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Yuichi Hashimoto - One of the best experts on this subject based on the ideXlab platform.
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structure activity relationship study of non steroidal npc1l1 ligands identified through cell based assay using Pharmacological Chaperone effect as a readout
Bioorganic & Medicinal Chemistry, 2014Co-Authors: Fumika Karaki, Hiromitsu Fukuda, Kenji Ohgane, Kosuke Dodo, Masahiko Nakamura, Yuichi HashimotoAbstract:Abstract Niemann-Pick type C1-like 1 (NPC1L1) is an intestinal cholesterol transporter that is known to be the target of the cholesterol absorption inhibitor ezetimibe. We previously discovered steroidal NPC1L1 ligands by using a novel cell-based assay that employs Pharmacological Chaperone effect as a readout. Those steroid derivatives bound to a site different from both the sterol-binding domain and the ezetimibe-binding site, implying that they may be a novel class of NPC1L1 inhibitors with a distinct mode of action. As an extension of that work, we aimed here to find non-steroidal NPC1L1 ligands, which may be better candidates for clinical application than steroidal ligands, by using the same assay to screen our focused library of ligands for liver X receptor (LXR), a nuclear receptor that recognizes oxysterols as endogenous ligands. Here we describe identification of a novel class of NPC1L1 ligands with a ring-fused quinolinone scaffold, and an analysis of the structure–activity relationships of their derivatives as NPC1L1 ligands.
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structure activity relationship studies of niemann pick type c1 like 1 npc1l1 ligands identified by screening assay monitoring Pharmacological Chaperone effect
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Fumika Karaki, Kenji Ohgane, Kosuke Dodo, Yuichi HashimotoAbstract:Abstract A number of hereditary diseases are caused by defective protein trafficking due to a folding defect resulting from point mutations in proteins. Ligands that bind to the folding intermediates of such mutant proteins and rescue their trafficking defects, known as Pharmacological Chaperones, have promise for the treatment of certain genetic diseases, including Fabry disease, cystic fibrosis, and Niemann-Pick disease type C. Here we show that this Pharmacological Chaperone effect can be used for ligand screening, that is, binding of candidate ligands can be detected by monitoring the ligand-mediated correction of a localization defect caused by artificially introduced point mutations of the protein of interest. Using this method, we discovered novel steroidal ligands of Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter that is the target of the cholesterol absorption inhibitor ezetimibe, and conducted structure–activity relationship studies. We also present data indicating that the binding site of the new ligands is distinct from both the N-terminal sterol-binding domain and the ezetimibe-binding site.
Kenneth J Valenzano - One of the best experts on this subject based on the ideXlab platform.
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Pharmacological Chaperone therapy preclinical development clinical translation and prospects for the treatment of lysosomal storage disorders
Molecular Therapy, 2015Co-Authors: Giancarlo Parenti, Generoso Andria, Kenneth J ValenzanoAbstract:Lysosomal storage disorders (LSDs) are a group of inborn metabolic diseases caused by mutations in genes that encode proteins involved in different lysosomal functions, in most instances acidic hydrolases. Different therapeutic approaches have been developed to treat these disorders. Pharmacological Chaperone therapy (PCT) is an emerging approach based on small-molecule ligands that selectively bind and stabilize mutant enzymes, increase their cellular levels, and improve lysosomal trafficking and activity. Compared to other approaches, PCT shows advantages, particularly in terms of oral administration, broad biodistribution, and positive impact on patients' quality of life. After preclinical in vitro and in vivo studies, PCT is now being translated in the first clinical trials, either as monotherapy or in combination with enzyme replacement therapy, for some of the most prevalent LSDs. For some LSDs, the results of the first clinical trials are encouraging and warrant further development. Future research in the field of PCT will be directed toward the identification of novel Chaperones, including new allosteric drugs, and the exploitation of synergies between Chaperone treatment and other therapeutic approaches.
