WO2009040816A1

WO2009040816A1 - Methods of treating lysosomal storage disorders

Info

Publication number: WO2009040816A1
Application number: PCT/IL2008/001291
Authority: WO
Inventors: Mia Horowitz; Idit Ron Ronen
Original assignee: Ramot At Tel Aviv University Ltd.
Priority date: 2007-09-26
Filing date: 2008-09-24
Publication date: 2009-04-02
Also published as: GB0718824D0

Abstract

The sphingolipid storage disease (SLSD) is selected from the group consisting of Gaucher' s Disease, Niemann-Pick Disease, Krabbe' s Disease (Globoid leukodystrophy), Metachromatic leukodystrophy, Ceramide lactoside lipidosis, Fabrys Disease, Tay-Sachs Disease, Sandhoffs Disease and Landing's Disease. The cholesterol synthesis inhibitor is selected from the group consisting of mevastatin, lovastatin, pravastatin, fluvastatin, simvastatin, rosuvastatin, cerivastatin and atorvastatin.

Description

METHODS OF TREATING LYSOSOMAL STORAGE DISORDERS

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to treatment of sphingolipid storage diseases (SLSD) including, but not limited to, Gaucher's disease.

Gaucher's disease (GD) is a sphingolipid storage disorder resulting from an inborn reduced activity or deficiency of glucocerebrosidase due to mutations in the glucocerebrosidase gene [Beutler (1995) Adv Genet 32:17-49] or, rarely, in the gene encoding the glucocerebrosidase activator (saposin C), designated prosaposin [Christomanou et al. (1986) Biol Chem Hoppe Seyler 367: 879-890; Qi and Grabowski (2001) Prog Nucleic Acid Res MoI Biol 66:203-239; Sandhoff et al. (1995) Sphingolipid activator proteins, in: S. Scriber, Beaudet, A., SIy₁W., VaIIe₁D. (ed): The Metabolic and Molecular Basis of Inherited Disease. McGrew HiII₁ 1995, 2427-2441]. Decreased glucocerebrosidase activity leads to accumulation of the substrate, glucocerebroside, in lysosomes (mainly in macrophages). In addition, disease of the nervous system can arise from accumulation of endogenous glycosphingolipid metabolites in brain tissue [Beutler (1995, Ibid) and Grabowski et al (1996) Crit Rev Oncol Hematol 23: 25-55].

GD is very heterogeneous in etiology due to the presence of more than 260 Gaucher causing mutations in the glucocerebrosidase gene [Mutation Database (HGMD): worldwidewebdothgmddotcfdotacdotuk]. Due to this heterogeneity, GD has been sub-divided into three different clinical types: the adult chronic non-neuronopathic type 1 disease (GD I [MIM 230800]) the infantile, acute neuronopathic type 2 disease (GD Il [MIM 230900]) and the juvenile sub-acute neuronopathic type 3 GD (GD III [MIM 231000]). There is much diversity in the clinical manifestations of the disease within all genotypes. Even siblings with the same mutations, including monozygotic twins, may exhibit markedly different phenotype [Eyal et al. (2004) Hum Genet 87: 328-332; Lachmann et al. (2004) Qjm 97: 199-204].

It has been shown that Endoplasmic Reticulum Associated Protein Degradation (ERAD) contributes to the phenotypic severity of GD [Ron and Horowitz (2005) Hum MoI Genet 14: 2387- 2398]. In ERAD, misfolded or unassembled proteins are recognized by endoplasmic reticulum (ER) quality control machinery, transposed from the ER to the cytosol by a retrograde transport and are eliminated by the ubiquitin-proteasome system (UPS) [C. Hammond, A. Helenius, Quality control in the secretory pathway, Curr Opin Cell Biol 7 (1995) 523-529; .Kopito (1997) Cell 88: 427-430; Bonifacino, and Weissman (1998) Annu Rev Cell Dev Biol 14: 19-57; Brodsky and McCracken (1999) Semin Cell Dev Biol 10: 507-513 and Sitia and Braakman (2003) Nature 426 891-894].

It has been shown in cultured peritoneal macrophages, that excess intracellular unesterified (free) cholesterol affects the ER quality control machinery. Moreover, the accumulation of free cholesterol in the ER membrane induces ER stress and triggers the unfolded protein response (UPR), leading to depletion of ER calcium stores [Feng et al. (2003) Nat Cell Biol 5: 781-792].

It has been shown recently that in cells of Niemann-Pick disease type C (NPC), characterized by impaired cholesterol processing and trafficking, glucocerebrosidase is mislocalized and its activity and levels are decreased and can be restored by incubating the cells in LPDS [lipoprotein depleted serum, Salvioli et al (2004) J Biol Chem 279: 17674-1768]. Salvioli et al. have also shown [Salvioli et al (2005) Biochem J 390:95-103] that in cells that were derived from type 1 Gaucher patients there was higher cholesterol level, observed by Filipin staining.

Currently, Gaucher's disease patients are treated according to three principles. First, by enzyme replacement therapy (ERT, e.g. by administration of recombinant enzyme by injections). By this mode of action, the enzyme enters the affected cells (e.g. macrophages) through the mannose receptor and is trafficked to the lysosomes, where it degrades the accumulating substrate [Barton et al. (1991) N Engl J Med 324, 1464-1470; Barton et al. (1993) N Engl J Med 328, 1564-1565, author reply 1567-1568; Weinreb et al., (2002) Am J Med 113, 112-119]. Second, by partial inhibition of the rate of biosynthesis of the substrate, known as substrate reduction therapy [SRT, Cox (2005) Acta Paediatr Suppl. 94(447):69-75]. The third principle, currently in clinical trials, is the use of chaperone based therapy (CBT). The chaperone binds the mutant enzyme in the endoplasmic reticulum (ER), stabilizes the native fold of a protein in the ER and enables the protein-chemical chaperone complex to be trafficked to the proper protein destination environment where it can degrade the substrate [Morello, et al. (2000) Trends Pharmacol Sci 21 , 466-469; Perlmutter (2002) Pediatr Res 52, 832-836].

Cox [Cox, supra] and Platt et al. [Platt et al., (1994) J Biol Chem 269, No. 8362-5] suggest the use of the glucosylceramide inhibitor N-butyl-deoxynojirimycin (NB-DNJ, miglustat), a semi-selective iminosugar which inhibits the activity of various glycohydrolases, for substrate reduction therapy in the treatment of lysosomal storage diseases, including Gaucher disease.

