Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease.

Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type of the disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases.

Iminosugar-based inhibitors of glucosylceramide synthase prolong survival but paradoxically increase brain glucosylceramide levels in Niemann-Pick C mice.

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disease caused by mutations in NPC1 or NPC2, the gene products of which are involved in cholesterol transport in late endosomes. NPC is characterized by an accumulation of cholesterol, sphingomyelin and glycosphingolipids in the visceral organs, primarily the liver and spleen. In the brain, there is a redistribution of unesterified cholesterol and a concomitant accumulation of glycosphingolipids. It has been suggested that reducing the aberrant lysosomal storage of glycosphingolipids in the brain by a substrate reduction therapy (SRT) approach may prove beneficial. Inhibiting glucosylceramide synthase (GCS) using the iminosugar-based inhibitor miglustat (NB-DNJ) has been reported to increase the survival of NPC mice. Here, we tested the effects of Genz-529468, a more potent iminosugar-based inhibitor of GCS, in the NPC mouse. Oral administration of Genz-529468 or NB-DNJ to NPC mice improved their motor function, reduced CNS inflammation, and increased their longevity. However, Genz-529468 offered a wider therapeutic window and better therapeutic index than NB-DNJ. Analysis of the glycolipids in the CNS of the iminosugar-treated NPC mouse revealed that the glucosylceramide (GL1) but not the ganglioside levels were highly elevated. This increase in GL1 was likely caused by the off-target inhibition of the murine non-lysosomal glucosylceramidase, Gba2. Hence, the basis for the observed effects of these inhibitors in NPC mice might be related to their inhibition of Gba2 or another unintended target rather than a result of substrate reduction.

Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease.

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects.

A glucosylceramide synthase inhibitor protects rats against the cytotoxic effects of shiga toxin 2.

Postdiarrhea hemolytic uremic syndrome is the most common cause of acute renal failure in children in Argentina. Renal damage has been strongly associated with Shiga toxin (Stx), which binds to the globotriaosylceramide (Gb3) receptor on the plasma membrane of target cells. The purpose of the study was to evaluate the in vivo effects of C-9, a potent inhibitor of glucosylceramide synthase and Gb3 synthesis, on kidney and colon in an experimental model of hemolytic uremic syndrome in rats. Rats were i.p. injected with supernatant from recombinant Escherichia coli expressing Stx2 (sStx2). A group of these rats were orally treated with C-9 during 6 d, from 2 d prior until 4 d after sStx2 injection. The injection of sStx2 caused renal damage as well as a loss of goblet cells in colonic mucosa. Oral treatment with C-9 significantly decreased rat mortality to 50% and reduced the extension of renal and intestinal injuries in the surviving rats. The C-9 also decreased Gb3 and glucosylceramide expression levels in rat kidneys. It is particularly interesting that an improvement was seen when C-9 was administered 2 d before challenge, which makes it potentially useful for prophylaxis.

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.

Substrate reduction augments the efficacy of enzyme therapy in a mouse model of Fabry disease.

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.

Improved management of lysosomal glucosylceramide levels in a mouse model of type 1 Gaucher disease using enzyme and substrate reduction therapy.

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.

Dual-action lipophilic iminosugar improves glycemic control in obese rodents by reduction of visceral glycosphingolipids and buffering of carbohydrate assimilation.

The lipophilic iminosugar N-[5-(adamantan-1-ylmethoxy)pentyl]-1-deoxynojirimycin (2, AMP-DNM) potently controls hyperglycemia in obese rodent models of insulin resistance. The reduction of visceral glycosphingolipids by 2 is thought to underlie its beneficial action. It cannot, however, be excluded that concomitant inhibition of intestinal glycosidases and associated buffering of carbohydrate assimilation add to this. To firmly establish the mode of action of 2, we developed a panel of lipophilic iminosugars varying in configuration at C-4/C-5 and N-substitution of the iminosugar. From these we identified the l-ido derivative of 2, l-ido-AMP-DNM (4), as a selective inhibitor of glycosphingolipid synthesis. Compound 4 lowered visceral glycosphingolipids in ob/ob mice and ZDF rats on a par with 2. In contrast to 2, 4 did not inhibit sucrase activity or sucrose assimilation. Treatment with 4 was significantly less effective in reducing blood glucose and HbA1c. We conclude that the combination of reduction of glycosphingolipids in tissue and buffering of carbohydrate assimilation by 2 produces a superior glucose homeostasis.

