eliglustat and Disease-Models--Animal

Fabry disease results from loss of activity of the lysosomal enzyme α-galactosidase A (GLA), leading to the accumulation of globoseries glycosphingolipids in vascular endothelial cells. Thrombosis and stroke are life-threatening complications of Fabry disease; however, the mechanism of the vasculopathy remains unclear. We explored the relationship between GLA deficiency and endothelial cell von Willebrand factor (VWF) secretion in in vivo and in vitro models of Fabry disease. Plasma VWF was significantly higher at two months and increased with age in Gla-null compared to wild-type mice. Disruption of GLA in a human endothelial cell line by siRNA and CRISPR/Cas9 resulted in a 3-fold and 5-fold increase in VWF secretion, respectively. The increase in VWF levels was associated with decreased endothelial nitric oxide synthase (eNOS) activity in both in vitro models. Pharmacological approaches that increase nitric oxide bioavailability or decrease reactive oxygen species completely normalized the elevated VWF secretion in GLA deficient cells. In contrast, the abnormality was not readily reversed by recombinant human GLA or by inhibition of glycosphingolipid synthesis with eliglustat. These results suggest that GLA deficiency promotes VWF secretion through eNOS dysregulation, which may contribute to the vasculopathy of Fabry disease.

Topics: alpha-Galactosidase; Animals; Cell Line; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Fabry Disease; Glucosyltransferases; Glycosphingolipids; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type III; Pyrrolidines; Reactive Oxygen Species; RNA, Small Interfering; von Willebrand Factor

Clonal B-cell proliferation is a frequent manifestation of Gaucher disease - a sphingolipidosis associated with a high risk of multiple myeloma and non-Hodgkin lymphoma. Gaucher disease is caused by genetic deficiency of acid β-glucosidase, the natural substrates of which (β-d-glucosylceramide and β-d-glucosylsphingosine) accumulate, principally in macrophages. Mice with inducible deficiency of β-glucosidase [Gba(tm1Karl/tm1Karl)Tg(MX1-cre)1Cgn/0] serve as an authentic model of human Gaucher disease; we have recently reported clonal B-cell proliferation accompanied by monoclonal serum paraproteins and cognate tumours in these animals. To explore the relationship between B-cell malignancy and the biochemical defect, we treated Gaucher mice with eliglustat tartrate (GENZ 112638), a potent and selective inhibitor of the first committed step in glycosphingolipid biosynthesis. Twenty-two Gaucher mice received 300 mg/kg of GENZ 112638 daily for 3-10 months from 6 weeks of age. Plasma concentrations of β-d-glucosylceramide and the unacylated glycosphingolipid, β-d-glucosylsphingosine, declined. After administration of GENZ 112638 to Gaucher mice for 3-10 months, serum paraproteins were not detected and there was a striking reduction in the malignant lymphoproliferation: neither lymphomas nor plasmacytomas were found in animals that had received the investigational agent. In contrast, 14 out of 60 Gaucher mice without GENZ 112638 treatment developed these tumours; monoclonal paraproteins were detected in plasma from 18 of the 44 age-matched mice with Gaucher disease that had not received GENZ 112638. Long-term inhibition of glycosphingolipid biosynthesis suppresses the development of spontaneous B-cell lymphoma and myeloma in Gaucher mice.

Topics: Animals; Disease Models, Animal; Female; Gaucher Disease; Glucosyltransferases; Lymphoma, B-Cell; Male; Mice; Pyrrolidines; Uridine Diphosphate

Gaucher disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (acid beta-glucosidase), with consequent cellular accumulation of glucosylceramide (GL-1). The disease is managed by intravenous administrations of recombinant glucocerebrosidase (imiglucerase), although symptomatic patients with mild to moderate type 1 Gaucher disease for whom enzyme replacement therapy (ERT) is not an option may also be treated by substrate reduction therapy (SRT) with miglustat. To determine whether the sequential use of both ERT and SRT may provide additional benefits, we compared the relative pharmacodynamic efficacies of separate and sequential therapies in a murine model of Gaucher disease (D409V/null). As expected, ERT with recombinant glucocerebrosidase was effective in reducing the burden of GL-1 storage in the liver, spleen, and lung of 3-month-old Gaucher mice. SRT using a novel inhibitor of glucosylceramide synthase (Genz-112638) was also effective, albeit to a lesser degree than ERT. Animals administered recombinant glucocerebrosidase and then Genz-112638 showed the lowest levels of GL-1 in all the visceral organs and a reduced number of Gaucher cells in the liver. This was likely because the additional deployment of SRT following enzyme therapy slowed the rate of reaccumulation of GL-1 in the affected organs. Hence, in patients whose disease has been stabilized by intravenously administered recombinant glucocerebrosidase, orally administered SRT with Genz-112638 could potentially be used as a convenient maintenance therapy. In patients naïve to treatment, ERT followed by SRT could potentially accelerate clearance of the offending substrate.

Topics: Animals; Disease Models, Animal; Enzyme Replacement Therapy; Female; Gaucher Disease; Glucosylceramidase; Glucosylceramides; Immunohistochemistry; Lysosomes; Male; Mice; Pyrrolidines; Recombinant Proteins; Tissue Distribution

Fabry disease is an X-linked glycosphingolipid storage disorder caused by a deficiency in the activity of the lysosomal hydrolase α-galactosidase A (α-gal). This deficiency results in accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in lysosomes. Endothelial cell storage of GL-3 frequently leads to kidney dysfunction, cardiac and cerebrovascular disease. The current treatment for Fabry disease is through infusions of recombinant α-gal (enzyme-replacement therapy; ERT). Although ERT can markedly reduce the lysosomal burden of GL-3 in endothelial cells, variability is seen in the clearance from several other cell types. This suggests that alternative and adjuvant therapies may be desirable. Use of glucosylceramide synthase inhibitors to abate the biosynthesis of glycosphingolipids (substrate reduction therapy, SRT) has been shown to be effective at reducing substrate levels in the related glycosphingolipidosis, Gaucher disease. Here, we show that such an inhibitor (eliglustat tartrate, Genz-112638) was effective at lowering GL-3 accumulation in a mouse model of Fabry disease. Relative efficacy of SRT and ERT at reducing GL-3 levels in Fabry mouse tissues differed with SRT being more effective in the kidney, and ERT more efficacious in the heart and liver. Combination therapy with ERT and SRT provided the most complete clearance of GL-3 from all the tissues. Furthermore, treatment normalized urine volume and uromodulin levels and significantly delayed the loss of a nociceptive response. The differential efficacies of SRT and ERT in the different tissues indicate that the combination approach is both additive and complementary suggesting the possibility of an improved therapeutic paradigm in the management of Fabry disease.

Topics: alpha-Galactosidase; Animals; Chromatography, High Pressure Liquid; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Enzyme Replacement Therapy; Fabry Disease; Female; Glucosyltransferases; Humans; Male; Mass Spectrometry; Mice; Mice, 129 Strain; Mice, Knockout; Pyrrolidines; Treatment Outcome; Trihexosylceramides; Uromodulin