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p 17 8 the co formulation of Pharmacological Chaperone at2220 with recombinant human acid alpha glucosidase improves enzyme uptake and glycogen reduction in a mouse model of pompe disease
Neuromuscular Disorders, 2013Co-Authors: Richie Khanna, Rebecca Soska, Yi Lun, Jessie Feng, Michelle Frascella, Lee Pellegrino, David J Lockhart, Anadina Garcia, John Flanagan, Kenneth J ValenzanoAbstract:Pompe disease is an inherited lysosomal storage disease that results from deficiency in acid alpha-glucosidase (GAA) activity, and is characterized by progressive accumulation of lysosomal glycogen in heart and skeletal muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) is the only approved treatment available for Pompe disease. While rhGAA provides some clinical benefits, the infused enzyme tends to be unstable at neutral pH/body temperature, shows insufficient uptake in key tissues, and can elicit immune responses that affect tolerability and efficacy. We have shown previously that oral pre-administration of the Pharmacological Chaperone AT2220 (1-deoxynojirimycin HCl, duvoglustat HCl) improves the Pharmacological properties of rhGAA via binding and stabilization, leading to increased enzyme uptake and glycogen reduction in GAA knock-out (KO) mice. In this study we tested the effects of intravenous (IV) and subcutaneous (SQ) administration of co-formulated AT2220 and rhGAA (AT2220 + rhGAA) as an alternative to giving AT2220 orally prior to rhGAA IV. In rats, IV or SQ administration of co-formulated AT2220 + rhGAA increased the circulating half-life of rhGAA up to twofold, though the maximal rhGAA plasma levels achieved via the SQ route were significantly lower than those seen following IV administration. In GAA KO mice, four IV administrations of co-formulated AT2220 + rhGAA resulted in up to 2.5-fold greater enzyme uptake and glycogen reduction in disease-relevant tissues compared to rhGAA alone; histological staining confirmed reduced skeletal muscle glycogen. Interestingly, four SQ administrations of co-formulated AT2220 + rhGAA increased rhGAA uptake in GAA KO mice, which was not significantly different from those seen following four IV administrations of rhGAA alone. Collectively, these data highlight the potential effects of co-formulated AT2220 + rhGAA using IV or SQ administration, thus warranting further preclinical investigation.
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t p 44 the Pharmacological Chaperone at2220 increases the stability of recombinant human acid α glucosidase and leads to greater tissue uptake and glycogen reduction in a mouse model of pompe disease
Neuromuscular Disorders, 2012Co-Authors: Kenneth J Valenzano, Rebecca Soska, Yi Lun, Jessie Feng, Michelle Frascella, Lee Pellegrino, David J Lockhart, Darlene Guillen, John Flanagan, Richie KhannaAbstract:Abstract Pompe disease is a lysosomal storage disease caused by deficiency of acid α-glucosidase (GAA) activity, and is characterized by impaired lysosomal glycogen catabolism, progressive skeletal muscle weakness, reduced cardiac function, and respiratory insufficiency. Recombinant human GAA (rhGAA, Genzyme) is the only approved enzyme replacement therapy (ERT) for Pompe, and is administered biweekly via intravenous infusion. While rhGAA does provide clinical benefit, it suffers from low stability at neutral pH/body temperature, shows modest tissue uptake and glycogen reduction, and can elicit immune responses that affect tolerability and efficacy. AT2220 (1-deoxynojirimycin HCl, duvoglustat hydrochloride) is a small molecule Pharmacological Chaperone that binds and stabilizes endogenous GAA in cells and tissues, resulting in increased lysosomal GAA activity. We hypothesized that AT2220 might also improve the Pharmacological properties of exogenous rhGAA. In human plasma, AT2220 co-incubation increased the stability and prevented denaturation of rhGAA at neutral pH/ 37 °C for up to 24 h. In rats, a single oral administration of AT2220 followed 30 min later by intravenous bolus administration of rhGAA resulted in a dose-dependent increase of up to 2-fold in the circulating half-life of rhGAA. A similar effect was seen on the circulating half-life of rhGAA when administered via intravenous infusion. In mice lacking endogenous GAA, oral administration of AT2220 resulted in up to 2.5-fold greater rhGAA uptake and glycogen reduction compared to administration of rhGAA alone in disease-relevant tissues. Collectively, these data indicate that AT2220 increases the stability of rhGAA, and that greater enzyme activity and substrate turnover can be achieved in muscle when co-administered with rhGAA. Based on these findings, a Phase 2 study exploring AT2220 co-administration with rhGAA has been initiated.
