- Email: [email protected]
34 Restored CFTR dependent sweat secretion in a cystic fibrosis patient treated with ataluren
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Posters / Journal of Cystic Fibrosis 15 (2016) S51–S120
biodistribution to the lung showing a >4 fold increase in lung:plasma partitioning in rat biodistribution studies compared to lumacaftor. Conclusion: FDL169 is a novel F508del-CFTR corrector that increases the amount of F508del-CFTR at the cell surface, has low risk of reduced activity upon chronic treatment and distributes well into lung tissue. 33 Rescue of premature stop mutations R1162X and W1282X by a combination of small molecule correctors L. Cebotaru1 , C. Boinot1 , I. Sabirzhanova1 , W.B. Guggino1 . 1 Johns Hopkins University, Baltimore, United States Aminoglycosides rescue Class 1 CFTR mutations. CFBE41o- cells expressing R1162X or W1282X display small amounts of truncated proteins. Proteasome inhibition increased R1162X and W1282X protein by 20 and 6 fold. Correctors C4+18 increased protein levels of R1162X and W1282X 2–3 fold. C4+18+gentamicin increased CFTR protein by 5 and 10 fold. C4+18 increased CFTR currents by 2 fold for both. Similar results occurred with gentamicin. C4+18 plus gentamicin increased currents only in cells containing W1282X 3 fold. Thus, R1162X and W1282X generate truncated proteins that are rapidly degraded. Correctors increase the levels of the mutant proteins. Functional studies showed that gentamycin plus correctors may be the preferred treatment. 34 Restored CFTR dependent sweat secretion in a cystic fibrosis patient treated with ataluren G. Bergamini1 , G. Tridello2 , E. Calcaterra1 , C. Sorio1 , B. Assael2 , P. Melotti2 . 1 Dept. of Medicine, General Pathology Section, University of Verona, Verona, Italy; 2 Azienda Ospedaliera Universitaria Integrata Verona, Cystic Fibrosis Center, Verona, Italy Objectives: A bioassay for CFTR function in vivo based on that published by Wine et al. (Plos ONE 2013) focused on the ratio between CFTRindependent (M-sweat, evoked by methacholine) and CFTR-dependent (C-sweat, induced by a beta-adrenergic cocktail) sweat secretion rates by multiple individual glands. Methods: Image analysis showed changes of volume of sweat drops secreted in an oil layer, including a water-soluble dye. Secretion rates were reproducing linearity of these measurements. Wine et al. (Plos ONE 2014) reported improvements of CFTR function in CF patients treated with ivacaftor undetectable by evaporimetry (Gonska et al. Ped Pulmonol 2013). High variability of response to ataluren has been described among CF patients. Results: CFTR-dependent sweating was improved in a CF patient with CFTR genotype R1162X/R1162X treated with ataluren 40 mg/kg/day enrolled in the clinical trial PTC124-GD-CF-023 (PTC Therapeutics, USA). After treatment 33% individual sweat glands responded to the CFTR dependent stimulation by the beta-adrenergic cocktail with increasing mean C-sweat/M-sweat ratio from 0 to 0.037. Conclusion: According to these data we suggest that ataluren might have effects on sweat glands and that our test could be capable of measuring CFTR function restoration. Correlation of these functional results with the overall clinical benefits and evaluation of other cases currently enrolled in ataluren clinical trials (PTC Therapeutics, USA) might promote an individualized medicine approach in CF. Acknowledgement: Supported by Lega Italiana Fibrosi Cistica – Associazione Veneta Onlus. 35 Standardised cultivation-independent methods to understand the effect of OligoG on Burkholderia and microbial diversity in the CF lung during a clinical trial R. Weiser1 , E. Mahenthiralingham1 , J. Marchesi1,2 , P. Opitz3 , M. Trentmann4 , P.D. Rye5 , E. Onsøyen5 , A. Dessen5 , A. Hilde Myrset5 , C. Schwartz4 , R. Fischer3 . 1 Cardiff University, Cardiff, United Kingdom; 2 Imperial College London, London, United Kingdom; 3 M¨ unchen, Pasing, Germany; 4 Christiane Herzog Zentrum, Berlin, Germany; 5 AlgiPharma AS, Sandvika, Norway Objectives: Lung infection is a major cause of morbidity and mortality in cystic fibrosis (CF) patients and Burkholderia infections particularly difficult to treat. Therapeutics are urgently needed to combat the high
