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Novel molecular tools to advance the evaluation of Gaucher disease therapeutics in live cells
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Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
dose from the SAD study (N=13). After single oral administration to JHS and NHS, dose-proportionality was observed from 50 mg to 450 mg, and from 50 mg to 675 mg, respectively. When comparing exposures from a single 150-mg dose between JHS and either NHS or NFP who received 150 mg QOD, no appreciable differences are observed. This similarity was further apparent when normalizing exposures by body weight. Based on these evaluations, characterization of PK in JHS is similar to NHS and to NFP; therefore, the PK of migalastat in JFP should not be different from JHS, NHS, or NFP.
doi:10.1016/j.ymgme.2016.11.168
160 Respiratory muscle training in late-onset Pompe disease Harrison N. Jones, Kaylea Nicholson, Kelly D. Crisp, Duke University, Durham, NC, United States Progressive respiratory muscle weakness remains the primary cause of morbidity and mortality in late-onset Pompe disease (LOPD). In response, we have programmatically investigated the utility of respiratory muscle training (RMT) as a treatment for respiratory weakness. In 2011, we first reported the results of a clinical RMT regimen in 2 adults with LOPD and severe respiratory weakness. RMT was well-tolerated and participants demonstrated substantial increases in inspiratory and expiratory strength. Next, we investigated the effects of our 12-week RMT regimen with a singlesubject A-B-A design replicated across 8 adults. RMT was followed by a 3-month detraining period. Pretest to posttest, maximum inspiratory pressure (MIP) improved in all 8 subjects and 7 of 8 showed increases in maximum expiratory pressure (MEP). Effect size calculations (Cohen’s d) revealed increases in both MIP and MEP that were large (d ≥1.0) to very large (d≥2.0) in magnitude. Across participants, pretest to posttest MIP increased by a mean of 20% and MEP increased by a mean of 15%. Furthermore, respiratory strength enhancements were generally durable to detraining. We will next conduct an exploratory, double-blind, placebo-controlled, randomized clinical trial of our 12-week RMT program in 28 adults with LOPD (NIH 1R21AR069880-01). Participants will be randomized to treatment (RMT) or control (sham-RMT) arms. Assessments will be conducted at pretest, posttest, and after 3- and 6-months detraining. Our primary outcome is MIP. Secondary outcomes include MEP, 6 Minute Walk Test (6MWT), Gait, Stairs, Gower, and Chair (GSGC) scale, peak cough flow (PCF), and patient-reported life activity/social participation. Exploratory outcomes target sleep and sleep breathing as assessed with standard questionnaires and laboratory polysomnography. Our aims are to 1) determine the utility and feasibility of sham-RMT as a control condition for RMT, and 2) identify the outcomes that are clinically meaningful to patients and clinicians for future efficacy testing.
doi:10.1016/j.ymgme.2016.11.169
161 Novel molecular tools to advance the evaluation of Gaucher disease therapeutics in live cells OIive Junga, Samarjit Patnaikb, Wendy Westbroekc, Shawn Stacheld, Michael Breslind, Paige Cramerd, Steve Titusb, Ty Vossb, Juan Maruganb, Ellen Sidranskyc, aNational Human Genome Research Institute, National Center for Advancing Translational Sciences, Bethesda, MD, United States,
b
National Center for Advancing Translational Sciences, Rockville, MD, United States, cNational Human Genome Research Institute, Bethesda, MD, United States, dMerck Research Laboratories, Rockville, MD, United States
Gaucher disease (GD), a common lysosomal disease, results from mutations in GBA1, which is the gene that encodes for the enzyme glucocerebrosidase (GCase). In GD, the natural substrate glucosylceramide (GlcCer) cannot be broken down into glucose and ceramide, leading to subsequent lysosomal accumulation of GlcCer. Current treatments for GD do not cross the blood-brain barrier, and subsequently, alternate therapeutic strategy for GD has been to identify small, non-inhibitory GCase chaperones through high throughput screening (HTS) assays. Since the mutant enzyme generally retains some catalytic activity, non-inhibitory chaperones can translocate the mutant enzyme to the lysosome. However, there is currently no efficient and/or precise method to evaluate noninhibitory chaperones for GD or other lysosomal diseases. We developed a substrate that can effectively measure GCase activity in lysosomes of live, intact cells. The substrate was synthesized using commercially available materials and analyzed by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The substrate-product pair was characterized in enzyme solution, cell lysates and live cells. Wild-type and patientderived (GD1-genotype N370S/N370S and GD2-genotype L444P/ RecNci) disease fibroblasts were treated with a known chaperone, and substrate