U.S. Pat. No. 6,890,941 discloses a therapeutically effective amount of a lipid regulating agent, such as a HMG-CoA reductase inhibitor, and compound that inhibits cholesterol synthesis (at a point between the formation of acetate and mevalonate) for treating a disorder related to elevated serum cholesterol concentration such as atherosclerosis, elevated LDL plasma levels, low HDL plasma levels, hypertriglyceridemia, hyperlipidemia, hypertension, hypercholesterolemia, cholesterol gallstones, lipid storage diseases, obesity, and diabetes.

U.S. Pat. No. 6,583,158 discloses imino sugars and related compounds for enhancing the intracellular activity of an enzyme (e.g. glucocerebrosidase) associated with a lysosomal storage disorder (e.g. Gaucher disease). Krivit et al. (Krivit et al., Bone Marrow Transplant. (1992) 10 Suppl 1 :97-101) discloses a method for treating Wolman disease (a fatal disorder characterized by absence of acid lipase and accumulation of cholesterol esters in cells) by reducing cellular cholesterol synthesis by use of lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. PCT Publication No. WO 98/31367 discloses a method for inhibiting or treating diseases associated with acid lipase deficiency, such as Wolman disease and/or cholesteryl ester storage disease, by administering a microsomal triglyceride transfer protein (MTP) inhibitor alone or in combination with another cholesterol lowering drug, such as pravastatin.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a use of a cholesterol synthesis inhibitor for the manufacture of a medicament identified for treating a sphingolipid storage disease (SLSD) in a subject in need thereof. According to an aspect of some embodiments of the present invention there is provided a method of treating a sphingolipid storage disease (SLSD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a cholesterol synthesis inhibitor, thereby inhibiting the sphingolipid storage disease in the subject.

According to an aspect of some embodiments of the present invention there is provided a method of treating a sphingolipid storage disease (SLSD) in a subject in need thereof, the method comprising: (a) determining in the subject at least one of an activity level of an enzyme associated with the SLSD and an intracellular cholesterol level; and (b) treating the subject with a cholesterol synthesis inhibitor according to the result of step (a), thereby treating the sphingolipid storage disease. According to some embodiments of the invention, the sphingolipid storage disease

(SLSD) comprises Gaucher's disease.

According to some embodiments of the invention, the sphingolipid storage disease (SLSD) is selected from the group consisting of Gaucher's Disease, Niemann-Pick Disease, Krabbe's Disease (Globoid leukodystrophy), Metachromatic leukodystrophy, Ceramide lactoside lipidosis, Fabry's Disease, Tay-Sachs Disease, Sandhoff s Disease and Landing's Disease.

According to some embodiments of the invention, the cholesterol synthesis inhibitor comprises an inhibitor of HMG CoA-reductase.

According to some embodiments of the invention, the inhibitor of HMG CoA-reductase comprises a statin. According to some embodiments of the invention, the statin comprises mevastatin.

According to some embodiments of the invention, the statin is selected from the group consisting of mevastatin, lovastatin, pravastatin, fluvastatin, simvastatin, rosuvastatin, cerivastatin and atorvastatin.

According to some embodiments of the invention, the subject has Gaucher's disease and a glucocerebrosidase enzymatic activity at least below 30 % of that of a normal subject free of the Gaucher's disease.

According to some embodiments of the invention, the subject has at least 30 % higher cholesterol level than a normal subject not having the sphingolipid storage disease. According to some embodiments of the invention, the use or method further comprising administering to the subject a therapeutically effective amount of an agent capable of treating the sphingolipid storage disease.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary non-limiting embodiments of the invention described in the following description, read with reference to the figures attached hereto. In the figures, identical and similar structures, elements or parts thereof that appear in more than one figure are generally labeled with the same or similar references in the figures in which they appear. Dimensions of components and features shown in the figures are chosen primarily for convenience and clarity of presentation and are not necessarily to scale. The attached figures are:

FIGs. 1A-B depict glucocerebrosidase levels in normal fibroblasts compared to fibroblasts derived from Gaucher disease (GD) affected brothers. Figure 1A depicts lysates

(containing the same amount of protein) which were prepared from skin fibroblasts of the two GD brothers (severely affected and mildly affected) and of foreskin fibroblasts (normal), which were treated with PNGase F. Following 10 % SDS-PAGE, Western blot analysis was performed using anti-glucocerebrosidase and anti erk antibodies; and Figure 1 B depicts normalization of the results of Figure 1 A. Intensity of the glucocerebrosidase band at each lane was divided by that of erk. The value obtained for the normal glucocerebrosidase was determined as 100 %. Results represent the mean + SEM of 13 independent experiments.

FIGs. 1C-D depict endo-H cleavage patterns in normal fibroblasts compared to fibroblasts derived from Gaucher disease (GD) affected brothers. Figure 1C depicts lysates (containing the same amount of protein) which were prepared from skin fibroblasts of the two GD brothers (severely affected and mildly affected) and of foreskin fibroblasts (normal) subjected to endo-H digestion and Western blot analysis with anti glucocerebrosidase and anti erk antibodies; and Figure 1 D depicts determination of endo-H resistant fraction via scanning of blots and measurement of band intensity. Glucocerebrosidase resistant fraction was calculated by dividing the intensity of the endo-H sensitive fraction (in the endo-H+ lane) by that of the total amount of glucocerebrosidase in the same lane. Results represent the mean + SEM of 4 independent experiments, as percentage of the endo-H resistant fraction.

