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WS18.1 Using CF colon organoids to study CFTR function of uncharacterized mutations and test potential therapies
Oral Presentations / Journal of Cystic Fibrosis 15 (2016) S1–S50
116 mg/g, p < 0.05). In addition, a significant association with increasing serum CRP concentrations (p < 0.0001) was observed. Conclusion: The majority of patients had increased FC concentrations, which were related to the severity of the CFTR mutation, pancreatic insufficiency, and serum CRP. WS17.5 Is there need for a multienzyme product substitution when using high calorie liquid diets in patients with exocrine pancreatic insufficiency? Studies on praecaecal digestibility in pigs with experimentally induced pancreatic exocrine insufficiency 1 1 A. Moßeler ¨ , B. Ahlfanger ¨ , J. Kamphues1 . 1 University of Veterinary Medicine, Foundation, Institute for Animal Nutrition, Hannover, Germany Objectives: Pancreatic exocrine insufficiency (PEI) is common in CF-patients causing maldigestion and malnutrition. High calorie supplementary food is often used to improve nutritional status. Recently a lipase device was presented (Grujic et al. 2015) to be used during PEG nutrition. The pancreatic duct ligated (PL) pig is an established model to study PEI. As PEI impairs digestion of fat but also of protein this study aimed to test the effect of a porcine pancreatic enzyme preparation (PPEP) containing lipases and proteases on digestibility of two liquid diets (>1.5 kcal/ml). Methods: 3 adult PL-minipigs fitted with ileo-caecal fistulas were used in this study. Praecaecal disappearance rate (DR) was determined in a screeningtest model. 2 commercially available balanced diets (A, B) were used. The diets were fed with or without addition of a PPEP at a dosage of 300,000 I.U. lipase. Results: PEI resulted in a low digestibility of fat (diet A: 31.4%, diet B: 54.3%) and protein (diet A: 24.0%, diet B: 22.6%) for both diets. PPEP resulted in a distinct increase of DR of fat (diet A: 97.7%, diet B: 96.9%) and protein (diet A: 81.2%, diet B: 79.8%). Conclusion: Although the benefits of lipase administration described by Grujic et al. 2015 are beyond dispute these results indicate that supplementation of proteolytic enzymes is beneficial and necessary as well. Therefore supplementary food should always be given with enzyme products containing lipase and protease (Shlieout et al. 2011) to optimise digestion of fat and protein. WS17.6 A randomized, controlled clinical trial of a liquid microbial lipase (NM-BL) in patients with exocrine pancreatic insufficiency due to cystic fibrosis J.E. Heubi1 , D. Schaeffer2 , R.C. Ahrens3 , N. Sollo4 , S. Strausbaugh5 , G. Graff6 , R. Jain7 , S. Witte8 , K. Forßmann8 . 1 Children’s Hospital Medical Center, Cincinnati, United States; 2 Nemours Children’s Clinic, Jacksonville, United States; 3 University of Iowa, Iowa City, United States; 4 Via Christi Research, Wichita, United States; 5 Rainbow Babies and Children’s Hospital, Cleveland, United States; 6 Penn State Milton S. Hershey Medical Center, Hershey, United States; 7 The University of Texas Southwestern Medical Center, Dallas, United States; 8 Nordmark Arzneimittel GmbH & Co. KG, Uetersen, Germany Objectives: Exocrine pancreatic insufficiency (EPI) commonly affects patients with cystic fibrosis (CF). Treatment of EPI with porcine derived pancreatic enzyme products improves fat malabsorption. However, there is a need for non-animal based products with higher lipase activity and formulations for use in infants, children, and adults using supplemental feeding. NM-BL is a novel microbial lipase with high specific activity in a liquid formulation for improved administration and mixing with food. Methods: This randomized, double-blind, crossover study included patients ≥12 years of age with CF and EPI. Eligible patients were placed on a high fat diet and hospitalized for two subsequent 72-hour fat balance studies. During study periods, standard enzyme replacement therapy was replaced by NM-BL or placebo. The primary outcome measure was coefficient of fat absorption (CFA), secondary outcome measures included the coefficient of nitrogen absorption (CNA), clinical symptoms, safety and tolerability. Results: Thirty-one patients were randomized into the study and 22 completed the study. CFA increased from 53.8% during placebo to
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72.7% after treatment with NM-BL (p < 0.001). CNA was unchanged with NM-BL (58.1% during placebo and 58.3% with treatment). Subjective assessment of stool fat and stool consistency improved with NM-BL. Adverse events were mostly gastrointestinal in nature and consistent with the underlying disease. Conclusion: The study provided proof-of-concept for the efficacy of the liquid microbial lipase NM-BL which was safe and well tolerated. Acknowledgement: This study was sponsored by Nordmark Arzneimittel GmbH & Co. KG.
