulmonary inflammation, and a high susceptibility of Cftr-deficient mice to pulmonary infections with P. aeruginosa. Normalisation of ceramide concentrations by genetic means normalised these changes, and pharmacological inhibition of acid sphingomyelinase prevented these changes. Ceramide accumulation was also observed in ciliated nasal epithelial cells, macrophages and lung transplant materials from CF patients (1-4). This finding suggests that the results of our murine studies also apply to humans with cystic fibrosis. Next, the investigators applied a panel of functional inhibitors of acid sphingomyelinase and treated Cftr-deficient mice by inhalation of amitriptyline, trimipramine, desipramine, chlorprothixene, fluoxetine, amlodipine, or sertraline, all of which are functional inhibitors of acid sphingomyelinase. This inhalation reduced the activity of acid sphingomyelinase specifically in the lung and normalised pulmonary ceramide concentrations, inflammation, and susceptibility to infection (2). Recent findings by C. Ward and associates confirmed the accumulation of ceramide in lung specimens from CF patients (5).

Clinical data:

1. Pilot study - Based on the amitriptyline-mediated protection of Cftr-deficient mice, the investigators initiated a small clinical cross over study with 4 cystic fibrosis patients that were treated with amitriptyline or placebo, respectively. The results revealed a clinical relevant increase of the lung function (determined as FEV1) in all cystic fibrosis patients after treatment with amitriptyline for 2 weeks.
2. Phase IIa - The positive data of the pilot study encouraged us to initiate a Phase IIa cross over study in 18 patients and to investigate a beneficial effect of amitriptyline on cystic fibrosis in a larger patient population to prove safety, biochemically prove the mechanisms of action, and for dose finding of amitriptyline (25 mg/day, 50 mg/day, 75 mg/day). Amitriptyline was well-tolerated in the patient group and no SAEs were recorded at the end of the 28-day-course. From the 80 AEs, 35 were related or possibly related to the medication. Two well-known AEs of amitriptyline, i.e. xerostomia and tiredness, were significantly different between placebo and the three amitriptyline treatment groups, but were mostly transient. FEV1 was analysed in the per protocol analysis in 13, 7, 8 and 8 available patient cycles, who had received placebo, 25 mg, 50 mg or 75 mg amitriptyline/day, respectively. After 14 days of treatment the primary endpoint FEV1 had improved significantly in the 25 mg/d amitriptyline group relative to placebo (FEV1: +5.0% compared to the placebo group; p = 0.048). No significant change of lung function was observed when patients were administered 50 mg and 75 mg of amitriptyline (6).
3. Phase IIb - To evaluate the positive data of the phase IIa-trial, the investigators initiated a phase IIb study in 2009 with a parallel group design.