FIGs. 1 E-F depict the effect of proteasome inhibition on glucocerebrosidase in normal fibroblasts compared to fibroblasts derived from Gaucher disease (GD) affected brothers. Figure 1 E depicts lysates (containing the same amount of protein) which were prepared from skin fibroblasts of the two GD brothers (severely affected and mildly affected) and of foreskin fibroblasts (normal) treated with 50 μM ALLN and 25 μM MG-132 for 20 hours and subjected to Western blot analysis. Blots were probed with anti glucocerebrosidase, anti erk and anti p53 antibodies; and Figure 1 F depicts normalization of results of Figure 1 E. Blots were scanned and glucocerebrosidase intensity at each lane was divided by that of erk and the protein ratio between treated and untreated cells was calculated. Results represent the mean + SEM of 4 independent experiments, as percentage of the fold increase in protein level due to the treatment of each variant. Significance was determined by West; ^*p<0.001. FIGs. 2A-F depict glucocerebrosidase stability in normal fibroblasts compared to fibroblasts derived from Gaucher disease (GD) affected brothers. Figures 2A-C depict normal cells and cells from the two GD affected brothers treated with 25 μg/ml chycloheximide (CHX) to inhibit de-novo protein synthesis, in the presence or absence of proteasome inhibitors (25 μM MG132 and 50 μM ALLN). At the indicated times, cell lysates were prepared, subjected to SDS- PAGE and blotted; the corresponding blot was interacted with anti glucocerebrosidase and anti erk (as a marker for protein amount) antibodies; and Figures 2D-F depict quantitation of glucocerebrosidase levels of Figures 2A-C after normalization according to the erk levels. Value obtained for the untreated glucocerebrosidase band (CHX chase at time 0) for each cell line was determined as 100 %. The numbers represent one experiment, which was repeated three times, with the same pattern.

FIGs. 3A-L depict localization of glucocerebrosidase in normal fibroblasts compared to fibroblasts derived from Gaucher disease (GD) affected brothers. Normal fibroblasts and fibroblasts from the two GD affected brothers were grown on coverslips, fixed, permeabilized with 0.1 % triton X-100 and interacted with anti glucocerebrosidase monoclonal antibody and anti calnexin polyclonal antibodies. Detection was performed with cy-3 conjugated goat anti-mouse antibodies to demonstrate glucocerebrosidase localization (depicted in red), and with cy-2 conjugated goat anti-rabbit antibodies to demonstrate endogenous calnexin (depicted in green). Colocalization was illustrated by merging cy-2 and cy-3 images (Merge). Results were visualized with a confocal microscope. Merge I- a lower magnification of cy-2 (calnexin) and cy-3 (glucocerebrosidase) merge shows wider field of cells.

FIGs. 3M-0 depict lysosmal localization of glucocerebrosidase in normal fibroblasts. Normal skin fibroblasts grown on coverslips, were loaded with Lysotracker (depicted in red), fixed with 4 % paraformaldehyde and treated as described above (for Figures 3A-L). Detection was performed with FITC conjugated goat anti-mouse antibodies (depicted in green). Colocalization was illustrated by merging FITC (depicted in green) and Lysotracker (depicted in red) images (Merge) [Scale bar: 10 μm].

FIGs. 4A-B depict intracellular levels of cholesterol in normal fibroblasts compared to fibroblasts derived from Gaucher disease (GD) affected brothers. Fibroblasts were grown in the presence of 20 % FCS or 10 % LPDS (lipoprotein depleted serum). Seven days later, the amount of total (Figure 4A) or free cellular (Figure 4B) cholesterol was measured in samples of cell lysates containing 20 mg of protein. The level of cellular cholesterol in normal untreated cells was considered as 100 % in each experiment. Mean and standard were calculated from results of three independent experiments, with two repetitions, each. Significance was determined by t- test; *p<0.001.

FIG. 4C depicts significantly higher levels of free cholesterol in cells from the severely affected GD brother compared to normal cells. Fibroblasts from normal or severely affected GD subject were grown in the presence of 20 % FCS or 10 % LPDS. Seven days later, cells were fixed with 4 % paraformaldehyde and stained with Filipin to visualize free cholesterol. FIGs. 4D-G depict glucocerebrosidase levels, endo-H sensitivity and intracellular localization following culture of fibroblasts (normal and of the GD affected brothers) with lipoprotein free medium (LPDS). Figure 4D depicts glucocerebrosidase levels by Western blot analysis with anti glucocerebrosidase and anti erk antibodies performed to test glucocerebrosidase processing. Cell lysates containing the same amount of protein were subjected to endo-H digestion and were loaded in each lane; Figure 4E depicts determination of the endo-H resistant fraction. Blots were scanned and glucocerebrosidase resistant fraction was calculated by dividing the intensity of the endo-H resistant fraction (in the endo-H+ lane) by that of the total amount of glucocerebrosidase in the same lane; results represent the mean +SEM of 4 independent experiments, as percentage; Figure 4F depicts determination of increase in glucocerebrosidase levels due to LPDS treatment. The intensity of glucocerebrosidase at each lane was divided by that of erk; the value obtained for the untreated glucocerebrosidase was determined as 100 %; Figure 4G depicts the intracellular localization of glucocerebrosidase in normal fibroblasts and fibroblasts from the severely affected brother. Cells were grown on cover- slips in the presence of 20 % FCS or 10 % LPDS for 7 days, fixed, permeabilized with 0.1 % triton X-100 and interacted with anti glucocerebrosidase monoclonal antibody and anti calnexin polyclonal antibodies. Detection was performed with cy-3 conjugated goat anti-mouse antibodies to demonstrate glucocerebrosidase localization (depicted in red), and with cy-2 conjugated goat anti-rabbit antibodies to demonstrate endogenous calnexin (depicted in green). Colocalization was illustrated by merging cy-2 and cy-3 images (merge) [Scale bar: 10 μm]; FIGs. 5A-C depict the effect of mevastatin on glucocerebrosidase levels and endo-H resistance in normal fibroblasts compared to fibroblasts derived from Gaucher disease (GD) affected brothers. Figure 5A depicts lysates, containing the same amount of protein, prepared from fibroblasts grown with or without mevastatin (20 μg/ml, 72 hours), and subjected to endo-H digestion and western blot analysis with anti glucocerebrosidase and anti erk antibodies; Figures 5B-C depict determination of the endo-H resistant fraction. The blots were scanned and the intensity of each band was measured. Glucocerebrosidase resistant fraction (Figure 5B) was calculated by dividing the intensity of the endo-H sensitive fraction (in the endo-H+ lane) by the intensity of the entire amount of the glucocerebrosidase in the same lane. The results represent the mean + SEM of 4 independent experiments, as percentage of the endo-H resistant fraction. To determine fold increase in glucocerebrosidase levels after mevastatin treatment (Figure 5C)₁ its intensity (normalized to erk) after treatment was divided by the glucocerebrosidase intensity (normalized to erk) before treatment. The results represent the mean + SEM of 6 different experiments. FIGs. 6A-C depict the effect of mevastatin on glucocerebrosidase levels and endo-H resistantance in two Gaucher patients homozygous for the N370S mutation. Figure 6A depicts lysates containing the same amount of protein, prepared from skin fibroblasts of the two GD patients, homozygotes for the N370S mutation, grown with or without mevastatin (20 μg/ml, 72 hours), subjected to endo-H digestion and western blot analysis with anti glucocerebrosidase and anti erk antibodies; Figures 6B-C depict determination of the endo-H resistant fraction. The blots were scanned and the intensity of each band was measured. Glucocerebrosidase resistant fraction (Figure 6B) was calculated by dividing the intensity of the endo-H sensitive fraction (in the endo-H+ lane) by intensity of the entire amount of the glucocerebrosidase in the same lane. The results represent the mean + SEM of 3 independent experiments, as percentage of the endo-H resistant fraction. To determine fold increase in glucocerebrosidase levels after mevastatin treatment (Figure 6C), its intensity (normalized to erk) after treatment was divided by the glucocerebrosidase intensity (normalized to erk) before treatment. The results represent the mean + SEM of 4 different experiments.