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the Pharmacological Chaperone at2220 increases recombinant human acid α glucosidase uptake and glycogen reduction in a mouse model of pompe disease
PLOS ONE, 2012Co-Authors: Richie Khanna, Rebecca Soska, Yi Lun, Jessie Feng, Michelle Frascella, Lee Pellegrino, John J Flanagan, David J Lockhart, Darlene Guillen, Kenneth J ValenzanoAbstract:Pompe disease is an inherited lysosomal storage disease that results from a deficiency in the enzyme acid α-glucosidase (GAA), and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. Recombinant human GAA (rhGAA) is the only approved enzyme replacement therapy (ERT) available for the treatment of Pompe disease. Although rhGAA has been shown to slow disease progression and improve some of the pathophysiogical manifestations, the infused enzyme tends to be unstable at neutral pH and body temperature, shows low uptake into some key target tissues, and may elicit immune responses that adversely affect tolerability and efficacy. We hypothesized that co-administration of the orally-available, small molecule Pharmacological Chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) may improve the Pharmacological properties of rhGAA via binding and stabilization. AT2220 co-incubation prevented rhGAA denaturation and loss of activity in vitro at neutral pH and 37°C in both buffer and blood. In addition, oral pre-administration of AT2220 to rats led to a greater than two-fold increase in the circulating half-life of intravenous rhGAA. Importantly, co-administration of AT2220 and rhGAA to GAA knock-out (KO) mice resulted in significantly greater rhGAA levels in plasma, and greater uptake and glycogen reduction in heart and skeletal muscles, compared to administration of rhGAA alone. Collectively, these preclinical data highlight the potentially beneficial effects of AT2220 on rhGAA in vitro and in vivo. As such, a Phase 2 clinical study has been initiated to investigate the effects of co-administered AT2220 on rhGAA in Pompe patients.
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a pharmacogenetic approach to identify mutant forms of α galactosidase a that respond to a Pharmacological Chaperone for fabry disease
Human Mutation, 2011Co-Authors: Evan Katz, Kenneth J Valenzano, John J Flanagan, David J Lockhart, Kirsten Mascioli, Maria Cecilia Della Valle, Jeffrey P Castelli, Raphael Schiffmann, Pol Boudes, Elfrida R BenjaminAbstract:Fabry disease is caused by mutations in the gene (GLA) that encodes α-galactosidase A (α-Gal A). The iminosugar AT1001 (GR181413A, migalastat hydrochloride, 1-deoxygalactonojirimycin) is a Pharmacological Chaperone that selectively binds and stabilizes α-Gal A, increasing total cellular levels and activity for some mutant forms (defined as “responsive”). In this study, we developed a cell-based assay in cultured HEK-293 cells to identify mutant forms of α-Gal A that are responsive to AT1001. Concentration-dependent increases in α-Gal A activity in response to AT1001 were shown for 49 (60%) of 81 mutant forms. The responses of α-Gal A mutant forms were generally consistent with the responses observed in male Fabry patient-derived lymphoblasts. Importantly, the HEK-293 cell responses of 19 α-Gal A mutant forms to a clinically achievable concentration of AT1001 (10 µM) were generally consistent with observed increases in α-Gal A activity in peripheral blood mononuclear cells from male Fabry patients orally administered AT1001 during Phase 2 clinical studies. This indicates that the cell-based responses can identify mutant forms of α-Gal A that are likely to respond to AT1001 in vivo. Thus, the HEK-293 cell-based assay may be a useful aid in the identification of Fabry patients with AT1001-responsive mutant forms. Hum Mutat 32:1–13, 2011. © 2011 Wiley-Liss, Inc.
Giancarlo Parenti - One of the best experts on this subject based on the ideXlab platform.
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Structure of human lysosomal acid α-glucosidase–a guide for the treatment of Pompe disease
Nature Communications, 2017Co-Authors: Veronique Roig-zamboni, Giancarlo Parenti, Marco Moracci, Beatrice Cobucci-ponzano, Roberta Iacono, Maria Carmina Ferrara, Stanley Germany, Yves Bourne, Gerlind SulzenbacherAbstract:Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric Pharmacological Chaperone N-acetylcysteine, which reveals the stabilizing function of this Chaperone at the structural level.
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Pharmacological Chaperone therapy preclinical development clinical translation and prospects for the treatment of lysosomal storage disorders
Molecular Therapy, 2015Co-Authors: Giancarlo Parenti, Generoso Andria, Kenneth J ValenzanoAbstract:Lysosomal storage disorders (LSDs) are a group of inborn metabolic diseases caused by mutations in genes that encode proteins involved in different lysosomal functions, in most instances acidic hydrolases. Different therapeutic approaches have been developed to treat these disorders. Pharmacological Chaperone therapy (PCT) is an emerging approach based on small-molecule ligands that selectively bind and stabilize mutant enzymes, increase their cellular levels, and improve lysosomal trafficking and activity. Compared to other approaches, PCT shows advantages, particularly in terms of oral administration, broad biodistribution, and positive impact on patients' quality of life. After preclinical in vitro and in vivo studies, PCT is now being translated in the first clinical trials, either as monotherapy or in combination with enzyme replacement therapy, for some of the most prevalent LSDs. For some LSDs, the results of the first clinical trials are encouraging and warrant further development. Future research in the field of PCT will be directed toward the identification of novel Chaperones, including new allosteric drugs, and the exploitation of synergies between Chaperone treatment and other therapeutic approaches.
