Melissa A. Ashlock

Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (Po) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl− secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by ≈10-fold, to ≈50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na+ and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway.

Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that impair the function of CFTR, an epithelial chloride channel required for proper function of the lung, pancreas, and other organs. Most patients with CF carry the F508del CFTR mutation, which causes defective CFTR protein folding and processing in the endoplasmic reticulum, resulting in minimal amounts of CFTR at the cell surface. One strategy to treat these patients is to correct the processing of F508del-CFTR with small molecules. Here we describe the in vitro pharmacology of VX-809, a CFTR corrector that was advanced into clinical development for the treatment of CF. In cultured human bronchial epithelial cells isolated from patients with CF homozygous for F508del, VX-809 improved F508del-CFTR processing in the endoplasmic reticulum and enhanced chloride secretion to approximately 14% of non-CF human bronchial epithelial cells (EC50, 81 ± 19 nM), a level associated with mild CF in patients with less disruptive CFTR mutations. F508del-CFTR corrected by VX-809 exhibited biochemical and functional characteristics similar to normal CFTR, including biochemical susceptibility to proteolysis, residence time in the plasma membrane, and single-channel open probability. VX-809 was more efficacious and selective for CFTR than previously reported CFTR correctors. VX-809 represents a class of CFTR corrector that specifically addresses the underlying processing defect in F508del-CFTR.

Fluorescent chloride indicators to assess the efficacy of CFTR cDNA delivery.

Cl(-)-sensitive fluorescent indicators have been used extensively in cell culture systems to measure the Cl(-)-transporting function of the cystic fibrosis transmembrane conductance regulator protein CFTR. These indicators have been used in establishing a surrogate end point to assess the efficacy of CFTR cDNA delivery in human gene therapy trials. The ability to measure Cl- transport with high sensitivity in small and heterogeneous tissue samples makes the use of Cl- indicators potentially attractive in gene delivery studies. In this review article, the important technical aspects of Cl- transport measurements by fluorescent indicators such as SPQ are described, applications of Cl- indicators to assay CFTR function are critically evaluated, and new methodological developments are discussed. The available Cl- indicators have been effective in quantifying Cl- transport rates in cell culture models and in vitro systems such as isolated membrane vesicles and liposomes. However, the imperfect photophysical properties of existing Cl- indicators limit their utility in performing measurements in airway tissues, where gene transfer vectors are delivered in CF gene therapy trials. The low efficiency of gene transfer and the cellular heterogeneity in airway samples pose substantial obstacles to functional measurements of CFTR expression. Significant new developments in generating long-wavelength and dual-wavelength halide indicators are described, and recommendations are proposed for the use of the indicators in gene therapy trials.

Therapeutics Development for Cystic Fibrosis: A Successful Model for a Multisystem Genetic Disease

Cystic fibrosis (CF) is a progressive genetic disease primarily involving the respiratory and gastrointestinal tracts. Multiple therapies directed at CF symptoms and clinical management strategies have emerged from iterative cycles of therapeutics development, helping to change the face of CF from a fatal childhood affliction to a disease in which nearly 50% of U.S. patients are adults. However, as a consequence of therapeutic advances, the burden of CF care is high, and despite progress, most patients succumb to respiratory failure. Addressing the basic defect in CF with systemic small molecules is evolving as a promising approach. A successful collaboration between a voluntary health organization and a pharmaceutical company, complemented by academic investigators and patients, has led to the clinical development of investigational drugs that restore function to defective CFTR protein in various tissues in CF patients. Important activities, leverage points, and challenges in this exemplary collaboration are reviewed with hope that the CF and other genetic disease communities can benefit from the lessons learned in generating new therapeutic approaches in CF.

Molecular pathophysiologic hints into Niemann-Pick Type C disease using cDNA microarray technology

Molecular pathophysiologic hints into Niemann-Pick Type C disease using cDNA microarray technology

Abstract 3685: A strategy to identify therapeutic candidates for chronic lymphocytic leukemia from a library of FDA approved drugs

Chronic lymphocytic leukemia (CLL) is an adult lymphoid malignancy with a variable clinical course. There is considerable interest in the identification of new treatments, as most current approaches are not curative. While most patients respond to initial chemotherapy, relapsed disease is often resistant to the drugs commonly used in CLL and are left without limited therapeutic options. In this study, we used a luminescent cell viability assay based on ATP levels (CellTiter-Glo, Promega) to find compounds that were potent and efficacious in killing CLL cells. We employed an in-house process of quantitative high throughput screening (qHTS) to assess 8-9 concentrations of each member of a 2,816 compound library (including FDA-approved drugs and those known to be bio-active from commercial suppliers). Using qHTS we generated potency values on each compound in lymphocytes donated from each of six individuals with CLL and five unaffected individuals. We found 102 compounds efficacious against cells from all six individuals with CLL (“consensus” drugs) with seven of these showing no efficacy on lymphocytes from a majority of unaffected donors, suggesting some degree of specificity for the leukemic cells. Furthermore, to determine if we could save time and cost we screened the same library using CLL cells pooled from these individuals. We found that over 90% of the 102 drugs identified as efficacious on cells from individuals were also efficacious in the pooled experiment. Importantly, a near perfect correlation was observed in potency values between the single and pooled studies. The results of this “pooling” approach suggests it is rationale to apply pooling and qHTS when screening CLL cells against much larger collections (>300,000 unapproved compounds) where novel compounds could be identified as starting points for drug discovery and development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3685.