Pilot study of adjunctive albuterol in late-onset Pompe disease reveals safety and efficacy from...
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GM2 gangliosidoses, including Tay-Sachs disease (TSD) and Sandhoff disease (SD), are neurodegenerative lysosomal diseases (LSD) associated with excessive accumulation of GM2 ganglioside (GM2) in the brain and neurological symptoms. TSD and SD are the lysosomal β-hexosamini- dase A (HexA) deficiencies caused by gene mutations in HEXA and HEXB loci, which encode the α- and β-subunit of HexA (αβ heterodimer), respectively. Hence there are no effective treatments for GM2 gan- gliosidoses, although several therapeutic approaches have been devel- oped and reported including gene therapy, enzyme replacement therapy, substrate reduction therapy, etc. Previously we constructed a human modified HEXB cDNA encoding altered β-subunit containing partial amino acid sequence of the α-subunit, and succeeded in production of the modified HexB (modB) by CHO cell line with the GM2-degrading activity in cooperation with intracellular GM2 activator protein (GM2A) in the brains by intracerebroventricular (icv) adminis- tration to SD model mice. The modB carrying N-glycans with terminal mannose 6-phosphate (M6P) residues is expected to be incorporated into the neural cells with cation-independent M6P receptors (CI-M6PR) and act as a low-antigenic camouflaged enzyme for TSD patients who have the normal Hex β-subunits. In this study we established a novel purification system to obtain selectively the recombinant modB with M6P-containing N-glycans (M6P-modB) by three-step column chroma- tography, including an affinity chromatography with Phos-Tag resin, which is usually used to capture the phosphorylated proteins. Purified M6P-modB was delivered more efficiently to lysosomes in the target cells derived from TSD patients and SD mice through binding with cell surface CI-M6PR, and reduced the GM2 accumulated in the target cells. We also developed two novel acidic pH-activatable fluorescent probes, including HMRodolB-βGlcNAc as a substrate for Hex and RhPs as a tag conjugated to Hex proteins. Cytoplasmic fluorescence (Ex. 514 nm, Em. 530 nm) due to HMRodolB released from HMRodolB-βGlcNAc by Hex activity in lysosomes was observed in the cultured cells and brain tissues derived from normal subject and the wild-type mice after incubation at 37°C in the presence of HMRodolB-βGlcNAc. In contrast, little fluores- cence was detected in the cells and tissues derived from SD model mice. These results indicate Hex-specific intracellular degradation of HMRodolB-βGlcNAc, which should be applicable for vital imaging of intralysosomal degradation by Hex. We also prepared the conjugates (modB-RhPs) between modB and RhPs without loss of Hex activity, and administered to the cultured cells derived from TSD patient and SD model mice. Restoration of Hex activity and punctate fluorescence were observed due to delivery of the modB-RhPs to lysosomes at which the pH condition is acidic. Imaging with the latter novel fluorescent probe is useful to visualize intralysosomal distribution of modB protein in the cultured cells and tissues with Hex deficiencies and evaluate enzyme replacement effects of not only modB but also other recombinant lysosomal enzymes. doi:10.1016/j.ymgme.2013.12.136 125 Pilot study of adjunctive albuterol in late-onset Pompe disease reveals safety and efficacy from upregulated Mannose-6-phosphate receptor expression Dwight Koeberl, Stephanie Austin, Laura Case, Edward C. Smith, Anne Buckley, Sarah P. Young, Deeksha Bali, Priya S. Kishnani, Duke University Medical Center, Durham, NC, USA Effective dosages for enzyme replacement therapy (ERT) in Pompe disease are much greater than for other lysosomal disorders, which has been attributed to low cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle. We have previously demonstrated the benefit of increased CI-MPR-mediated uptake of recombinant human acid-α-glucosidase during ERT in mice with Pompe disease following addition of albuterol therapy. Currently we have completed a pilot study of albuterol in patients with late-onset Pompe disease ERT for greater than two years, who were not improving further. This was an open label study of albuterol in adult patients with Pompe disease, during which subjects underwent a baseline evaluation including blood safety testing, EKG, pulmonary function testing, muscle function and strength testing, 6 minute walk test (6MWT), and muscle biopsy at Baseline and Week 12. Patients returned at Week 6 and 24 for physical examination, safety testing, muscle function and strength testing, and 6MWT. The initial dose for albuterol extended release (ER) was 4 mg BID. At Week 6 the dose of albuterol was increased to 8 mg BID, barring dose-limiting adverse events (AE). This study was approved by the Duke University Institutional Review Board, and written consent was obtained at study entry. The 6MWT distance increased in all 7 subjects at Week 6 (+30 +/- 13 meters; p = 0.002), Week 12 (+34 +/- 14 meters; p = 0.004), and Week 24 (+42 +/- 37 meters; p = 0.02), in comparison with Baseline. Grip strength was improved significantly for both hands at Week 12. Furthermore, individual subjects reported benefits, including: 1) a woman could now stand up from sitting the floor much more easily (time for supine to standing decreased from 30 seconds to 11 seconds); and 2) a man can now readily swing his legs out of his van seat (hip