Inhibiting glycosphingolipid synthesis ameliorates hepatic steatosis in obese mice.

UNLABELLED: Steatosis in the liver is a common feature of obesity and type 2 diabetes and the precursor to the development of nonalcoholic steatohepatitis (NASH), cirrhosis, and liver failure. It has been shown previously that inhibiting glycosphingolipid (GSL) synthesis increases insulin sensitivity and lowers glucose levels in diabetic rodent models. Here we demonstrate that inhibiting GSL synthesis in ob/ob mice not only improved glucose homeostasis but also markedly reduced the development of hepatic steatosis. The ob/ob mice were treated for 7 weeks with a specific inhibitor of glucosylceramide synthase, the initial enzyme involved in the synthesis of GSLs. Besides lowering glucose and hemoglobin A1c (HbA1c) levels, drug treatment also significantly reduced the liver/body weight ratio, decreased the accumulation of triglycerides, and improved several markers of liver pathology. Drug treatment reduced liver glucosylceramide (GL1) levels in the ob/ob mouse. Treatment also reduced the expression of several genes associated with hepatic steatosis, including those involved in lipogenesis, gluconeogenesis, and inflammation. In addition, inhibiting GSL synthesis in diet-induced obese mice both prevented the development of steatosis and partially reversed preexisting steatosis. CONCLUSION: These data indicate that inhibiting GSL synthesis ameliorates the liver pathology associated with obesity and diabetes, and may represent a novel strategy for treating fatty liver disease and NASH.

A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease.

An approach to treating Gaucher disease is substrate inhibition therapy which seeks to abate the aberrant lysosomal accumulation of glucosylceramide. We have identified a novel inhibitor of glucosylceramide synthase (Genz-112638) and assessed its activity in a murine model of Gaucher disease (D409V/null). Biochemical characterization of Genz-112638 showed good potency (IC(50) approximately 24nM) and specificity against the target enzyme. Mice that received drug prior to significant accumulation of substrate (10 weeks of age) showed reduced levels of glucosylceramide and number of Gaucher cells in the spleen, lung and liver when compared to age-matched control animals. Treatment of older mice that already displayed significant amounts of tissue glucosylceramide (7 months old) resulted in arrest of further accumulation of the substrate and appearance of additional Gaucher cells in affected organs. These data indicate that substrate inhibition therapy with Genz-112638 represents a viable alternate approach to enzyme therapy to treat the visceral pathology in Gaucher disease.

Measurement of lysosomal glucocerebrosidase activity in mouse liver using a fluorescence-activated cell sorter assay.

Lysosomal acid beta-glucocerebrosidase hydrolyzes glucocerebroside to glucose ceramide. Patients diagnosed with Gaucher disease, however, lack this enzyme, leading to the accumulation of glucocerebroside in tissue macrophages within multiple organs. Such patients can receive enzyme replacement therapy during which a human placental-derived or recombinant form of acid beta-glucocerebrosidase is targeted to the macrophages. As part of evaluating the effectiveness of such therapies, currently available methodologies for measuring acid beta-glucocerebrosidase activity are primarily conducted in cultured cell lines or tissue culture. However, these in vitro assays are limited by their ability to evaluate the efficacy of in vivo acid beta-glucocerebrosidase replacement therapy in animal models. In particular, there is an unmet need to simultaneously define cellular localization and evaluate enzyme activity following treatment in vivo. In addition, results of commonly used fluorescent-based assays for enzyme activity are difficult to compare day to day and/or across laboratories due to the variability inherent in flow cytometric measurement. In this article, we describe a reproducible and consistent quantitative method for the combined measurement of fluorescein intensity from enzyme-substrate conversion and cell localization by phenotype-specific phycoerythrin-antibody staining. Following infusion of recombinant human acid beta-glucocerebrosidase in mice, nonparenchymal cells are prepared from the livers of treated and control animals. Acid beta-glucocerebrosidase activity is measured in molecules of equivalent soluble fluorophore units within Kupffer cell populations as defined by phenotype-specific monoclonal antibodies. This assay should be applicable to investigations of other Gaucher disease treatments in both human and animal models.