Workshop 18. CFTR: Functional tests for therapeutic interventions WS18.1 Using CF colon organoids to study CFTR function of uncharacterized mutations and test potential therapies A.S. Ramalho1 , F. Vermeulen2 , M. Proesmans2 , K. De Boeck2 . 1 KULeuven, Organ Systems, Leuven, Belgium; 2 UZ Leuven, Pediatric Pulmonology, Dept Pediatrics, Leuven, Belgium More than 1,900 mutations are reported to the CFTR Mutation Database most being disease-causing. However, many have not yet been characterized. The forskolin induced swelling (FIS) of organoids establish in Utrecht (Dekkers et al Nat Med.2013;19(7):939–45) can be used to quantify CFTR function ex vivo in patients’ own tissue and to study how therapeutic compounds can increase this function. We grew intestinal organoid cultures from rectal biopsies of patients with uncharacterized mutations. The area of the organoids at time 0 before FIS was considered 100% and was further monitored during 60 minutes. Organoids were incubated with VX-809 3mM overnight, baseline organoid surface area was determined and VX-770 3mM was then added acutely in combination with forskolin 0.08mM. The results correspond to the mean±SEM organoid area after 60 min, using 2 wells per test condition and at least 20 organoids per well. In the FIS assay, organoids from patients with genotypes N1303K/K464E; E60X/G85R; c.2988+1G>A/c.1545–1546delTA did not respond to VXcompounds. But for two other genotypes, F508del/Q237E and F508del/L159S, there was a marked response to VX-770 plus VX-809VX i.e. swelling to 152%±5% and 180%±3% respectively. Overall, organoids allow assessment of CFTR function in individual patients. This can thus be a promising tool to preselect the ideal candidates for CFTR modulator therapy enabling personalized and precision medicine, especially in patients with very rare CFTR mutations. Acknowledgements: To J Beekman lab (Utrecht University) for the help with the FIS assay. This work was funded by Belgium IWT grant 130663 Cystic Fibrosis. WS18.2 Prospective selection of potential CFTR-modifying treatments using intestinal organoids G. Berkers1 , J.F. Dekkers1 , E. Kruisselbrink1 , A.M. Vonk1 , S. HeidaMichel1 , M. Geerdink1 , K.M. de Winter-de Groot1 , H.G.M. Arets1 , I. Bronsveld2 , E.A. van de Graaf2 , C.J. Majoor3 , G.H. Koppelman4 , J. Roukema5 , H.M. Janssens6 , H.G.M. Heijerman7 , R.G.J. Vries8 , J.C. Clevers8 , H. de Jonge9 , J.M. Beekman1 , C.K. van der Ent1 . 1 University Medical Center Utrecht – Wilhelmina Children’s Hospital, Utrecht, Netherlands; 2 University Medical Center Utrecht, Utrecht, Netherlands; 3 Academic Medical Center, Amsterdam, Netherlands; 4 University Medical Center Groningen, Groningen, Netherlands; 5 Radboud University Medical Center, Nijmegen, Netherlands; 6 Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands; 7 Haga Teaching Hospital, The Hague, Netherlands; 8 Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, Netherlands; 9 Erasmus Medical Center, Rotterdam, Netherlands Objectives: We have set up a functional CFTR assay using patientderived intestinal stem cell cultures, termed organoids (Dekkers et
116 mg/g, p < 0.05). In addition, a significant association with increasing serum CRP concentrations (p < 0.0001) was observed. Conclusion: The majority of patients had increased FC concentrations, which were related to the severity of the CFTR mutation, pancreatic insufficiency, and serum CRP. WS17.5 Is there need for a multienzyme product substitution when using high calorie liquid diets in patients with exocrine pancreatic insufficiency? Studies on praecaecal digestibility in pigs with experimentally induced pancreatic exocrine insufficiency 1 1 A. Moßeler ¨ , B. Ahlfanger ¨ , J. Kamphues1 . 