abduction increased from 1 to 2 + on manual muscle testing). Finally, analysis of the quadriceps biopsies revealed a significant increase in CI- MPR at Week 12 (p = 0.03). Only mild adverse events have been reported, including tremor, transient difficulty falling asleep, and mild urinary retention (requiring early morning voiding). Therefore, this pilot study revealed initial safety and efficacy in an open label study of adjunctive albuterol therapy in patients with late-onset Pompe disease on ERT. doi:10.1016/j.ymgme.2013.12.137 126 Does hyperferritinemia reflect iron overload in Gaucher disease? Tiago B. Koppe, Filipp Vairo, Divair Doneda, Cristina B. Netto, Ida D. Schwartz, UFRGS, Porto Alegre, Brazil Background: Gaucher disease is characterized by decreased β- glucosidase activity, leading to an intracellular accumulation of glycosphingolipids and bone/hematological/visceral disease. Since 1983, there have been reports of hyperferritinemia in Gaucher patients, but the impact of this finding on iron homeostasis is unknown. Objective: To evaluate iron metabolism in Gaucher patients seen at the Reference Center of Hospital de Clínicas de Porto Alegre, Brazil. METHODS: A retrospective chart review study. The following parameters of iron metabolism were evaluated: serum iron, transferrin saturation, total iron binding capacity and serum ferritin ([SF]). Several statistical approaches were performed using SPSS software (IBM©), comparing serum ferritin to different variables. Statistic level was α = 0.05. Results: 39 patients were included (mean age = 33.35 ± 16.49 years and mean treatment time = 7.02 ± 6.29 years). 8/19 women and 15/20 men presented hyperferritinemia. Presence or absence of splenectomy, splenomegaly and hepatomegaly did not influence [SF], but there as a borderline effect of enzyme replacement therapy (ERT) (n = 19, t = 1.745, p = 0.098). Among Pearson correlations, the Zimran score and the GS3 score did not correlated to [SF], as well as to the plasma activity of chitotriosidase and hemoglobinemia, but there was a tendency regarding treatment time and [SF] (n = 33, r = -0.313, p = 0.056). There was a strong correlation between [SF] and age (r = 0.718, p b 0.001). Only two hiperferritinemic patients had evidence of iron overload. Abstracts S62
Transcript of Pilot study of adjunctive albuterol in late-onset Pompe disease reveals safety and efficacy from...
GM2 gangliosidoses, including Tay-Sachs disease (TSD) and Sandhoffdisease (SD), are neurodegenerative lysosomal diseases (LSD) associatedwith excessive accumulation of GM2 ganglioside (GM2) in the brain andneurological symptoms. TSD and SD are the lysosomal β-hexosamini-dase A (HexA) deficiencies caused by genemutations in HEXA andHEXBloci, which encode the α- and β-subunit of HexA (αβ heterodimer),respectively. Hence there are no effective treatments for GM2 gan-gliosidoses, although several therapeutic approaches have been devel-oped and reported including gene therapy, enzyme replacementtherapy, substrate reduction therapy, etc. Previously we constructed ahuman modified HEXB cDNA encoding altered β-subunit containingpartial amino acid sequence of the α-subunit, and succeeded inproduction of the modified HexB (modB) by CHO cell line with theGM2-degrading activity in cooperation with intracellular GM2 activatorprotein (GM2A) in the brains by intracerebroventricular (icv) adminis-tration to SD model mice. The modB carrying N-glycans with terminalmannose 6-phosphate (M6P) residues is expected to be incorporatedinto the neural cells with cation-independent M6P receptors (CI-M6PR)and act as a low-antigenic camouflaged enzyme for TSD patients whohave the normal Hex β-subunits. In this study we established a novelpurification system to obtain selectively the recombinant modB withM6P-containing N-glycans (M6P-modB) by three-step column chroma-tography, including an affinity chromatography with Phos-Tag resin,which is usually used to capture the phosphorylated proteins. PurifiedM6P-modB was delivered more efficiently to lysosomes in the targetcells derived from TSD patients and SD mice through binding with cellsurface CI-M6PR, and reduced the GM2 accumulated in the target cells.We also developed two novel acidic pH-activatable fluorescent probes,including HMRodolB-βGlcNAc as a substrate for Hex and RhPs as a tagconjugated to Hex proteins. Cytoplasmic fluorescence (Ex. 514 nm, Em.530 nm) due to HMRodolB released from HMRodolB-βGlcNAc by Hexactivity in lysosomeswas observed in the cultured cells and brain tissuesderived from normal subject and the wild-type mice after incubation at37°C in the presence of HMRodolB-βGlcNAc. In contrast, little fluores-cence was detected in the cells and tissues derived from SDmodel mice.These results indicate Hex-specific intracellular degradation ofHMRodolB-βGlcNAc, which should be applicable for vital imaging ofintralysosomal degradation by Hex. We also prepared the conjugates(modB-RhPs) between modB and RhPs without loss of Hex activity, andadministered to the cultured cells derived from TSD patient and SDmodel mice. Restoration of Hex activity and punctate fluorescence wereobserved due to delivery of themodB-RhPs to lysosomes atwhich the pHcondition is acidic. Imaging with the latter novel fluorescent probe isuseful to visualize intralysosomal distribution of modB protein in thecultured cells and tissues with Hex deficiencies and evaluate enzymereplacement effects of not only modB but also other recombinantlysosomal enzymes.
