Migalastat HCl reduces globotriaosylsphingosine (lyso-Gb3) in Fabry transgenic mice and in the plasma of Fabry patients

Fabry disease (FD) results from mutations in the gene ( GLA ) that encodes the lysosomal enzyme α-galactosidase A (α-Gal A), and involves pathological accumulation of globotriaosylceramide (GL-3) and globotriaosylsphingosine (lyso-Gb 3 ). Migalastat hydrochloride (GR181413A) is a pharmacological chaperone that selectively binds, stabilizes, and increases cellular levels of α-Gal A. Oral administration of migalastat HCl reduces tissue GL-3 in Fabry transgenic mice, and in urine and kidneys of some FD patients. A liquid chromatography-tandem mass spectrometry method was developed to measure lyso-Gb 3 in mouse tissues and human plasma. Oral administration of migalastat HCl to transgenic mice reduced elevated lyso-Gb 3 levels up to 64%, 59%, and 81% in kidney, heart, and skin, respectively, generally equal to or greater than observed for GL-3. Furthermore, baseline plasma lyso-Gb 3 levels were markedly elevated in six male FD patients enrolled in Phase 2 studies. Oral administration of migalastat HCl (150 mg QOD) reduced urine GL-3 and plasma lyso-Gb 3 in three subjects (range: 15% to 46% within 48 weeks of treatment). In contrast, three showed no reductions in either substrate. These results suggest that measurement of tissue and/or plasma lyso-Gb 3 is feasible and may be warranted in future studies of migalastat HCl or other new potential therapies for FD

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

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

<div><p>Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the <i>GAA</i> 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, <i>in vitro</i> and <i>in vivo</i>, 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 <i>in vitro</i>. 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 <i>Gaa</i> 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 <i>in situ</i>. As such, AT2220 may warrant further evaluation as a treatment for Pompe disease.</p></div

Inter-assay % Bias and precision (% CV) of lyso-Gb₃ quality control human plasma samples.

<p>Quality control samples were prepared in normal human plasma at five concentration levels and analyzed on four separate days. Twenty replicates at each concentration level were used for inter-assay determination. Conc., concentration (ng/mL); SD, Standard Deviation.</p

Effect of AT2220 incubation on enzyme activity and protein levels of secreted wild-type and mutant GAA.

<p>COS-7 cells were transfected with wild-type or various mutant forms of GAA, followed by incubation with or without 100 µM AT2220. Increases in the enzymatic activity and total protein levels of secreted GAA were measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102092#s2" target="_blank">Materials and Methods</a>’, and as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102092#pone-0102092-g002" target="_blank"><b>Fig. 2</b></a>. AT2220-mediated increases in the relative specific activities of the various mutant forms of GAA were calculated as the ratio of GAA enzyme activity to the total GAA protein levels. Activity and protein values represent the mean ± SD from four independent experiments.</p

Inter-assay % Bias and precision (% CV) of lyso-Gb₃ quality control mouse tissue samples.

<p>Quality control samples were prepared in wild-type mouse plasma or tissue homogenates at four concentration levels and analyzed on three separate days. Six to 10 replicates at each concentration level were used for inter-assay determination. Conc., concentration; SD, Standard Deviation.</p

AT2220 promotes the export of P545L GAA from the ER.

<p>COS-7 cells were transiently transfected with wild-type or P545L GAA, immediately followed by 3-day incubation with and without 100 µM AT2220. Cells were then fixed and stained with antibodies against GAA (green) and calnexin, a marker for the endoplasmic reticulum (ER; red) as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102092#s2" target="_blank">Materials and Methods</a>’. Co-localization of the GAA and calnexin signals appears yellow. Puncta (indicative of lysosomal localization) were seen throughout wild-type cells, as well as in P545L cells incubated with AT2220 (arrowheads). Scale bars: 50 µm.</p

AT2220 increases the stability of mature P545L GAA in lysosomes of Pompe fibroblasts.

<p>(<b>A</b> and <b>B</b>) P545L GAA fibroblasts were incubated with 100 µCi ³⁵S-cysteine/methionine for 3 days, chased for 0 to 4 days, then harvested and lysed. Incubation with 100 µM AT2220 during the pulse only, the pulse and the chase, or neither was performed as indicated. Isoforms of GAA (denoted as the 110, 95, 76, and 70 kDa species) were immunoprecipitated from cell lysates, separated by SDS-PAGE, and imaged with a phosphorimager. As controls, immunoprecipitates from metabolically labeled wild-type (denoted as ‘WT’ in the figure) and R854× fibroblasts (both in the absence of AT2220) were also evaluated. (<b>C</b>) Levels of the mature 76 kDa isoform of P545L GAA were quantified, normalized against the levels present at the 1-day chase time point, and plotted as a function of time. The data were fitted with an exponential function to estimate the half-life of the 76 kDa isoform of P545L GAA in the presence or absence of AT2220. The 1-day chase time point was chosen as time 0 for the exponential fit in order to eliminate the potential contribution of labeled 110 kDa precursor to the 76 kDa pool.</p

hP545L GAA Tg/KO mice have reduced tissue GAA activity, and elevated glycogen and LAMP1 levels.

<p>(<b>A</b>) GAA and glycogen levels were assessed in tissue lysates prepared from heart and gastrocnemius of 12-week old male <i>Gaa</i> KO, hP545L GAA Tg/KO, and wild-type (denoted as ‘WT’ in the figure) mice. Significant differences in GAA activity and glycogen levels in hP545L GAA Tg/KO and <i>Gaa</i> KO mice compared to wild-type mice are reported as *p<0.05, t-test. Significant differences in GAA activity and glycogen levels in hP545L GAA Tg/KO mice compared to <i>Gaa</i> KO mice are reported as ^#p<0.05, t-test. Each bar represents the mean ± SEM from four mice per group analyzed in triplicate. Glycogen accumulation (<b>B</b>) and lysosomal proliferation (assessed by the quantity of LAMP1) (<b>C</b>) were assessed histochemically in <i>Gaa</i> KO, hP545L GAA Tg/KO, and wild-type (denoted as ‘WT’ in the figure) mouse cardiomyocytes and skeletal muscles fibers of the gastrocnemius. Glycogen staining is represented as dark pink spots, and LAMP1 staining as dark brown spots (each denoted with black arrows). The data shown are representative photomicrographs from four mice using a 40× (scale bar: 50 µm) or 20× (scale bar: 100 µm) objective in (<b>B</b>) and (<b>C</b>), respectively.</p