Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency in alpha-galactosidase A (alpha-Gal A) activity and subsequent accumulation of the substrate globotriaosylceramide (GL-3), which contributes to disease pathology. The pharmacological chaperone (PC) DGJ (1-deoxygalactonojirimycin) binds and stabilizes alpha-Gal A, increasing enzyme levels in cultured cells and in vivo. The ability of DGJ to reduce GL-3 in vivo was investigated using transgenic (Tg) mice that express a mutant form of human alpha-Gal A (R301Q) on a knockout background (Tg/KO), which leads to GL-3 accumulation in disease-relevant tissues. Four-week daily oral administration of DGJ to Tg/KO mice resulted in significant and dose-dependent increases in alpha-Gal A activity, with concomitant GL-3 reduction in skin, heart, kidney, brain, and plasma; 24-week administration resulted in even greater reductions. Compared to daily administration, less frequent DGJ administration, including repeated cycles of 4 days with DGJ followed by 3 days without or every other day with DGJ, resulted in even greater GL-3 reductions that were comparable to those obtained with Fabrazyme. Collectively, these data indicate that oral administration of DGJ increases mutant alpha-Gal A activity and reduces GL-3 in disease-relevant tissues in Tg/KO mice, and thus merits further evaluation as a treatment for Fabry disease.
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.
A semi-automated high-throughput liquid/liquid extraction (LLE) assay was developed for RAD001 and cyclosporin A (CsA) in human blood. After addition of internal standard and ammonium hydroxide, samples were extracted twice with methyl tert-butyl ether (MTBE). The organic extract was evaporated to dryness and reconstituted in mobile phase. Where possible, sample transfer and LLE steps were automated using a Tomtec Quadra 96 workstation. Samples were analyzed using ESI-LC/MS/MS employing the transitions of ([M + NH(4)](+) --> [M + H](+)) for CsA and ([M + NH(4)](+) --> [M + H-(CH(3)OH + H(2)O)](+)) for RAD001, under isocratic chromatographic conditions (75:25, (v/v), acetonitrile/20 mM ammonium acetate) with a run time of 3.6 min. A lower limit of quantitation (LLOQ) of 0.368 ng/mL and 5.23 ng/mL was achieved for RAD001 and CsA, respectively, using a sample volume of 0.3 mL for the analysis. The method was validated over a 3-day period and the resulting calibration curves had a correlation coefficient >0.99 over the concentration range 0.368 to 409 ng/mL and 5.24 to 1748 ng/mL for RAD001 and CsA, respectively. The inter-day coefficient of variation (CV) was less than 15% at the LLOQ for both compounds. The method was applied to the analysis of clinical samples. Under normal working conditions four 96-well plates could be extracted and LC/MS analysis completed in less than 28 h. A marked improvement in sample throughput efficiency was realized with this LLE method when compared to existing solid phase extraction (SPE) methods which deal with both RAD001 and CsA.
A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of terbinafine in human and minipig plasma has been developed and validated. The method used positive-ion mode for monitoring terbinafine, and used a stable isotope labelled terbinafine as the internal standard. Subsequent to acetonitrile protein precipitation, the supernatant was directly (unfiltered) injected onto the LC column (retention time approximately 4.3 min) for analysis. Interday and intraday accuracy and precision were assessed from the relative recoveries (observed concentration in percent of the nominal value) of spiked samples analyzed on three different days. The lower limit of quantitation (LLOQ) was 0.0679 ng/mL in human and minipig using a plasma sample volume of 0.08 mL. The method was fast, specific, and exhibited ruggedness. Furthermore, the use of turbulent flow chromatography (TurboFlow LC/MS/MS) coupled to mass spectrometry for direct analysis of terbinafine in plasma is discussed. The technique allowed direct introduction of plasma with satisfactory chromatographic peak shape and increased throughput.
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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