FIGs. 7A-C depict the effect of U18666A on glucocerebrosidase in cells derived from patients with GD. Figure 7A depicts cells from normal and GD skin fibroblasts grown in the presence or absence of 12 μg/ml U18666A. Forty eight hours later, cell lysates containing the same amounts of protein were subjected to endo-H digestion and Western blot analysis with anti glucocerebrosidase and anti erk antibodies; Figure 7B depicts determination of the endo-H resistant fraction. The blots were scanned and the intensity of each band was measured. Glucocerebrosidase resistant fraction was calculated by dividing the intensity of the endo-H sensitive fraction (in the endo-H+ lane) by that of the total amount of glucocerebrosidase in the same lane. Results represent means + SEM of 4 independent experiments, as percentage of the endo-H resistant fraction; Figure 7C depicts fold decrease in glucocerebrosidase activity as calculated for each cell line by dividing glucocerebrosidase activity in untreated cells by that of the U18666A treated cells. The results represent means + SEM, measured in 3 experiments with 2 repetitions for each one.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present invention relates to treatment of sphingolipid storage diseases (SLSD) including, but not limited to, Gaucher's disease.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. While reducing the present invention to practice, the present inventors have surprisingly uncovered that downregulation of intracellular cholesterol synthesis in Gaucher's disease (GD) patients allows correct trafficking of glucocerebrosidase to the lysosome, probably by decreasing the degree of endoplasmic reticulum (ER) associated degradation (ERAD) of the enzyme. This is followed by a significant improvement in stabilization, maturation, lysosomal localization and activity of the mutant glucocerebrosidase enzymes in these patients.

As is shown hereinbelow and in the Examples section which follows, the present inventors have shown that mutant glucocerebrosidase variants in GD subjects present variable degrees of endoplasmic reticulum (ER) retention and undergo ER associated degradation (ERAD) in the proteasomes (Figures 1A-F). Furthermore, the glucocerebrosidase variants have a short half life (Figures 2A-F)₁ are retained in the ER and do not reach the lysosomes (Figures 3A-O). These results are more prevalent in a severely affected GD subject in comparison to a mildly affected GD subject. Thus, the degree of ERAD is one of the major factors that determine GD severity.

Moreover, it is shown herein that elevated levels of total and free intracellular cholesterol are present in cells that were derived from GD patients whose clinical disease was more severe than expected by their genotype (Figures 4A-C). Growth of the GD derived cells in cholesterol depleted medium led to a decrease in the degree of ERAD, to an improvement in stabilization, maturation, lysosomal localization and activity of the mutant glucocerebrosidase variants (Figures 4D-G). The same effect was achieved by treating the GD derived cells with the HMG CoA reductase inhibitor mevastatin (Figures 5A-C and Figures 6A-C). All these findings substantiate the use of inhibitors of cholesterol synthesis for the treatment of sphingolipid storage diseases (SLSD), such as Gaucher's disease.

Thus, according to one aspect of the present invention there is provided a use of a cholesterol synthesis inhibitor for the manufacture of a medicament identified for treating a sphingolipid storage disease (SLSD) in a subject in need thereof.

As used herein the term "subject in need thereof refers to a mammal, preferably a human subject who is diagnosed with a sphingolipid storage disease. In an exemplary embodiment the subject is free of atherosclerosis caused by high blood cholesterol levels.

As used herein the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a sphingolipid storage disease.

As used herein, the term "sphingolipid storage disease" (SLSD) or sphingolipidosis refers to a medical condition characterized by intra-lysosomal deposit of glycosphingolipids and phosphosphingolipids. Typically such a disease is an inherited metabolic disorder classified by an enzyme defect in the degradation pathway and substrate accumulation (or storage). Typically the abnormal storage of sphingolipids is in the central nervous system or in the visceral organs of the subject. Moreover, clinical features may vary between subjects and may range from mild to severe. Examples of sphingolipid storage diseases include, but are not limited to, Gaucher's

Disease (Types I, Il and III), Niemann-Pick Disease (types A and B), Krabbe's Disease (also known as Globoid leukodystrophy or galactosylceramide lipidosis), Metachromatic leukodystrophy (MLD, also called Arylsulfatase A deficiency), Ceramide lactoside lipidosis, Fabry's Disease (also known as Anderson-Fabry disease, Angiokeratoma corporis diffusum, Ruiter-Pompen-Wyers syndrome, Ceramide trihexosidosis, and Sweeley-Klionsky disease), GM2 gangliosidosis (Tay-Sachs and Sandhoff forms), GM1 gangliosidosis (e.g. Landing's Disease).

