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. 2012;7(7):e40776.
doi: 10.1371/journal.pone.0040776. Epub 2012 Jul 18.

The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease

Richie Khanna  1 , John J Flanagan, Jessie Feng, Rebecca Soska, Michelle Frascella, Lee J Pellegrino, Yi Lun, Darlene Guillen, David J Lockhart, Kenneth J Valenzano
Affiliations

The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease

Richie Khanna et al. PLoS One. 2012.

Abstract

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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Conflict of interest statement

Competing Interests: All authors are full-time employees and shareholders of Amicus Therapeutics. The authors have read and adhere to all PLoS ONE policies on sharing the data and materials in this study.

Figures

Figure 1
Figure 1. AT2220 increases the physical stability of rhGAA in vitro.
(A) Time course of rhGAA denaturation in neutral and acidic buffer at 37°C in the absence and presence of 50 µM AT2220. Denaturation was monitored by changes in the fluorescence of SYPRO Orange as a function of time. (B) Time course of rhGAA inactivation (i.e. loss of activity) in neutral and acidic buffer at 37°C in the absence and presence of 50 µM AT2220. (C) Time course of rhGAA inactivation (i.e. loss of activity) in human whole blood at 37°C in the absence and presence of 50 µM AT2220. In both (B) and (C), GAA enzyme activity was determined at the indicated time points using the fluorogenic substrate 4-MUG. To obtain relative enzyme activity levels, measurements at the various time points were compared to the activity at the zero time point.
Figure 2
Figure 2. AT2220 increases the circulating half-life of rhGAA in rats.
(A) Eight-week old male Sprague Dawley rats were administered vehicle (water) or AT2220 (3 or 30 mg/kg) via oral gavage. Thirty minutes later, vehicle (saline) or rhGAA (10 mg/kg) was administered via bolus tail vein injection. Blood was collected at the indicated time points, and GAA activity (upper panel) and protein levels (lower panel) were measured in plasma as described in ‘Materials and Methods’. (B) Eight-week old male Sprague Dawley rats were administered vehicle (water) or AT2220 (3 or 30 mg/kg) via oral gavage. Thirty minutes later, vehicle (saline) or rhGAA (10 mg/kg) was administered via 60-minute intravenous infusion. Blood was collected at the indicated time points, and GAA activity (upper panel) and protein levels (lower panel) were measured in plasma. PS: post-start infusion; PE: post-end infusion. In both (A) and (B), each time point represents the mean±SEM of the activity measured from 3 rats; each lane on the Western blot contains plasma from a single rat, and is representative of two rats in each group.
Figure 3
Figure 3. AT2220 increases the circulating levels of rhGAA in GAA KO mice.
Twelve-week old male GAA KO mice were administered vehicle (water) or AT2220 (30 mg/kg) via oral gavage once every other week for 8 weeks. Thirty minutes after each AT2220 oral administration, vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail vein injection. Blood was collected after the last (i.e., 4th) rhGAA administration and, GAA activity (upper panel) and protein levels (lower panel) were measured in plasma as described in ‘Materials and Methods’. Each bar represents the mean±SEM of the GAA activity measured from 5 mice per group. Statistically significant increases were seen in plasma GAA activity compared to baseline (*p<0.05, t-test) and compared to rhGAA administration alone (#p<0.05, t-test). Each lane on the Western blot contains plasma from a single mouse, and is representative of two mice in each group.
Figure 4
Figure 4. Co-administration of AT2220 promotes greater tissue uptake of rhGAA in GAA KO mice.
Twelve-week old male GAA KO mice were administered vehicle (water) or AT2220 (30 mg/kg) via oral gavage once every other week for 8 weeks. Thirty minutes after each AT2220 oral administration, vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail vein injection. Mice were euthanized 7 days after the last (i.e., 4th) rhGAA administration and tissue GAA activity was measured as described in ‘Materials and Methods’. Each bar represents the mean±SEM of the GAA activity measured from 5 mice per group. Statistically significant increases were seen in GAA activity compared to baseline (*p<0.05, t-test) and compared to rhGAA administration alone (#p<0.05, t-test). For comparison, GAA levels in wild-type C57BL/6 mice were 15±2, 16±0.6, 21±3, 18±2, 11±2, and 25±3 nmol/mg protein/hr in heart, diaphragm, gastrocnemius, quadriceps, triceps, and tongue, respectively (mean±SEM of 7 mice).
Figure 5
Figure 5. Co-administration of AT2220 promotes greater tissue glycogen reduction in GAA KO mice.
Twelve-week old male GAA KO mice were administered vehicle (water) or AT2220 (10 or 30 mg/kg) via oral gavage once every other week for 8 weeks. Thirty minutes after each AT2220 administration, vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail vein injection. Mice were euthanized 21 days after the last (i.e., 4th) rhGAA administration and tissue glycogen levels were measured as described in ‘Materials and Methods’. Dotted lines show glycogen levels in the respective tissues of wild-type C57BL/6 mice. The data presented are an average of two independent studies with each bar representing the mean±SEM of the activity measured from 12 mice per group. Statistically significant reductions were seen in glycogen levels compared to baseline (*p<0.05, t-test) and compared to rhGAA administration alone (#p<0.05, t-test). In addition, the effect of AT2220 co-administration was also found to be significant for a linear trend (p<0.05; except in triceps), indicating a dose-dependent effect.
Figure 6
Figure 6. Co-administration of AT2220 promotes cell type-specific reduction of glycogen in GAA KO mice.
Twelve-week old male GAA KO mice were administered vehicle (water) or AT2220 (30 mg/kg) via oral gavage once every other week for 8 weeks. Thirty minutes after each AT2220 oral administration, vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail vein injection. Mice were euthanized 21 days after the last (i.e., 4th) rhGAA administration and glycogen levels in heart and quadriceps were measured immunohistochemically as described in ‘Materials and Methods’. A strong glycogen signal, represented as dark blue or pink spots (denoted with arrows) in heart and quadriceps, respectively, was observed. (*) indicates glycogen reduction in individual skeletal muscle fibers of quadriceps. The data shown are representative photomicrographs from 6 mice/group (magnification: 20X).
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References

    1. Hirschhorn R, Reuser AJJ. Scriver C, Beaudet A, Sly W, Valle D, editors. Glycogen storage disease type ii: Acid alpha-glucosidase (acid maltase) deficiency. 2001. pp. 3389–3420. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill.
    1. van der Ploeg AT, Reuser AJJ. Pompe's disease. Lancet. 2008;372:1342–1353. - PubMed
    1. van den Hout HM, Hop W, van Diggelen OP, Smeitink JA, Smit GP, et al. The natural course of infantile pompe's disease: 20 original cases compared with 133 cases from the literature. Pediatrics. 2003;112:332–340. - PubMed
    1. Kishnani PS, Howell RR. Pompe disease in infants and children. J Pediatr. 2004;144:S35–43. - PubMed
    1. Kishnani PS, Nicolino M, Voit T, Rogers RC, Tsai AC, et al. Chinese hamster ovary cell-derived recombinant human acid alpha-glucosidase in infantile-onset pompe disease. J Pediatr. 2006;149:89–97. - PMC - PubMed

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