1 University of Veterinary Medicine, Foundation, Institute for Animal Nutrition, Hannover, Germany Objectives: Pancreatic exocrine insufficiency (PEI) is common in CF-patients causing maldigestion and malnutrition. High calorie supplementary food is often used to improve nutritional status. Recently a lipase device was presented (Grujic et al. 2015) to be used during PEG nutrition. The pancreatic duct ligated (PL) pig is an established model to study PEI. As PEI impairs digestion of fat but also of protein this study aimed to test the effect of a porcine pancreatic enzyme preparation (PPEP) containing lipases and proteases on digestibility of two liquid diets (>1.5 kcal/ml). Methods: 3 adult PL-minipigs fitted with ileo-caecal fistulas were used in this study. Praecaecal disappearance rate (DR) was determined in a screeningtest model. 2 commercially available balanced diets (A, B) were used. The diets were fed with or without addition of a PPEP at a dosage of 300,000 I.U. lipase. Results: PEI resulted in a low digestibility of fat (diet A: 31.4%, diet B: 54.3%) and protein (diet A: 24.0%, diet B: 22.6%) for both diets. PPEP resulted in a distinct increase of DR of fat (diet A: 97.7%, diet B: 96.9%) and protein (diet A: 81.2%, diet B: 79.8%). Conclusion: Although the benefits of lipase administration described by Grujic et al. 2015 are beyond dispute these results indicate that supplementation of proteolytic enzymes is beneficial and necessary as well. Therefore supplementary food should always be given with enzyme products containing lipase and protease (Shlieout et al. 2011) to optimise digestion of fat and protein. WS17.6 A randomized, controlled clinical trial of a liquid microbial lipase (NM-BL) in patients with exocrine pancreatic insufficiency due to cystic fibrosis J.E. Heubi1 , D. Schaeffer2 , R.C. Ahrens3 , N. Sollo4 , S. Strausbaugh5 , G. Graff6 , R. Jain7 , S. Witte8 , K. Forßmann8 . 1 Children’s Hospital Medical Center, Cincinnati, United States; 2 Nemours Children’s Clinic, Jacksonville, United States; 3 University of Iowa, Iowa City, United States; 4 Via Christi Research, Wichita, United States; 5 Rainbow Babies and Children’s Hospital, Cleveland, United States; 6 Penn State Milton S. Hershey Medical Center, Hershey, United States; 7 The University of Texas Southwestern Medical Center, Dallas, United States; 8 Nordmark Arzneimittel GmbH & Co. KG, Uetersen, Germany Objectives: Exocrine pancreatic insufficiency (EPI) commonly affects patients with cystic fibrosis (CF). Treatment of EPI with porcine derived pancreatic enzyme products improves fat malabsorption. However, there is a need for non-animal based products with higher lipase activity and formulations for use in infants, children, and adults using supplemental feeding. NM-BL is a novel microbial lipase with high specific activity in a liquid formulation for improved administration and mixing with food. Methods: This randomized, double-blind, crossover study included patients ≥12 years of age with CF and EPI. Eligible patients were placed on a high fat diet and hospitalized for two subsequent 72-hour fat balance studies. During study periods, standard enzyme replacement therapy was replaced by NM-BL or placebo. The primary outcome measure was coefficient of fat absorption (CFA), secondary outcome measures included the coefficient of nitrogen absorption (CNA), clinical symptoms, safety and tolerability. Results: Thirty-one patients were randomized into the study and 22 completed the study. CFA increased from 53.8% during placebo to
S29
72.7% after treatment with NM-BL (p < 0.001). CNA was unchanged with NM-BL (58.1% during placebo and 58.3% with treatment). Subjective assessment of stool fat and stool consistency improved with NM-BL. Adverse events were mostly gastrointestinal in nature and consistent with the underlying disease. Conclusion: The study provided proof-of-concept for the efficacy of the liquid microbial lipase NM-BL which was safe and well tolerated. Acknowledgement: This study was sponsored by Nordmark Arzneimittel GmbH & Co. KG.