turnover was compared to non-treated cells on both plate- and image-based platforms. The cells were co-stained with Lysotracker Deep Red (LTDR) to identify localization of substrate turnover in lysosomes at the end of the time course. Preliminary results indicate an increased substrate turnover in chaperone-treated cells on both platforms and a significant co-localization between LTDR and the formed product. Our study indicates that this novel live-cell substrate can potentially be used in HTS to measure lysosome-specific GCase enzyme activity in control and GD fibroblasts. Live-cell substrates will ultimately have far-reaching implications for the evaluation of other LSDs as well as related disorders, such as Parkinson disease, and further facilitate drug discovery for such affected lysosomal diseases. doi:10.1016/j.ymgme.2016.11.170
162 Quantitative systems pharmacology model of acid sphingomyelinase deficiency and the enzyme replacement therapy olipudase alfa is an innovative tool for linking pathophysiology and pharmacology Chanchala Kaddia, Rena Baekb, Paul Jasperc, Bradley Niesnerc, John Pappasc, John Tolsmac, Jing Lia, Shayne Watsona, Sharon Tanb, Ana Cristina Pugab, Edward Schuchmand, Jeffrey S. Barretta, Karim Azera, a Sanofi, Bridgewater, NJ, United States, bSanofi Genzyme, Cambridge, MA, United States, cRES Group Inc., Needham, MA, United States, dMount Sinai School of Medicine, New York, NY, United States Olipudase alfa (rhASM) is an investigational enzyme replacement therapy in development for the treatment of nonneurological manifestations of acid sphingomyelinase deficiency (ASMD), traditionally known as Niemann-Pick disease types A and B. ASMD is clinically heterogeneous, affecting multiple organ systems, with clinical manifestations including hepatosplenomegaly, infiltrative pulmonary disease, and hyperlipidemia [1]. A phase 1b trial of olipudase alfa treated five adults with chronic visceral ASMD (Niemann-Pick disease type B), and demonstrated improvements in clinical markers including spleen and liver volume, infiltrative lung disease, and stored sphingomyelin [2, 3]. Phase 2/3 adult and phase
Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
dose from the SAD study (N=13). After single oral administration to JHS and NHS, dose-proportionality was observed from 50 mg to 450 mg, and from 50 mg to 675 mg, respectively. When comparing exposures from a single 150-mg dose between JHS and either NHS or NFP who received 150 mg QOD, no appreciable differences are observed. This similarity was further apparent when normalizing exposures by body weight. Based on these evaluations, characterization of PK in JHS is similar to NHS and to NFP; therefore, the PK of migalastat in JFP should not be different from JHS, NHS, or NFP.
doi:10.1016/j.ymgme.2016.11.168
160 Respiratory muscle training in late-onset Pompe disease Harrison N. Jones, Kaylea Nicholson, Kelly D. Crisp, Duke University, Durham, NC, United States Progressive respiratory muscle weakness remains the primary cause of morbidity and mortality in late-onset Pompe disease (LOPD). In response, we have programmatically investigated the utility of respiratory muscle training (RMT) as a treatment for respiratory weakness. In 2011, we first reported the results of a clinical RMT regimen in 2 adults with LOPD and severe respiratory weakness. RMT was well-tolerated and participants demonstrated substantial increases in inspiratory and expiratory strength. Next, we investigated the effects of our 12-week RMT regimen with a singlesubject A-B-A design replicated across 8 adults. RMT was followed by a 3-month detraining period. Pretest to posttest, maximum inspiratory pressure (MIP) improved in all 8 subjects and 7 of 8 showed increases in maximum expiratory pressure (MEP). Effect size calculations (Cohen’s d) revealed increases in both MIP and MEP that were large (d ≥1.0) to very large (d≥2.0) in magnitude. Across participants, pretest to posttest MIP increased by a mean of 20% and MEP increased by a mean of 15%. Furthermore, respiratory strength enhancements were generally durable to detraining. We will next conduct an exploratory, double-blind, placebo-controlled, randomized clinical trial of our 12-week RMT program in 28 adults with LOPD (NIH 1R21AR069880-01). Participants will be randomized to treatment (RMT) or control (sham-RMT) arms. Assessments will be conducted at pretest, posttest, and after 3- and 6-months detraining. Our primary outcome is MIP. Secondary outcomes include MEP, 6 Minute Walk Test (6MWT), Gait, Stairs, Gower, and Chair (GSGC) scale, peak cough flow (PCF), and patient-reported life activity/social participation. Exploratory outcomes target sleep and sleep breathing as assessed with standard questionnaires and laboratory polysomnography. Our aims are to 1) determine the utility and feasibility of sham-RMT as a control condition for RMT, and 2) identify the outcomes that are clinically meaningful to patients and clinicians for future efficacy testing.