doi:10.1016/j.ymgme.2013.12.136
125Pilot study of adjunctive albuterol in late-onset Pompedisease reveals safety and efficacy from upregulatedMannose-6-phosphate receptor expression
Dwight Koeberl, Stephanie Austin, Laura Case, Edward C. Smith, AnneBuckley, Sarah P. Young, Deeksha Bali, Priya S. Kishnani, DukeUniversity Medical Center, Durham, NC, USA
Effective dosages for enzyme replacement therapy (ERT) in Pompedisease are much greater than for other lysosomal disorders, which hasbeen attributed to low cation-independent mannose-6-phosphatereceptor (CI-MPR) in skeletal muscle. We have previously demonstratedthebenefit of increased CI-MPR-mediateduptake of recombinant human
acid-α-glucosidase during ERT in mice with Pompe disease followingaddition of albuterol therapy. Currently we have completed a pilot studyof albuterol in patients with late-onset Pompe disease ERT for greaterthan two years, who were not improving further. This was an open labelstudy of albuterol in adult patients with Pompe disease, during whichsubjects underwent a baseline evaluation including blood safety testing,EKG, pulmonary function testing,muscle function and strength testing, 6minute walk test (6MWT), and muscle biopsy at Baseline and Week 12.Patients returned at Week 6 and 24 for physical examination, safetytesting,muscle function and strength testing, and 6MWT. The initial dosefor albuterol extended release (ER) was 4 mg BID. AtWeek 6 the dose ofalbuterol was increased to 8 mg BID, barring dose-limiting adverseevents (AE). This study was approved by the Duke UniversityInstitutional Review Board, and written consent was obtained at studyentry. The 6MWT distance increased in all 7 subjects at Week 6 (+30+/- 13 meters; p = 0.002), Week 12 (+34 +/- 14 meters; p = 0.004),and Week 24 (+42 +/- 37 meters; p = 0.02), in comparison withBaseline. Grip strength was improved significantly for both hands atWeek 12. Furthermore, individual subjects reported benefits, including:1) a woman could now stand up from sitting the floor muchmore easily(time for supine to standing decreased from 30 seconds to 11 seconds);and 2) a man can now readily swing his legs out of his van seat (hipabduction increased from 1 to 2+ on manual muscle testing). Finally,analysis of the quadriceps biopsies revealed a significant increase in CI-MPR at Week 12 (p= 0.03). Only mild adverse events have beenreported, including tremor, transient difficulty falling asleep, and mildurinary retention (requiring earlymorning voiding). Therefore, this pilotstudy revealed initial safety and efficacy in an open label study ofadjunctive albuterol therapy in patients with late-onset Pompe diseaseon ERT.
doi:10.1016/j.ymgme.2013.12.137
126Does hyperferritinemia reflect iron overload in Gaucher disease?
Tiago B. Koppe, Filipp Vairo, Divair Doneda, Cristina B. Netto, Ida D.Schwartz, UFRGS, Porto Alegre, Brazil
Background: Gaucher disease is characterized by decreased β-glucosidase activity, leading to an intracellular accumulation ofglycosphingolipids and bone/hematological/visceral disease. Since1983, there have been reports of hyperferritinemia in Gaucher patients,but the impact of this finding on iron homeostasis is unknown.
Objective: To evaluate iron metabolism in Gaucher patients seenat the Reference Center of Hospital de Clínicas de Porto Alegre, Brazil.
METHODS: A retrospective chart review study. The followingparameters of iron metabolism were evaluated: serum iron, transferrinsaturation, total iron binding capacity and serum ferritin ([SF]). Severalstatistical approaches were performed using SPSS software (IBM©),comparing serum ferritin to different variables. Statistic level wasα=0.05.
Results: 39 patients were included (mean age = 33.35 ± 16.49 yearsandmean treatment time= 7.02 ± 6.29 years). 8/19women and 15/20men presented hyperferritinemia. Presence or absence of splenectomy,splenomegaly and hepatomegaly did not influence [SF], but there as aborderline effect of enzyme replacement therapy (ERT) (n = 19,t = 1.745, p = 0.098). Among Pearson correlations, the Zimran scoreand the GS3 score did not correlated to [SF], as well as to the plasmaactivity of chitotriosidase and hemoglobinemia, but there was atendency regarding treatment time and [SF] (n= 33, r = -0.313,p = 0.056). There was a strong correlation between [SF] and age(r = 0.718, p b 0.001). Only two hiperferritinemic patients had evidenceof iron overload.
AbstractsS62
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