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Pharmacological Chaperone therapy for lysosomal storage diseases.
Future medicinal chemistry, 2014Co-Authors: Giancarlo Parenti, Marco Moracci, Simona Fecarotta, Generoso AndriaAbstract:Pharmacological Chaperone therapy is an emerging approach to treat lysosomal storage diseases. Small-molecule Chaperones interact with mutant enzymes, favor their correct conformation and enhance their stability. This approach shows significant advantages when compared with existing therapies, particularly in terms of the bioavailability of drugs, oral administration and positive impact on the quality of patients' lives. On the other hand, future research in this field must confront important challenges. The identification of novel Chaperones is indispensable to expanding the number of patients amenable to this treatment and to optimize therapeutic efficacy. It is important to develop new allosteric drugs, to address the risk of inhibiting target enzymes. Future research must also be directed towards the exploitation of synergies between Chaperone treatment and other therapeutic approaches.
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Synergy between the Pharmacological Chaperone 1-deoxygalactonojirimycin and agalsidase alpha in cultured fibroblasts from patients with Fabry disease.
Journal of Inherited Metabolic Disease, 2013Co-Authors: Antonio Pisani, Generoso Andria, Caterina Porto, Giancarlo ParentiAbstract:Dear Editor, We recently published an article in the Journal of Inherited Metabolic Disorders on the synergy between the Pharmacological Chaperone 1-deoxygalactonojirimycin (DGJ) and the recombinant human alpha-galactosidase A (rh-alphaGal A) in Fabry disease (FD) [Porto et al 2012]. These studies showed that in cultured fibroblasts from FD patients the coadministration of rh-alpha-Gal A and DGJ translated into greater correction of enzyme activity and in improved lyso-Gb3 clearance, compared to the results obtained with the recombinant enzyme alone. The enhancing effect of DGJ on rh-alpha-Gal A appeared promising and in line with previous studies performed by us in another lysosomal disease, Pompe disease [Porto et al 2009]. This effect has the potential to translate into improved clinical efficacy of the recombinant enzymes used for enzyme replacement therapy. Our studies were performed using agalsidase beta, one of the two rh-alpha-Gal A preparations currently available for the treatment of FD, that is purified from Chinese hamster ovary cells. Since then, due to a shortage in the availability of agalsidase beta, a large number of FD patients were switched to agalsidase alpha, the other recombinant enzyme approved for enzyme replacement therapy in FD, that is purified from a human fibroblast cell line. Although in principle there are no reasons to believe that the effect of DGJ should be specific for one of these preparations, we thought that it would be advisable to replicate our studies also with agalsidase alpha. To this purpose we studied three FD fibroblast cell lines (patients 1, 2, and 4, in Porto et al 2012) under similar experimental conditions. The cells were incubated with agalsidase alpha (5 nmol/l) for 24 h, in the absence or in the presence of 20 μmol/l DGJ. After harvesting the cells, alpha-Gal A activity was measured as described [Porto et al 2012]. Untreated cells and cells incubated with DGJ alone were used for comparison. Alpha-Gal A activity in the cells incubated with agalsidase alpha alone varied among the different cell lines (9.71±2.47, 5.86±1.22, and 19.32±8.44 nmoles 4-methylumbelliferone/ mg/hour, respectively) (Fig. S1). When the cells where co-incubated with DGJ and agalsidase alpha the correction of intracellular enzyme activity was highly improved (194.22±32.41, 88.10±13.78, 341.96±24.92, respectively). Increases in alpha-Gal A activity ranged from 15 to 20-fold. These results are comparable to those obtained with agalsidase beta, and show for the first time that the synergistic effect between DGJ and rh-alpha-Gal A is observed with either of the two recombinant enzyme preparations presently available. These data may provide information that may be useful for future clinical trials on the combination of Chaperones and enzyme replacement therapy.