doi:10.1016/j.ymgme.2016.11.169
161 Novel molecular tools to advance the evaluation of Gaucher disease therapeutics in live cells OIive Junga, Samarjit Patnaikb, Wendy Westbroekc, Shawn Stacheld, Michael Breslind, Paige Cramerd, Steve Titusb, Ty Vossb, Juan Maruganb, Ellen Sidranskyc, aNational Human Genome Research Institute, National Center for Advancing Translational Sciences, Bethesda, MD, United States,
b
National Center for Advancing Translational Sciences, Rockville, MD, United States, cNational Human Genome Research Institute, Bethesda, MD, United States, dMerck Research Laboratories, Rockville, MD, United States
Gaucher disease (GD), a common lysosomal disease, results from mutations in GBA1, which is the gene that encodes for the enzyme glucocerebrosidase (GCase). In GD, the natural substrate glucosylceramide (GlcCer) cannot be broken down into glucose and ceramide, leading to subsequent lysosomal accumulation of GlcCer. Current treatments for GD do not cross the blood-brain barrier, and subsequently, alternate therapeutic strategy for GD has been to identify small, non-inhibitory GCase chaperones through high throughput screening (HTS) assays. Since the mutant enzyme generally retains some catalytic activity, non-inhibitory chaperones can translocate the mutant enzyme to the lysosome. However, there is currently no efficient and/or precise method to evaluate noninhibitory chaperones for GD or other lysosomal diseases. We developed a substrate that can effectively measure GCase activity in lysosomes of live, intact cells. The substrate was synthesized using commercially available materials and analyzed by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The substrate-product pair was characterized in enzyme solution, cell lysates and live cells. Wild-type and patientderived (GD1-genotype N370S/N370S and GD2-genotype L444P/ RecNci) disease fibroblasts were treated with a known chaperone, and substrate turnover was compared to non-treated cells on both plate- and image-based platforms. The cells were co-stained with Lysotracker Deep Red (LTDR) to identify localization of substrate turnover in lysosomes at the end of the time course. Preliminary results indicate an increased substrate turnover in chaperone-treated cells on both platforms and a significant co-localization between LTDR and the formed product. Our study indicates that this novel live-cell substrate can potentially be used in HTS to measure lysosome-specific GCase enzyme activity in control and GD fibroblasts. Live-cell substrates will ultimately have far-reaching implications for the evaluation of other LSDs as well as related disorders, such as Parkinson disease, and further facilitate drug discovery for such affected lysosomal diseases. doi:10.1016/j.ymgme.2016.11.170
162 Quantitative systems pharmacology model of acid sphingomyelinase deficiency and the enzyme replacement therapy olipudase alfa is an innovative tool for linking pathophysiology and pharmacology Chanchala Kaddia, Rena Baekb, Paul Jasperc, Bradley Niesnerc, John Pappasc, John Tolsmac, Jing Lia, Shayne Watsona, Sharon Tanb, Ana Cristina Pugab, Edward Schuchmand, Jeffrey S. Barretta, Karim Azera, a Sanofi, Bridgewater, NJ, United States, bSanofi Genzyme, Cambridge, MA, United States, cRES Group Inc., Needham, MA, United States, dMount Sinai School of Medicine, New York, NY, United States Olipudase alfa (rhASM) is an investigational enzyme replacement therapy in development for the treatment of nonneurological manifestations of acid sphingomyelinase deficiency (ASMD), traditionally known as Niemann-Pick disease types A and B. ASMD is clinically heterogeneous, affecting multiple organ systems, with clinical manifestations including hepatosplenomegaly, infiltrative pulmonary disease, and hyperlipidemia [1]. A phase 1b trial of olipudase alfa treated five adults with chronic visceral ASMD (Niemann-Pick disease type B), and demonstrated improvements in clinical markers including spleen and liver volume, infiltrative lung disease, and stored sphingomyelin [2, 3]. Phase 2/3 adult and phase