Different enzymes are associated with the different SLSDs. These enzymes are directly involved in the etiology of the different sphingolipid storage diseases. For example, in Gaucher's disease, only a fraction of the mutant glucocerebrosidase enzyme reaches the lysosomes leading to an accumulation of its substrate, the fatty substance glucocerebroside, in the lysosomes (see the Example section below). In Niemann-Pick Disease types A and B, a mutation in the enzyme acid sphingomyelinase (ASM) causes an abnormal accumulation of sphingomyelin. In Krabbe's Disease, a mutation in galactosylceramidase causes imperfect growth and development of myelin. In Metachromatic leukodystrophy, a mutation in arylsulfatase A results in build up of sulfatides in many tissues of the body, eventually destroying the myelin of the nervous system. In Ceramide lactoside lipidosis, a mutation of ceramide lactosidase results in an accumulation of ceramide lactoside. In Fabry's Disease, a mutation in alpha galactosidase A causes accumulation of the glycolipid globotriaosylceramide. In Tay-Sachs the mutation is in the enzyme beta-hexosaminidase A and in Sandhoff disease the mutation is in the enzyme beta- hexosaminidase A and B. GM 1 gangliosidosis is caused by a mutation of beta-galactosidase, with resulting abnormal storage of acidic lipid material.

According to an exemplary embodiment of the present invention, the enzymatic activity and/or level of the enzyme associated with the SLSD is determined prior to treatment. An enzymatic activity and/or level may be measured using any method known to one of ordinary skill in the art. For example, by:

In situ activity assay: According to this method, a chromogenic substrate is applied on the cells containing an active enzyme and the enzyme catalyzes a reaction in which the substrate is decomposed to produce a chromogenic product visible by a light or a fluorescent microscope. In vitro activity assays: In these methods the activity of a particular enzyme is measured in a protein mixture extracted from the cells. The activity can be measured in a spectrophotometer while using colorimetric methods or can be measured in a non-denaturing acrylamide gel (i.e., activity gel). Following electrophoresis, the gel is soaked in a solution containing a substrate and colorimetric reagents. The resulting stained band corresponds to the enzymatic activity of the protein of interest. If well calibrated and within the linear range of response, the amount of enzyme present in the sample is proportional to the amount of color produced. An enzyme standard is generally employed to improve quantitative accuracy.

For example, measuring enzymatic activity in cells (e.g. primary skin fibroblasts) of Gaucher's disease subjects may be effected by measuring glucocerebrosidase activity using the artificial substrate 4-MUG (Genzyme CorBoston Corp., MA, USA). The enzymatic reaction may be performed, as described in the Examples section which follows, in 0.2 ml of 100 mM potassium phosphate buffer, pH 5.8, 0.15 %Triton X-100 (v/v, Sigma, Israel) and 0.125 % taurocholate (w/v, Calbiochem, grade A, Merck, Darmstadt, Germany) in the presence of 1.5 mM 4-MUG₁ for 60 minutes at 37 ⁰C. The reaction may then be stopped by the addition of 1 ml 0.1 M glycine in 0.1 M NaOH pH 10. The amount of 4-methyl-umbelliferone (4-MU) is then quantified using a PerkinElmer Luminescence Spectrometer LS 50 (excitation length: 340 nm; emission: 448 nm).

Measuring expression level of any of the aforementioned enzymes can be effected using methods which are well known in the art. For example, by: Enzyme linked immunosorbent assay (ELISA): This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.

Western blot: This method involves separation of a substrate from other proteins by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents. Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.

Radioimmunoassay (RIA): In one version, this method involves precipitation of the desired protein (i.e., the substrate) with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labeled with I¹²⁵) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.

In an alternate version of the RIA, a labeled substrate and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of substrate is added in varying amounts. The decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.

Fluorescence activated cell sorting (FACS): This method involves detection of a substrate in situ in cells by substrate specific antibodies. The substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously. lmmunohistochemical analysis: This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies. The substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or Giemsa stain.

It will be appreciated that abnormal enzymatic activity and/or level (i.e. abnormally low levels) will be determined by comparison of the enzymatic activity and/or level in the SLSD subject to those of an unaffected, healthy individual who is not afflicted with SLSD.

According to an exemplary embodiment, in a Gaucher's disease subject, the glucocerebrosidase enzymatic activity is typically below 80%, 50 %, 30 %, 20 % of that of healthy individuals. In exemplary embodiment the activity is below 30 %. As depicted in detail in the examples section which follows, Gaucher's disease subjects were found to have significantly higher levels of intracellular total and free cholesterol compared to normal subjects (Example 6). These levels were about 65-70 % higher in a severely affected GD subject compared to normal levels (in subjects free of the disease, Figures 4A-B).

As used herein the phrase "intracellular cholesterol" refers to cholesterol synthesized and found within the cell of a subject. Typically intracellular cholesterol refers to both total and free cholesterol levels.

Measurements of intracellular cholesterol levels may be accomplished using any method known in the art, as for example, using the fluorometric assay - Amplex Red Cholesterol kit from

Molecular Probes (Invitrogen, Eugene, OR, USA); by staining with Filipin, an intrinsically fluorescent, sterol-binding, antibiotic which binds selectively to cholesterol (and not to cholesterol esters); by cholesterol oxidase assay; or by an enzymatic density shift method.

High intracellular cholesterol levels of the SLSD afflicted subject may be in exemplary embodiments at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, or at least 100 % higher than normal levels. In exemplary embodiment the intracellular cholesterol level is higher than 30 %.

It will be appreciated that if the level of intracellular cholesterol is above a threshold and/or level or activity of the enzyme associated with the SLSD, therapy with a cholesterol synthesis inhibitor is undertaken. According to embodiments of the present invention, treatment can be undertaken even if serum cholesterol is of about normal values. As used herein, the phrase "cholesterol synthesis inhibitor" refers to any agent which at least partially prevents the synthesis of cholesterol within the cell. Examples of such inhibitors include, but are not limited to, inhibitors of HMG-CoA reductase (such as statins), inhibitors of prenylating enzymes (such as Farnesyl Transferase) and distal inhibitors of cholesterol biosynthesis (such as zaragozic acid, SKF 104976, SR 31747, BM 15766, and AY 9944).

In an exemplary embodiment of the invention, the cholesterol synthesis inhibitor is an HMG-CoA reductase inhibitor. HMG-CoA (E.C. 1.1.1.88) also termed 3-hydroxy-3-methyl- glutaryl-CoA reductase or HMGR is the key enzyme in cholesterol synthesis. HMG-CoA reductase inhibitors are a class of hypolipidemic agents, used to lower cholesterol levels. Cholesterol is lowered as HMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway of cholesterol synthesis. Lowering of cholesterol levels is often apparent after one week of therapy and maximum effect can be achieved within 4-6 weeks.