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the Pharmacological Chaperone n butyldeoxynojirimycin enhances enzyme replacement therapy in pompe disease fibroblasts
Molecular Therapy, 2009Co-Authors: Caterina Porto, Generoso Andria, Maria Rosaria Tuzzi, Barbara Rossi, Federica Fontana, Monica Cardone, Antonietta Tarallo, Maria Vittoria Barone, Giancarlo ParentiAbstract:In spite of the progress in the treatment of lysosomal storage diseases (LSDs), in some of these disorders the available therapies show limited efficacy and a need exists to identify novel therapeutic strategies. We studied the combination of enzyme replacement and enzyme enhancement by Pharmacological Chaperones in Pompe disease (PD), a metabolic myopathy caused by the deficiency of the lysosomal acid α-glucosidase. We showed that coincubation of Pompe fibroblasts with recombinant human α-glucosidase and the Chaperone N-butyldeoxynojirimycin (NB-DNJ) resulted in more efficient correction of enzyme activity. The Chaperone improved α-glucosidase delivery to lysosomes, enhanced enzyme maturation, and increased enzyme stability. Improved enzyme correction was also found in vivo in a mouse model of PD treated with coadministration of single infusions of recombinant human α-glucosidase and oral NB-DNJ. The enhancing effect of Chaperones on recombinant enzymes was also observed in fibroblasts from another lysosomal disease, Fabry disease, treated with recombinant α-galactosidase A and the specific Chaperone 1-deoxygalactonojirimycin (DGJ). These results have important clinical implications, as they demonstrate synergy between Pharmacological Chaperones and enzyme replacement. A synergistic effect of these treatments may result particularly useful in patients responding poorly to therapy and in tissues in which sufficient enzyme levels are difficult to obtain.
Rebecca Soska - One of the best experts on this subject based on the ideXlab platform.
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coformulation of a novel human α galactosidase a with the Pharmacological Chaperone at1001 leads to improved substrate reduction in fabry mice
Molecular Therapy, 2015Co-Authors: Yi Lun, Rebecca Soska, Michelle Frascella, Nastry Brignol, Adriane Schilling, Rick Hamler, Sean Sullivan, Robert Boyd, Kate Chang, Anadina GarciaAbstract:Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene that encodes α-galactosidase A and is characterized by pathological accumulation of globotriaosylceramide and globotriaosylsphingosine. Earlier, the authors demonstrated that oral coadministration of the Pharmacological Chaperone AT1001 (migalastat HCl; 1-deoxygalactonojirimycin HCl) prior to intravenous administration of enzyme replacement therapy improved the Pharmacological properties of the enzyme. In this study, the authors investigated the effects of coformulating AT1001 with a proprietary recombinant human α-galactosidase A (ATB100) into a single intravenous formulation. AT1001 increased the physical stability and reduced aggregation of ATB100 at neutral pH in vitro, and increased the potency for ATB100-mediated globotriaosylceramide reduction in cultured Fabry fibroblasts. In Fabry mice, AT1001 coformulation increased the total exposure of active enzyme, and increased ATB100 levels in cardiomyocytes, cardiac vascular endothelial cells, renal distal tubular epithelial cells, and glomerular cells, cell types that do not show substantial uptake with enzyme replacement therapy alone. Notably, AT1001 coformulation also leads to greater tissue globotriaosylceramide reduction when compared with ATB100 alone, which was positively correlated with reductions in plasma globotriaosylsphingosine. Collectively, these data indicate that intravenous administration of ATB100 coformulated with AT1001 may provide an improved therapy for Fabry disease and thus warrants further investigation.
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the Pharmacological Chaperone at2220 increases the specific activity and lysosomal delivery of mutant acid alpha glucosidase and promotes glycogen reduction in a transgenic mouse model of pompe disease
PLOS ONE, 2014Co-Authors: Richie Khanna, Rebecca Soska, Yi Lun, Jessie Feng, Michelle Frascella, Lee Pellegrino, Anadina Garcia, Allan C Powe, Rohini Dhulipala, Nastry BrignolAbstract:Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule Pharmacological Chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may warrant further evaluation as a treatment for Pompe disease.
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p 17 8 the co formulation of Pharmacological Chaperone at2220 with recombinant human acid alpha glucosidase improves enzyme uptake and glycogen reduction in a mouse model of pompe disease
Neuromuscular Disorders, 2013Co-Authors: Richie Khanna, Rebecca Soska, Yi Lun, Jessie Feng, Michelle Frascella, Lee Pellegrino, David J Lockhart, Anadina Garcia, John Flanagan, Kenneth J ValenzanoAbstract:Pompe disease is an inherited lysosomal storage disease that results from deficiency in acid alpha-glucosidase (GAA) activity, and is characterized by progressive accumulation of lysosomal glycogen in heart and skeletal muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) is the only approved treatment available for Pompe disease. While rhGAA provides some clinical benefits, the infused enzyme tends to be unstable at neutral pH/body temperature, shows insufficient uptake in key tissues, and can elicit immune responses that affect tolerability and efficacy. We have shown previously that oral pre-administration of the Pharmacological Chaperone AT2220 (1-deoxynojirimycin HCl, duvoglustat HCl) improves the Pharmacological properties of rhGAA via binding and stabilization, leading to increased enzyme uptake and glycogen reduction in GAA knock-out (KO) mice. In this study we tested the effects of intravenous (IV) and subcutaneous (SQ) administration of co-formulated AT2220 and rhGAA (AT2220 + rhGAA) as an alternative to giving AT2220 orally prior to rhGAA IV. In rats, IV or SQ administration of co-formulated AT2220 + rhGAA increased the circulating half-life of rhGAA up to twofold, though the maximal rhGAA plasma levels achieved via the SQ route were significantly lower than those seen following IV administration. In GAA KO mice, four IV administrations of co-formulated AT2220 + rhGAA resulted in up to 2.5-fold greater enzyme uptake and glycogen reduction in disease-relevant tissues compared to rhGAA alone; histological staining confirmed reduced skeletal muscle glycogen. Interestingly, four SQ administrations of co-formulated AT2220 + rhGAA increased rhGAA uptake in GAA KO mice, which was not significantly different from those seen following four IV administrations of rhGAA alone. Collectively, these data highlight the potential effects of co-formulated AT2220 + rhGAA using IV or SQ administration, thus warranting further preclinical investigation.
