Correlation between nasal potential difference measurements, genotype and clinical condition in patients with cystic fibrosis.

In cystic fibrosis (CF), the clinical condition of patients correlates poorly with genotype. One possible explanation is that clinical status is influenced by net preserved chloride secretion rather than the CF mutation. We tested the relationships between residual chloride secretion, as measured by nasal potential difference (PD) and the type of mutation (genotypes expressing apical cystic fibrosis transmembrane conductance regulator (CFTR) protein versus those that do not) and clinical status. Twenty two CF patients (mean age 25.7 yrs, 11 females and 11 males, mean forced expiratory volume in one second (FEV1) 53.1% of predicted) with defined genotypes were recruited. Nasal PD was measured using a standard protocol involving the perfusion of the nasal epithelium with a sodium channel blocker (amiloride), followed by a solution of low chloride and finally with isoprenaline. Patients with apical CFTR protein showed higher residual chloride secretion than those without (amiloride to isoprenaline value of 4.59 and 0.56 mV, respectively, p = 0.01). There was no correlation between mutation type and clinical condition. When these patients were recategorized as "high" (> 10 mV amiloride to isoprenaline response) or "low" (10 mV or less) chloride secretors, we found that the former group had a significantly higher FEV1 (67.7 versus 48.3% pred) and a better pulmonary radiological score (4.14 versus 7.07, by Northern scoring system). These results suggest that some cystic fibrosis patients, regardless of genotype, have an ability to secrete chloride when stimulated with chloride secretatagogues, and this is correlated with a better lung function. These results also have implications for the use of potential difference measurements in novel cystic fibrosis transmembrane conductance regulator replacement trials.

Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.

In cystic fibrosis (CF), mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in defective transepithelial ion transport, leading to life shortening inflammatory lung disease. Before lung studies, we tested the safety and efficacy of gene delivery to the nasal epithelium of CF patients using pCMV-CFTR-DOTAP cationic liposome complex. A single dose of 400 micrograms pCMV-CFTR:2.4 mg DOTAP was administered in a randomised, double-blinded fashion to the nasal epithelium of eight CF patients, with a further eight receiving buffer only. Patients were monitored for signs and symptoms for 2 weeks before treatment and 4 weeks after treatment. Inflammatory cells were quantified in a nasal biopsy taken 3 days after treatment. There was no evidence for excess nasal inflammation, circulating inflammatory markers or other adverse events ascribable to active treatment. Gene transfer and expression were assayed by the polymerase chain reaction. Transgene DNA was detected in seven of the eight treated patients up to 28 days after treatment and vector derived CFTR mRNA in two of the seven patients at +3 and +7 days. Transepithelial ion transport was assayed before and after treatment by nasal potential difference during drug perfusion and by SPQ fluorescence halide ion conductance. Partial, sustained correction of CFTR-related functional changes toward normal values were detected in two treated patients. The level of gene transfer and functional correction were comparable to those reported previously using adenoviral vectors or another DNA-liposome complex, but here were sustained and uncompromised by false positives. These results justify further studies with pCMV-CFTR-DOTAP aimed at treating CF lung disease.

Laboratory and clinical studies in support of cystic fibrosis gene therapy using pCMV-CFTR-DOTAP.

The first phase I study of cystic fibrosis gene therapy using cationic liposomes to deliver the cystic fibrosis conductance regulator gene to the nose reported partial and transient correction of the nasal transepithelial ion transport defect, While encouraging, further improvements will be required if this form of treatment is to be of therapeutic value. We tested a new formulation, pCMV-CFTR-DOTAP. The complex is stable for 10 days and effective at correcting the electrophysiological deficit in the trachea of CF mutant mice at 8 or 9 days after intratracheal instillation. Reliable protocols for consistent detection of as few as 10 molecules of CFTR mRNA and DNA in nasal brushing samples are described, Both vector and DNA have been produced to Good Manufacturing Practice standard, Nasal potential difference measurements developed at the National Heart and Lung Institute to assess the CFTR ion channel activity in CF patients replicated well at the Scottish Adult Cystic Fibrosis Service. The SPO fluorescence assay for halide ion conductance in nasal brushings has also been tested. These establish baseline conditions in the Scottish CF cohort from which evidence for correction can be judged under clinical trial conditions. These studies formed the basis for regulatory approval of a randomised, placebo controlled double-blind phase I research study.

Systemic haemodynamic changes in patients with cystic fibrosis with and without chronic liver disease.

BACKGROUND/AIMS: There are well-documented systemic haemodynamic changes associated with chronic liver disease. Patients with cystic fibrosis may develop chronic liver disease, but it is not known whether these systemic haemodynamic changes develop and, if they do, whether they are influenced by the associated chronic lung disease. We therefore undertook a study to document the circulatory status of cystic fibrosis patients with and without chronic liver disease. METHODS: Fifty-six subjects with cystic fibrosis were studied. Systemic haemodynamic and pulmonary parameters, in sub-groups both with (LD) and without (NLD) liver disease, were measured at rest and during measured exertion. Cystic fibrosis-related chronic liver disease was diagnosed using previously validated ultrasound criteria. Patients underwent assessment at rest and in the fourth minute of seated bicycle exercise at 25W. Heart rate (ECG), blood pressure (semiautomated sphygmomanometer), aortic blood velocity (pulsed Doppler suprasternal probe), arterial oxygen saturation (pulse oximeter) and respiratory variables (pneumotachometer with expired gas analysis by an automatic system) were measured. RESULTS: A complete data set was available for 45 patients (22 LD) at rest and 40 patients (19 LD) on exercise. The patients were well matched for age, sex, height, weight, and pulmonary function. Patients with chronic liver disease had a hyperkinetic circulation while ventilatory variables before and during exercise were similar for the two groups. There was evidence that the circulatory changes were exacerbated by both deteriorating hepatic and pulmonary function. CONCLUSIONS: Cystic fibrosis patients with chronic liver disease have a hyperdynamic circulation similar to that documented in other forms of chronic liver disease. These circulatory changes are exacerbated by deteriorating hepatic and pulmonary function.