Workshop 18. CFTR: Functional tests for therapeutic interventions WS18.1 Using CF colon organoids to study CFTR function of uncharacterized mutations and test potential therapies A.S. Ramalho1 , F. Vermeulen2 , M. Proesmans2 , K. De Boeck2 . 1 KULeuven, Organ Systems, Leuven, Belgium; 2 UZ Leuven, Pediatric Pulmonology, Dept Pediatrics, Leuven, Belgium More than 1,900 mutations are reported to the CFTR Mutation Database most being disease-causing. However, many have not yet been characterized. The forskolin induced swelling (FIS) of organoids establish in Utrecht (Dekkers et al Nat Med.2013;19(7):939–45) can be used to quantify CFTR function ex vivo in patients’ own tissue and to study how therapeutic compounds can increase this function. We grew intestinal organoid cultures from rectal biopsies of patients with uncharacterized mutations. The area of the organoids at time 0 before FIS was considered 100% and was further monitored during 60 minutes. Organoids were incubated with VX-809 3mM overnight, baseline organoid surface area was determined and VX-770 3mM was then added acutely in combination with forskolin 0.08mM. The results correspond to the mean±SEM organoid area after 60 min, using 2 wells per test condition and at least 20 organoids per well. In the FIS assay, organoids from patients with genotypes N1303K/K464E; E60X/G85R; c.2988+1G>A/c.1545–1546delTA did not respond to VXcompounds. But for two other genotypes, F508del/Q237E and F508del/L159S, there was a marked response to VX-770 plus VX-809VX i.e. swelling to 152%±5% and 180%±3% respectively. Overall, organoids allow assessment of CFTR function in individual patients. This can thus be a promising tool to preselect the ideal candidates for CFTR modulator therapy enabling personalized and precision medicine, especially in patients with very rare CFTR mutations. Acknowledgements: To J Beekman lab (Utrecht University) for the help with the FIS assay. This work was funded by Belgium IWT grant 130663 Cystic Fibrosis. WS18.2 Prospective selection of potential CFTR-modifying treatments using intestinal organoids G. Berkers1 , J.F. Dekkers1 , E. Kruisselbrink1 , A.M. Vonk1 , S. HeidaMichel1 , M. Geerdink1 , K.M. de Winter-de Groot1 , H.G.M. Arets1 , I. Bronsveld2 , E.A. van de Graaf2 , C.J. Majoor3 , G.H. Koppelman4 , J. Roukema5 , H.M. Janssens6 , H.G.M. Heijerman7 , R.G.J. Vries8 , J.C. Clevers8 , H. de Jonge9 , J.M. Beekman1 , C.K. van der Ent1 . 1 University Medical Center Utrecht – Wilhelmina Children’s Hospital, Utrecht, Netherlands; 2 University Medical Center Utrecht, Utrecht, Netherlands; 3 Academic Medical Center, Amsterdam, Netherlands; 4 University Medical Center Groningen, Groningen, Netherlands; 5 Radboud University Medical Center, Nijmegen, Netherlands; 6 Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, Netherlands; 7 Haga Teaching Hospital, The Hague, Netherlands; 8 Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, Netherlands; 9 Erasmus Medical Center, Rotterdam, Netherlands Objectives: We have set up a functional CFTR assay using patientderived intestinal stem cell cultures, termed organoids (Dekkers et