intrinsic antibiotic resistance of this pathogen. OligoG CF-5/20 is a short chain alginate oligomer. Studies have shown OligoG to release stagnant mucin, disrupt bacterial biofilms in vitro, potentiate antibiotics and reduce sputum viscosity. Our aim was to develop standardised cultureindependent methods to monitor Burkholderia and bacterial diversity in CF sputum. Methods: The clinical trial (NCT02453789) is a randomized, doubleblind, placebo-controlled cross-over study of inhaled OligoG for 28 days in 12 CF patients with chronic Burkholderia infection. Paired sputum samples were taken at 6 time-points for analysis. A standardised sputum DNA extraction procedure was developed, enabling i) Identification of Burkholderia spp. via PCR amplification, sequencing and analysis of recA and gyrB genes, ii) Enumeration of total bacteria and Burkholderia via qPCR, and iii) Investigation of bacterial diversity using ribosomal intergenic spacer analysis (RISA) and 16S rRNA gene sequencing. Results: Molecular techniques were effective for enumeration and identification of bacteria in sputum and revealed striking differences in comparison to culture methods. There was high concordance between RISA and 16S rRNA sequencing results for paired sputum samples indicating temporal stability of bacterial diversity. Conclusion: The integration of culture-independent techniques into the trial has provided a comprehensive view of the baseline diversity and effects on the microbiota. 36 A phase I study investigating the delivery of tobramycin using the TobrAir® device compared with (TOBI® ) PARI LC® PLUS and PARI TurboBOY® Podhaler™ using pharmacokinetic and pharmacoscintigraphic methods M. Beckert1 , W. de Kruijf2 , T. Norling3 . 1 CaRACS, Berlin, Germany; 2 Medspray BV, Enschede, Netherlands; 3 Pharmaero ApS, Copenhagen, Denmark Tobramycin is a well-known, effective antibiotic for the management of P. aeruginosa infections in CF patients. Pharmaero has developed TobrAir, a fixed drug device combination product, providing a liquid tobramycin formulation for inhalation. Tobramycin as TobrAir is formulated as a 15% solution, administered as 75 mg b.i.d. with 10 inhalations per dosing, thus resulting in a total daily dose of 150 mg. We investigated (1) safety and tolerability, (2) lung deposition, and (3) pharmacokinetics of tobramycin delivered via TobrAir, compared to TOBI/PARI LC PLUS (as an example for a nebulizer) and TOBI Podhaler (as an example for a dry powder inhaler). This randomized crossover study assessed the aerosol delivery and lung deposition of tobramycin by pharmacoscintigraphy using TobrAir compared to TOBI/PARI LC PLUS. Tobramycin plasma levels were determined for all three devices. Twelve healthy volunteers received the three treatment regimens in a randomized order: a single dose of 75 mg tobramycin radiolabelled with 99m Tc delivered to the lungs via TobrAir, 300 mg TOBI radiolabelled with 99m Tc delivered by PARI LC PLUS and 112 mg tobramycin delivered by TOBI Podhaler, not radiolabelled. As a result, the delivery of tobramycin via Pharmaero’s new drug-device combination product was safe and well-tolerated. TobrAir demonstrated a higher lung deposition with significantly reduced treatment time compared to the delivery via TOBI/PARI LC PLUS as well as a superior relative bioavailability compared to both TOBI/PARI LC PLUS and TOBI Podhaler. TobrAir therefore may become an efficacious treatment for CF patients infected with P. aeruginosa. 37 Microbiologic changes observed over 6 months in a randomized, open-label comparison of inhaled levofloxacin and inhaled tobramycin in persons with cystic fibrosis and chronic P. aeruginosa (Pa) airway infection D.R. VanDevanter1 , J.S. Elborn2 , P. Flume3 , K. Polu4 , J.J. LiPuma5 . 1 Case Western Reserve University School of Medicine, Cleveland, United States; 2 Queen’s University, Belfast, United Kingdom; 3 University of South Carolina, Charleston, United States; 4 Raptor Pharma, Novato, United States; 5 University of Michigan, Ann Arbor, United States Objectives: Levofloxacin inhalation solution (LIS; Quinsair® ) was approved by the EMA after a three on/off cycle 168-day study