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t p 44 the Pharmacological Chaperone at2220 increases the stability of recombinant human acid α glucosidase and leads to greater tissue uptake and glycogen reduction in a mouse model of pompe disease
Neuromuscular Disorders, 2012Co-Authors: Kenneth J Valenzano, Rebecca Soska, Yi Lun, Jessie Feng, Michelle Frascella, Lee Pellegrino, David J Lockhart, Darlene Guillen, John Flanagan, Richie KhannaAbstract:Abstract Pompe disease is a lysosomal storage disease caused by deficiency of acid α-glucosidase (GAA) activity, and is characterized by impaired lysosomal glycogen catabolism, progressive skeletal muscle weakness, reduced cardiac function, and respiratory insufficiency. Recombinant human GAA (rhGAA, Genzyme) is the only approved enzyme replacement therapy (ERT) for Pompe, and is administered biweekly via intravenous infusion. While rhGAA does provide clinical benefit, it suffers from low stability at neutral pH/body temperature, shows modest tissue uptake and glycogen reduction, and can elicit immune responses that affect tolerability and efficacy. AT2220 (1-deoxynojirimycin HCl, duvoglustat hydrochloride) is a small molecule Pharmacological Chaperone that binds and stabilizes endogenous GAA in cells and tissues, resulting in increased lysosomal GAA activity. We hypothesized that AT2220 might also improve the Pharmacological properties of exogenous rhGAA. In human plasma, AT2220 co-incubation increased the stability and prevented denaturation of rhGAA at neutral pH/ 37 °C for up to 24 h. In rats, a single oral administration of AT2220 followed 30 min later by intravenous bolus administration of rhGAA resulted in a dose-dependent increase of up to 2-fold in the circulating half-life of rhGAA. A similar effect was seen on the circulating half-life of rhGAA when administered via intravenous infusion. In mice lacking endogenous GAA, oral administration of AT2220 resulted in up to 2.5-fold greater rhGAA uptake and glycogen reduction compared to administration of rhGAA alone in disease-relevant tissues. Collectively, these data indicate that AT2220 increases the stability of rhGAA, and that greater enzyme activity and substrate turnover can be achieved in muscle when co-administered with rhGAA. Based on these findings, a Phase 2 study exploring AT2220 co-administration with rhGAA has been initiated.
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the Pharmacological Chaperone at2220 increases recombinant human acid α glucosidase uptake and glycogen reduction in a mouse model of pompe disease
PLOS ONE, 2012Co-Authors: Richie Khanna, Rebecca Soska, Yi Lun, Jessie Feng, Michelle Frascella, Lee Pellegrino, John J Flanagan, David J Lockhart, Darlene Guillen, Kenneth J ValenzanoAbstract:Pompe disease is an inherited lysosomal storage disease that results from a deficiency in the enzyme acid α-glucosidase (GAA), and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. Recombinant human GAA (rhGAA) is the only approved enzyme replacement therapy (ERT) available for the treatment of Pompe disease. Although rhGAA has been shown to slow disease progression and improve some of the pathophysiogical manifestations, the infused enzyme tends to be unstable at neutral pH and body temperature, shows low uptake into some key target tissues, and may elicit immune responses that adversely affect tolerability and efficacy. We hypothesized that co-administration of the orally-available, small molecule Pharmacological Chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) may improve the Pharmacological properties of rhGAA via binding and stabilization. AT2220 co-incubation prevented rhGAA denaturation and loss of activity in vitro at neutral pH and 37°C in both buffer and blood. In addition, oral pre-administration of AT2220 to rats led to a greater than two-fold increase in the circulating half-life of intravenous rhGAA. Importantly, co-administration of AT2220 and rhGAA to GAA knock-out (KO) mice resulted in significantly greater rhGAA levels in plasma, and greater uptake and glycogen reduction in heart and skeletal muscles, compared to administration of rhGAA alone. Collectively, these preclinical data highlight the potentially beneficial effects of AT2220 on rhGAA in vitro and in vivo. As such, a Phase 2 clinical study has been initiated to investigate the effects of co-administered AT2220 on rhGAA in Pompe patients.