It will be appreciated that any inhibitor of HMG-CoA reductase may be used according to the present teachings. Typically the HMG-CoA reductase comprises a statin. According to an exemplary embodiment, the statin may comprise, without being limited to, mevastatin, lovastatin, pravastatin, fluvastatin, simvastatin, rosuvastatin, cerivastatin and atorvastatin and derivatives, analogs and combinations (e.g., Vytorin that combines simvastatin and ezetimibe) thereof. Some of these statins are naturally occurring compounds (such as mevastatin which is isolated from red yeast rice). Other statins are produced synthetically (e.g. atorvastatin and fluvastatin) or are fermentation derived (e.g. lovastatin and pravastatin).

The term inhibitor of HMG-CoA reductase is also intended to include all pharmaceutically acceptable salt, ester and lactone forms of compounds which have HMG-CoA reductase inhibitory activity, and therefore the use of such salts, esters and lactone forms is included within the scope of this invention. It will be appreciated, that the cholesterol synthesis inhibitor of the present invention can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the cholesterol synthesis inhibitor.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections. Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (cholesterol synthesis inhibitor) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., neuronal damage) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

In vitro models for Gaucher's disease include, but are not limited, cultures of primary skin fibroblasts and foreskin fibroblasts or the murine macrophage cell line WEHI-3B (cultured in the presence of an irreversible glucocerebrosidase inhibitor; Platt et al., supra). In vivo models for

Gaucher's disease and other SLSDs include, but are not limited to, animal models including mouse, rat, dog, cat, guinea pig, emu, quail, goat, cattle, sheep, and pig. For example, mouse knockout models are used for in vivo assessment of Metachromatic leukodystrophy, Niemann-

Pick types A and B, GM2 gangliosidosis/Tay-Sachs, GM2 gangliosidosis/Sandhoff and Fabry disease, and dog and cat models are used for in vivo assessment of GM1 gangliosidosis

[Ellinwood et al., (2004) J Gene Med 6: 481-506].

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, ef al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1). Dosage amount and interval may be adjusted individually to provide intracellular levels of the active ingredient which are sufficient to suppress cholesterol synthesis (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.

It will be appreciated that the dosage information for HMG-CoA reductase inhibitors is well known in the art, since several are marketed in the U.S. In particular, the daily dosage amounts of the HMG-CoA reductase inhibitor may be the same or similar to those amounts which are employed for anti-hypercholesterolemic treatment and which are described in the Physicians' Desk Reference (PDR). For example, see the 53.sup.th Ed. of the PDR, 1999 (Medical Economics Co); in particular, see at page 216 the heading "Hypolipidemics," subheading "HMG-CoA Reductase Inhibitors," and the reference pages cited therein. Typically, the oral dosage amount of HMG-CoA reductase inhibitors is from about 1 to 200 mg/day, and more preferably from about 5 to 160 mg/day. However, dosage amounts will vary depending on the potency of the specific HMG-CoA reductase inhibitor used as well as other factors as noted above. An HMG-CoA reductase inhibitor which has sufficiently greater potency may be given in sub-milligram daily dosages.

The cholesterol synthesis inhibitors will be given for a sufficient amount of time to enable a decrease in intracellular cholesterol levels, an increase in cellular enzymatic activity of the enzyme associated with SLSD, and preferably until there is alleviation in symptoms related to SLSD. It is advisable to measure intracellular cholesterol levels prior to treatment and during the treatment course. Furthermore, once a subject received a cholesterol synthesis inhibitor, it is advisable that they return for follow-up evaluation, which include, for example, hematologic and chemical tests for safety.

It will be appreciated that treatment of SLSD according to the present teachings may be combined with other possible SLSD treatments. Thus, the cholesterol synthesis inhibitor of the present invention may be administered in conjunction with, for example, enzymatic complementation therapy (e.g. glucocerebrosidase for Gaucher's disease; acid sphingomyelinase (ASM) for Niemann-Pick Disease; beta-hexosaminidase for Tay-Sachs and Sandhoff disease; beta-galactosidase for GM1 gangliosidosis; galactosylceramidase for Krabbe's Disease; arylsulfatase A for Metachromatic leukodystrophy; ceramide lactosidase for Ceramide lactoside lipidosis; and alpha galactosidase A for Fabry's Disease), with substrate reduction therapy (e.g. miglustat for inhibition of various glycohydrolases, for substrate reduction therapy in the treatment of lysosomal storage diseases, including Gaucher's disease), or with the use of chemical chaperones that bind the mutant enzyme in the endoplasmic reticulum (ER), stabilizes the native fold of a protein in the ER and enables the protein-chemical chaperone complex to be trafficked to the proper protein destination environment where it can degrade the substrate (e.g. the lysosomes).

It will be appreciated that the cholesterol synthesis inhibitors of the present invention may be administered prior to, concomitantly with, or following administration of other known SLSD treatment agents (as depicted above).

It is expected that during the life of this patent many relevant inhibitors of cholesterol synthesis will be developed and the scope of the term "cholesterol synthesis inhibitor" is intended to include all such newly developed inhibitors a priori. As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

The following materials and methods are presented in support of examples set forth hereinbelow:

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook ef a/., (1989); "Current Protocols in Molecular Biology" Volumes l-lll Ausubel, R. M., ed. (1994); Ausubel ef a/., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson ef a/., "Recombinant DNA", Scientific American Books, New York; Birren ef a/, (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801 ,531 ; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes l-lll CeIMs, J. E., ed. (1994); "Current Protocols in Immunology" Volumes l-lll Coligan J. E., ed. (1994); Stites ef a/, (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791 ,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901 ,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011 ,771 and 5,281 ,521 ; Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak ef a/., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. EXAMPLE 1

General Materials and Methods Antibodies

The following antibodies were used in this study: Primary antibodies Mouse monoclonal anti-glucocerebrosidase 8E4 and 2C7 (Barneveld et al. (1983) Eur J

Biochem 134: 585-589; kindly provided by Prof. H. Aerts E. C. Slater Institute for Biochemical Research, University of Amsterdam, the Netherlands);

Mouse monoclonal anti-p53 DO1 (kindly provided by Dr D. Lane, Department of Surgery and Molecular Oncology, University of Dundee, Dundee, United Kingdom.); Rabbit anti-erk (C16, Santa Cruz Biotechnology, Santa Cruz, CA, USA);

Rabbit anti-calnexin (SPA-860, Stressgen Biotechnologies, Victoria, BC, Canada);

Secondary antibodies:

FITC conjugated goat anti mouse; cy-3 conjugated goat anti mouse; cy-2 conjugated goat anti rabbit, horseradish peroxidase conjugated goat anti mouse and goat anti rabbit (purchased from Jackson lmmuno Research Laboratories, West Grove, PA, USA). Enzymes:

Endoglycosidase-H (Endo-H) and peptide N glycosidase F (PNGase F) were purchased from New England Biolabs (Beverly, MA, USA).