Posters / Journal of Cystic Fibrosis 15 (2016) S51–S120
biodistribution to the lung showing a >4 fold increase in lung:plasma partitioning in rat biodistribution studies compared to lumacaftor. Conclusion: FDL169 is a novel F508del-CFTR corrector that increases the amount of F508del-CFTR at the cell surface, has low risk of reduced activity upon chronic treatment and distributes well into lung tissue. 33 Rescue of premature stop mutations R1162X and W1282X by a combination of small molecule correctors L. Cebotaru1 , C. Boinot1 , I. Sabirzhanova1 , W.B. Guggino1 . 1 Johns Hopkins University, Baltimore, United States Aminoglycosides rescue Class 1 CFTR mutations. CFBE41o- cells expressing R1162X or W1282X display small amounts of truncated proteins. Proteasome inhibition increased R1162X and W1282X protein by 20 and 6 fold. Correctors C4+18 increased protein levels of R1162X and W1282X 2–3 fold. C4+18+gentamicin increased CFTR protein by 5 and 10 fold. C4+18 increased CFTR currents by 2 fold for both. Similar results occurred with gentamicin. C4+18 plus gentamicin increased currents only in cells containing W1282X 3 fold. Thus, R1162X and W1282X generate truncated proteins that are rapidly degraded. Correctors increase the levels of the mutant proteins. Functional studies showed that gentamycin plus correctors may be the preferred treatment. 34 Restored CFTR dependent sweat secretion in a cystic fibrosis patient treated with ataluren G. Bergamini1 , G. Tridello2 , E. Calcaterra1 , C. Sorio1 , B. Assael2 , P. Melotti2 . 1 Dept. of Medicine, General Pathology Section, University of Verona, Verona, Italy; 2 Azienda Ospedaliera Universitaria Integrata Verona, Cystic Fibrosis Center, Verona, Italy Objectives: A bioassay for CFTR function in vivo based on that published by Wine et al. (Plos ONE 2013) focused on the ratio between CFTRindependent (M-sweat, evoked by methacholine) and CFTR-dependent (C-sweat, induced by a beta-adrenergic cocktail) sweat secretion rates by multiple individual glands. Methods: Image analysis showed changes of volume of sweat drops secreted in an oil layer, including a water-soluble dye. Secretion rates were reproducing linearity of these measurements. Wine et al. (Plos ONE 2014) reported improvements of CFTR function in CF patients treated with ivacaftor undetectable by evaporimetry (Gonska et al. Ped Pulmonol 2013). High variability of response to ataluren has been described among CF patients. Results: CFTR-dependent sweating was improved in a CF patient with CFTR genotype R1162X/R1162X treated with ataluren 40 mg/kg/day enrolled in the clinical trial PTC124-GD-CF-023 (PTC Therapeutics, USA). After treatment 33% individual sweat glands responded to the CFTR dependent stimulation by the beta-adrenergic cocktail with increasing mean C-sweat/M-sweat ratio from 0 to 0.037. Conclusion: According to these data we suggest that ataluren might have effects on sweat glands and that our test could be capable of measuring CFTR function restoration. Correlation of these functional results with the overall clinical benefits and evaluation of other cases currently enrolled in ataluren clinical trials (PTC Therapeutics, USA) might promote an individualized medicine approach in CF. Acknowledgement: Supported by Lega Italiana Fibrosi Cistica – Associazione Veneta Onlus. 35 Standardised cultivation-independent methods to understand the effect of OligoG on Burkholderia and microbial diversity in the CF lung during a clinical trial R. Weiser1 , E. Mahenthiralingham1 , J. Marchesi1,2 , P. Opitz3 , M. Trentmann4 , P.D. Rye5 , E. Onsøyen5 , A. Dessen5 , A. Hilde Myrset5 , C. Schwartz4 , R. Fischer3 . 1 Cardiff University, Cardiff, United Kingdom; 2 Imperial College London, London, United Kingdom; 3 M¨ unchen, Pasing, Germany; 4 Christiane Herzog Zentrum, Berlin, Germany; 5 AlgiPharma AS, Sandvika, Norway Objectives: Lung infection is a major cause of morbidity and mortality in cystic fibrosis (CF) patients and Burkholderia infections particularly difficult to treat. Therapeutics are urgently needed to combat the high