Elfrida R Benjamin - One of the best experts on this subject based on the ideXlab platform.
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co administration with the Pharmacological Chaperone at1001 increases recombinant human α galactosidase a tissue uptake and improves substrate reduction in fabry mice
Molecular Therapy, 2012Co-Authors: Elfrida R Benjamin, Richie Khanna, Michelle Frascella, Nastry Brignol, Lee Pellegrino, John J Flanagan, Adriane Schilling, Brian Ranes, Darlene Guillen, Rebecca SoskaAbstract:Fabry disease is an X-linked lysosomal storage disorder (LSD) caused by mutations in the gene (GLA) that encodes the lysosomal hydrolase α-galactosidase A (α-Gal A), and is characterized by pathological accumulation of the substrate, globotriaosylceramide (GL-3). Regular infusion of recombinant human α-Gal A (rhα-Gal A), termed enzyme replacement therapy (ERT), is the primary treatment for Fabry disease. However, rhα-Gal A has low physical stability, a short circulating half-life, and variable uptake into different disease-relevant tissues. We hypothesized that coadministration of the orally available, small molecule Pharmacological Chaperone AT1001 (GR181413A, 1-deoxygalactonojirimycin, migalastat hydrochloride) may improve the Pharmacological properties of rhα-Gal A via binding and stabilization. AT1001 prevented rhα-Gal A denaturation and activity loss in vitro at neutral pH and 37 °C. Coincubation of Fabry fibroblasts with rhα-Gal A and AT1001 resulted in up to fourfold higher cellular α-Gal A and ~30% greater GL-3 reduction compared to rhα-Gal A alone. Furthermore, coadministration of AT1001 to rats increased the circulating half-life of rhα-Gal A by >2.5-fold, and in GLA knockout mice resulted in up to fivefold higher α-Gal A levels and fourfold greater GL-3 reduction than rhα-Gal A alone. Collectively, these data highlight the potentially beneficial effects of AT1001 on rhα-Gal A, thus warranting clinical investigation.
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a pharmacogenetic approach to identify mutant forms of α galactosidase a that respond to a Pharmacological Chaperone for fabry disease
Human Mutation, 2011Co-Authors: Evan Katz, Kenneth J Valenzano, John J Flanagan, David J Lockhart, Kirsten Mascioli, Maria Cecilia Della Valle, Jeffrey P Castelli, Raphael Schiffmann, Pol Boudes, Elfrida R BenjaminAbstract:Fabry disease is caused by mutations in the gene (GLA) that encodes α-galactosidase A (α-Gal A). The iminosugar AT1001 (GR181413A, migalastat hydrochloride, 1-deoxygalactonojirimycin) is a Pharmacological Chaperone that selectively binds and stabilizes α-Gal A, increasing total cellular levels and activity for some mutant forms (defined as “responsive”). In this study, we developed a cell-based assay in cultured HEK-293 cells to identify mutant forms of α-Gal A that are responsive to AT1001. Concentration-dependent increases in α-Gal A activity in response to AT1001 were shown for 49 (60%) of 81 mutant forms. The responses of α-Gal A mutant forms were generally consistent with the responses observed in male Fabry patient-derived lymphoblasts. Importantly, the HEK-293 cell responses of 19 α-Gal A mutant forms to a clinically achievable concentration of AT1001 (10 µM) were generally consistent with observed increases in α-Gal A activity in peripheral blood mononuclear cells from male Fabry patients orally administered AT1001 during Phase 2 clinical studies. This indicates that the cell-based responses can identify mutant forms of α-Gal A that are likely to respond to AT1001 in vivo. Thus, the HEK-293 cell-based assay may be a useful aid in the identification of Fabry patients with AT1001-responsive mutant forms. Hum Mutat 32:1–13, 2011. © 2011 Wiley-Liss, Inc.