Restriction enzymes were purchased from several companies and used according to the manufacturers' recommendations. Chemicals:

Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132) and N-acetyl-L-leucyl-L-leucyl-L- norleucinalAc (ALLN) (purchased from Calbiochem, San Diego, CA, USA).

Cycloheximide (CHX) was purchased from Sigma Aldrich, Israel.

LPDS was purchased from Sigma Aldrich, Israel. Four-methyl-umbelliferyl-glucopyranoside (4-MUG) was purchased from Genzyme Corp.

(Boston, MA, USA).

Leupeptin, phenylmethylsulfonyl fluoride (PMSF) and aprotinin were purchased from Sigma Aldrich, Israel.

Lysotracker Red DND-99 was purchased from Molecular probes, (Invitrogen, Eugene, OR, USA).

Cell Lines

Human primary skin fibroblasts and foreskin fibroblasts (FS11 ) were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 20 % Fetal Calf Serum (FCS). All cells were grown at 37 ⁰C in the presence of 5 % CO₂ This study complied with the ethical guidelines of Tel-Aviv University.

Protein amounts

Protein amounts were determined by the Bradford technique (Bradford (1976) Anal Biochem 72: 248-254).

Endo-H and PNGase F treatment Samples of cell lysates, containing the same amount of protein, were subjected to an overnight incubation with endo-H or PNGase F, according to the manufacturer's instructions.

Proteasome inhibition

Subconfluent human skin fibroblasts were treated with 25 μM MG132 and 50 μM ALLN for 20 hours after which protein lysates were prepared and samples, containing the same amount of protein, were subjected to Western blot analysis.

Cvcloheximide chase experiments

Subconfluent human skin fibroblasts were chased with 25 μg /ml CHX to inhibit de-novo protein synthesis with or without proteasome inhibitors (25 μM MG132 and 50 μM ALLN). At the indicated times, cell lysates were prepared, and the same amount of lysates were subjected to SDS-PAGE and blotted.

SDS-PAGE and Western Blotting

Cell monolayers were washed 3 times with ice-cold phosphate-buffered saline (PBS) and lysed at 4 ⁰C in 500 μl of lysis buffer (10 mM Hepes pH8.0, 100 mM NaCI, 1 mM MgCI₂, and 0.5

% NP-40) containing 10 μg/ml aprotinin, 0.1 mM PMSF and 10 μg/ml leupeptin. Lysates were incubated on ice for 30 minutes and centrifuged at 10,000 g for 15 minutes at 4 ⁰C. Samples containing the same amount of protein were subjected to 10 % SDS-PAGE and electroblotted onto a nitrocellulose membrane (Schleicher & Schuell BioSience, Keene, NH₁ USA). Membranes were blocked with 5 % skim milk and 0.1 % Tween 20 in Tris-buffered saline (TBS) for 2 hours at room temperature (RT) and incubated with the primary antibody for 1 hour at RT. The membranes were then washed 3 times in 0.1 % Tween 20 in TBS and incubated with the appropriate secondary antibody for 1 hour at RT. After washing, membranes were reacted with enhanced chemiluminol (ECL) detection reagents (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) and analyzed by luminescent image analyzer (Kodak X-OMAT 2000 Processor, Kodak

Rochester, New York, USA).

Free cholesterol depletion

Primary skin fibroblasts were grown in DMEM containing 10 % LPDS (lipoprotein depleted serum) or 20 % FCS (Fetal Calf Serum). Seven days later, cells were subjected to indirect immunoflourescence or lysates were prepared and tested for glucocerebrosidase levels, activity, cholesterol levels and endo-H sensitivity.

Quantitative measurement of total and free intracellular cholesterol

A fluorometric assay to measure both cholesterol and cholesteryl ester was performed using the Amplex Red Cholesterol kit from Molecular Probes (Invitrogen, Eugene, OR, USA), according to the manufacturer's protocol. The assay is based on an enzyme-coupled reaction, where cholesteryl ester is hydrolyzed by cholesterol esterase into cholesterol, which is then oxidized by cholesterol oxidase, yielding H₂O₂. The H₂O₂ is detected by Amplex Red reagent that reacts with it in the presence of horseradish peroxidase (HRP) to produce fluorescent resorufin.

The resorufin is detected by excitation at 530 nm and emission at 590 nm. Measurements of total cellular cholesterol were calibrated by incubating both cholesterol oxidase and cholesterol esterase with known concentrations of cholesterol. Measurements of free cholesterol were calibrated by incubating lysates with cholesterol oxidase only.

Immunocvtochemical analysis and confocal laser scanning microscopy

Subconfluent cells, grown on cover-slips, were washed twice with PBS, fixed for 5 minutes at 4 "C in methanol, following five minutes at 4 °C in methanol-acetone (at a ratio of 1 :1).

After washes, cells were permeabilized with 0.1 % Triton X-100 in PBS for 3 minutes at RT.

Cells were than washed three times with PBS, blocked by incubation with PBS containing 5 % bovine serum albumin (BSA) and 20 % normal goat serum for 30 minutes at RT, and then incubated for 1 hour with the corresponding primary antibody (1 :100 dilution for 2C7 and 1 :200 for rabbit anti-calnexin) in 1 % BSA in PBS at RT. Following 3 washes with PBS, cells were immunostained with rabbit-cy-2 or mouse-cy-3 conjugated secondary antibodies (1:200 dilution) in 1 % BSA in PBS for 45 minutes at RT. Following three washes with PBS, the cover-slips were mounted with galvanol. For lysostracker colocalization, cells, loaded for 1 hour with 25 nM lysotracker at 37 ⁰C, were fixed for 15 minutes in 4 % (v/v) paraformaldehyde/PBS at RT, treated as above and immunostained with mouse-FITC conjugated secondary antibodies. Cells were observed and analyzed with a LSM 510 confocal laser scanning microscope (Carl Zeiss, Germany).

