Co-administration With the Pharmacological Chaperone AT1001 Increases Recombinant Human α-Galactosidase A Tissue Uptake and Improves Substrate Reduction in Fabry Mice

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

Co-administration of AT2220 promotes greater tissue uptake of rhGAA in GAA KO mice.

<p>Twelve-week old male <i>GAA</i> 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>i.e.</i>, 4^th) rhGAA administration and tissue GAA activity was measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040776#s2" target="_blank">Materials and Methods</a>’. 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).</p

Co-administration of AT2220 promotes cell type-specific reduction of glycogen in GAA KO mice.

<p>Twelve-week old male <i>GAA</i> 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>i.e.,</i> 4^th) rhGAA administration and glycogen levels in heart and quadriceps were measured immunohistochemically as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040776#s2" target="_blank">Materials and Methods</a>’. 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).</p

AT2220 increases the physical stability of rhGAA in vitro.

<p>(<b>A</b>) 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>B</b>) Time course of rhGAA inactivation (<i>i.e.</i> loss of activity) in neutral and acidic buffer at 37°C in the absence and presence of 50 µM AT2220. (<b>C</b>) Time course of rhGAA inactivation (<i>i.e.</i> loss of activity) in human whole blood at 37°C in the absence and presence of 50 µM AT2220. In both (<b>B</b>) and (<b>C</b>), 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.</p

Effect of AT2220 co-administration on rhGAA-mediated glycogen reduction (% change from Baseline) in <i>GAA</i> KO mice.

<p>Twelve-week old male <i>GAA</i> KO mice were administered vehicle (water) or AT2220 via oral gavage once every other week for 8 weeks. Thirty minutes after each AT2220 oral administration, rhGAA (20 mg/kg) was administered via bolus tail vein injection. Mice were euthanized 21 days after the last (<i>i.e.,</i> 4^th) rhGAA administration, and tissue glycogen levels were measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040776#s2" target="_blank">Materials and Methods</a>’. Baseline glycogen levels in untreated <i>GAA</i> KO mice were 417±32, 340±24, 405±27, 353±21, 446±18, and 389±10 µg/mg protein in heart, diaphragm, quadriceps, gastrocnemius, triceps, and tongue, respectively, and in wild-type C57BL/6 mice were 23±2, 50±6, 40±5, 45±2, 35±7, and 32±2 µg/mg protein, respectively (mean±SEM of 7 mice). The data shown represent the percent glycogen change from baseline in each tissue as normalized between wild-type (0%) and <i>GAA</i> KO (100%) levels. Each value represents the mean±SEM of 12 mice. 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).</p

AT2220 increases the circulating half-life of rhGAA in rats.

<p>(<b>A</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 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 ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040776#s2" target="_blank">Materials and Methods</a>’. (<b>B</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. <i>PS</i>: post-start infusion; <i>PE</i>: post-end infusion. In both (<b>A</b>) and (<b>B</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.</p

Description of the different doses, routes, and regimens utilized for rhGAA and AT2220 administration.

<p>SD, Sprague Dawley.</p

AT2220 increases the circulating levels of rhGAA in GAA KO mice.

<p>Twelve-week old male <i>GAA</i> 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>i.e.,</i> 4^th) rhGAA administration and, GAA activity (upper panel) and protein levels (lower panel) were measured in plasma as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040776#s2" target="_blank">Materials and Methods</a>’. 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.</p

Co-administration of AT2220 promotes greater tissue glycogen reduction in GAA KO mice.

<p>Twelve-week old male <i>GAA</i> 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>i.e.</i>, 4^th) rhGAA administration and tissue glycogen levels were measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040776#s2" target="_blank">Materials and Methods</a>’. 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.</p