intrinsic antibiotic resistance of this pathogen. OligoG CF-5/20 is a short chain alginate oligomer. Studies have shown OligoG to release stagnant mucin, disrupt bacterial biofilms in vitro, potentiate antibiotics and reduce sputum viscosity. Our aim was to develop standardised cultureindependent methods to monitor Burkholderia and bacterial diversity in CF sputum. Methods: The clinical trial (NCT02453789) is a randomized, doubleblind, placebo-controlled cross-over study of inhaled OligoG for 28 days in 12 CF patients with chronic Burkholderia infection. Paired sputum samples were taken at 6 time-points for analysis. A standardised sputum DNA extraction procedure was developed, enabling i) Identification of Burkholderia spp. via PCR amplification, sequencing and analysis of recA and gyrB genes, ii) Enumeration of total bacteria and Burkholderia via qPCR, and iii) Investigation of bacterial diversity using ribosomal intergenic spacer analysis (RISA) and 16S rRNA gene sequencing. Results: Molecular techniques were effective for enumeration and identification of bacteria in sputum and revealed striking differences in comparison to culture methods. There was high concordance between RISA and 16S rRNA sequencing results for paired sputum samples indicating temporal stability of bacterial diversity. Conclusion: The integration of culture-independent techniques into the trial has provided a comprehensive view of the baseline diversity and effects on the microbiota. 36 A phase I study investigating the delivery of tobramycin using the TobrAir® device compared with (TOBI® ) PARI LC® PLUS and PARI TurboBOY® Podhaler™ using pharmacokinetic and pharmacoscintigraphic methods M. Beckert1 , W. de Kruijf2 , T. Norling3 . 1 CaRACS, Berlin, Germany; 2 Medspray BV, Enschede, Netherlands; 3 Pharmaero ApS, Copenhagen, Denmark Tobramycin is a well-known, effective antibiotic for the management of P. aeruginosa infections in CF patients. Pharmaero has developed TobrAir, a fixed drug device combination product, providing a liquid tobramycin formulation for inhalation. Tobramycin as TobrAir is formulated as a 15% solution, administered as 75 mg b.i.d. with 10 inhalations per dosing, thus resulting in a total daily dose of 150 mg. We investigated (1) safety and tolerability, (2) lung deposition, and (3) pharmacokinetics of tobramycin delivered via TobrAir, compared to TOBI/PARI LC PLUS (as an example for a nebulizer) and TOBI Podhaler (as an example for a dry powder inhaler). This randomized crossover study assessed the aerosol delivery and lung deposition of tobramycin by pharmacoscintigraphy using TobrAir compared to TOBI/PARI LC PLUS. Tobramycin plasma levels were determined for all three devices. Twelve healthy volunteers received the three treatment regimens in a randomized order: a single dose of 75 mg tobramycin radiolabelled with 99m Tc delivered to the lungs via TobrAir, 300 mg TOBI radiolabelled with 99m Tc delivered by PARI LC PLUS and 112 mg tobramycin delivered by TOBI Podhaler, not radiolabelled. As a result, the delivery of tobramycin via Pharmaero’s new drug-device combination product was safe and well-tolerated. TobrAir demonstrated a higher lung deposition with significantly reduced treatment time compared to the delivery via TOBI/PARI LC PLUS as well as a superior relative bioavailability compared to both TOBI/PARI LC PLUS and TOBI Podhaler. TobrAir therefore may become an efficacious treatment for CF patients infected with P. aeruginosa. 37 Microbiologic changes observed over 6 months in a randomized, open-label comparison of inhaled levofloxacin and inhaled tobramycin in persons with cystic fibrosis and chronic P. aeruginosa (Pa) airway infection D.R. VanDevanter1 , J.S. Elborn2 , P. Flume3 , K. Polu4 , J.J. LiPuma5 . 1 Case Western Reserve University School of Medicine, Cleveland, United States; 2 Queen’s University, Belfast, United Kingdom; 3 University of South Carolina, Charleston, United States; 4 Raptor Pharma, Novato, United States; 5 University of Michigan, Ann Arbor, United States Objectives: Levofloxacin inhalation solution (LIS; Quinsair® ) was approved by the EMA after a three on/off cycle 168-day study