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the Pharmacological Chaperone isofagomine increases the activity of the gaucher disease l444p mutant form of β glucosidase
FEBS Journal, 2010Co-Authors: Richie Khanna, Brigitte Rigat, Rebecca Soska, Jessie Feng, Lee Pellegrino, Elfrida R Benjamin, Adriane Schilling, Hadis Nafar, Brian Ranes, Yi LunAbstract:Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid beta-glucosidase (GCase). We have shown previously that the small molecule Pharmacological Chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity. In this study, we investigated the effect of IFG on L444P GCase. Incubation of Gaucher patient-derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5- and 1.3-fold increases in L444P GCase activity, respectively, as measured in cell lysates. The effect in fibroblasts was increased approximately 2-fold using glycoprotein-enrichment, GCase-immunocapture, or by incubating cells overnight in IFG-free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay. IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels. Importantly, this reduction was seen only following three-day incubation in IFG-free media, underscoring the importance of IFG removal to restore lysosomal GCase activity. In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2- to 5-fold) in GCase activity in disease-relevant tissues, including brain. Additionally, eight-week IFG administration significantly lowered plasma chitin III and IgG levels, and 24-week administration significantly reduced spleen and liver weights. Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues. Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non-neuronopathic Gaucher disease.
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the Pharmacological Chaperone 1 deoxynojirimycin increases the activity and lysosomal trafficking of multiple mutant forms of acid alpha glucosidase
Human Mutation, 2009Co-Authors: John J Flanagan, Caterina Porto, Maria Rosaria Tuzzi, Maria Vittoria Cubellis, Barbara Rossi, Katherine Tang, Kirsten Mascioli, Francesca Donaudy, Federica Fontana, Elfrida R BenjaminAbstract:Pompe disease is a lysosomal storage disorder (LSD) caused by mutations in the gene that encodes acid α-glucosidase (GAA). Recently, small molecule Pharmacological Chaperones have been shown to increase protein stability and cellular levels for mutant lysosomal enzymes and have emerged as a new therapeutic strategy for the treatment of LSDs. In this study, we characterized the Pharmacological Chaperone 1-deoxynojirimycin (DNJ) on 76 different mutant forms of GAA identified in Pompe disease. DNJ significantly increased enzyme activity and protein levels for 16 different GAA mutants in patient-derived fibroblasts and in transiently transfected COS-7 cells. Additionally, DNJ increased the processing of these GAA mutants to their mature lysosomal forms, suggesting facilitated trafficking through the secretory pathway. Immunofluorescence microscopy studies showed increased colocalization of GAA with the lysosomal marker LAMP2 after incubation with DNJ, confirming increased lysosomal trafficking. Lastly, a GAA structural model was constructed based on the related eukaryotic glucosidase maltase-glucoamylase. The mutated residues identified in responsive forms of GAA are located throughout most of the structural domains, with half of these residues located in two short regions within the catalytic domain. Taken together, these data support further evaluation of DNJ as a potential treatment for Pompe disease in patients that express responsive forms of GAA. Hum Mutat 30:1–10, 2009. © 2009 Wiley-Liss, Inc.
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the Pharmacological Chaperone 1 deoxygalactonojirimycin increases α galactosidase a levels in fabry patient cell lines
Journal of Inherited Metabolic Disease, 2009Co-Authors: Elfrida R Benjamin, Robert J Desnick, John J Flanagan, Adriane Schilling, Huihwa Chang, L Agarwal, Evan Katz, C W Pine, Brandon Wustman, David J LockhartAbstract:Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene encoding α-galactosidase A (α-Gal A), with consequent accumulation of its major glycosphingolipid substrate, globotriaosylceramide (GL-3). Over 500 Fabry mutations have been reported; approximately 60% are missense. The iminosugar 1-deoxygalactonojirimycin (DGJ, migalastat hydrochloride, AT1001) is a Pharmacological Chaperone that selectively binds α-Gal A, increasing physical stability, lysosomal trafficking, and cellular activity. To identify DGJ-responsive mutant forms of α-Gal A, the effect of DGJ incubation on α-Gal A levels was assessed in cultured lymphoblasts from males with Fabry disease representing 75 different missense mutations, one insertion, and one splice-site mutation. Baseline α-Gal A levels ranged from 0 to 52% of normal. Increases in α-Gal A levels (1.5- to 28-fold) after continuous DGJ incubation for 5 days were seen for 49 different missense mutant forms with varying EC50 values (820 nmol/L to >1 mmol/L). Amino acid substitutions in responsive forms were located throughout both structural domains of the enzyme. Half of the missense mutant forms associated with classic (early-onset) Fabry disease and a majority (90%) associated with later-onset Fabry disease were responsive. In cultured fibroblasts from males with Fabry disease, the responses to DGJ were comparable to those of lymphoblasts with the same mutation. Importantly, elevated GL-3 levels in responsive Fabry fibroblasts were reduced after DGJ incubation, indicating that increased mutant α-Gal A levels can reduce accumulated substrate. These data indicate that DGJ merits further evaluation as a treatment for patients with Fabry disease with various missense mutations.