Filipin staining of free cholesterol Filipin staining was performed essentially as described elsewhere (Naslavskyet al. (2007)

Biochem Biophys Res Commun 357: 792-799). Briefly, cells plated on glass coverslips, were fixed with 4 % paraformaldehyde. They were then blocked, permeabilized and stained simultaneously by incubation with PBS containing 5 % bovine serum albumin (BSA), 20 % normal goat serum and 5 μg/ml Filipin (Sigma, Israel) for 1 hour at room temperature. Unbound Filipin was washed extensively with PBS. Fluorescence of Filipin was detected by excitation with a 405 nm laser, and collecting emission with a 420-480 nm band pass filter using LSM 510 META confocal laser scanning microscope (Carl Zeiss, Germany). Enzymatic activity Confluent primary skin fibroblasts were washed twice with PBS, collected with a rubber policeman in 0.5 ml sterile water and frozen in aliquots at -80 ⁰C. Samples containing 20 μg of protein were assayed for glucocerebrosidase activity with 4-MUG (Genzyme CorBoston Corp., MA, USA). The enzymatic reaction was performed in 0.2 ml of 100 mM potassium phosphate buffer, pH5.8, 0.15 %Triton X-100 (v/v, Sigma, Israel) and 0.125 % taurocholate (w/v, Calbiochem, grade A, Merck, Darmstadt, Germany) in the presence of 1.5 mM 4-MUG, for 60 minutes at 37 °C. The reaction was stopped by the addition of 1 ml 0.1 M glycine in 0.1 M NaOH pH 10. The amount of 4-methyl-umbelliferone (4-MU) was quantified using a PerkinElmer Luminescence Spectrometer LS 50 (excitation length: 340 nm; emission: 448 nm). Quantitation Blots were scanned using Image Scan scanner (Amersham Pharmacia Biotech, GE Healthcare, Buckinghamshire, UK) and the intensity of each band was measured by the image master densitometer I Dprime (Amersham Pharmacia Biotech, GE Healthcare, Buckinghamshire, UK). EXAMPLE 2

Glucocerebrosidase activity in cell lysates from brothers with the same glucocerebrosidase mutations

In order to elucidate the physiologic mechanisms underlying phenotypic differences between genotypically similar individuals, a unique family with Gaucher disease (GD) was used to provide biological material for the present study.

In this family, two GD brothers were shown to have the same three point mutations in the glucocerebrosidase gene: D140H+E326K on one allele, that was derived from the mother and a K157Q on the allele that was derived from the father (Eyal et al. (1991) Hum Genet 87: 328-332). Despite the genotypic identity between these two brothers, they presented a significantly different disease severity.

The first brother (hereinafter "severely affected") was affected with myoclonic jerks at the age of 15 and thereafter his cognitive functions declined and his seizures became increasingly difficult to control. He was diagnosed as a Gaucher patient on the basis of the presence of Gaucher cells in his spleen, liver and marrow. Furthermore, his leukocyte glucocerebrosidase level was markedly low and his plasma glucocerebroside was increased by about two-fold (10.2 nmol/ml, whereas normal levels are 5.1 ± 1.5 nmol/ml). He became wheelchair-bound with nearly constant multifocal myoclonic jerks and died at the age of 28 of bacterial endocarditis. Autopsy of his brain disclosed degeneration of the lateral columns of the spinal cord and mild gliosis.

The second brother (hereinafter "mildly affected") was diagnosed as a Gaucher patient at the age of 26, on the basis of leukocyte glucocerebrosidase determination and an elevated plasma glucocerebroside level (7 nmol/ml). He is essentially well, without palpable liver or spleen.

In order to establish that the observed clinical differences between the severely affected and mildly affected brothers were related to GD, \n-vitro glucocerebrosidase activity was tested in lysates of primary skin fibroblasts that were derived from each of the brothers as well as from normal foreskin fibroblasts, using the artificial substrate 4-MUG. Table 1 , hereinbelow, summarizes the endogenous activity of glucocerebrosidase in normal cells as well as cells of the two GD brothers toward 4-MUG. The results represented the mean ± SEM as percentage of the activity of normal glucocerebrosidase, measured in 3 experiments with 2 repetitions for each. The results indicated that lysates of both affected brothers exhibited low glucocerebrosidase activity compared to the normal levels. However, glucocerebrosidase activity in cells of the severely affected brother was about

3 fold lower than that presented by cells of his brother. These results indicated that the differences in the clinical features between the two brothers were most probably due to a significant change in glucocerebrosidase activity. Table 1 : In-vitro glucocerebrosidase activity in cell lysates Cell line % Activity of normal

Normal 100

Mildiy affected brother 36.5 ± 7.2

Severely affected brother 12.8 + 1.8

EXAMPLE 3 Comparison of ER retention and degradation of glucocerebrosidase in cells of the brothers

In order to establish whether the differences in glucocerebrosidase activity observed in Example 2 were due to a difference in glucocerebrosidase levels, Western blot analysis of PNGase F digested cell lysates derived from the two brothers and from normal foreskin fibroblasts was performed.

Since PNGase F is an endoglycosidase that removes all asparagine-linked glycans from glycoproteins (Maley et al. (1989) Anal Biochem 180: 195-204; Plummer et al (1984) J Biol Chem 259: 10700-10704; and Trimble and Tarentino (1991) J Biol Chem 266: 1646-1651), it generates one glucocerebrosidase isoform whose intensity can be readily measured.

As shown in Figures 1A and 1 B, both brothers presented a decrease in glucocerebrosidase levels. In cells of the severely affected brother only 26 % of the normal glucocerebrosidase levels were present, while in the mildly affected brother, 35 % of the normal glucocerebrosidase levels were present. To ensure that the decrease in glucocerebrosidase levels did not reflect a general decrease in the amount of lysosomal enzymes, β- hexosaminidase-A level was tested and no difference from normal levels was observed (data not shown), indicating that the decrease was specific to glucocerebrosidase.

In an effort to follow the intracellular fate of glucocerebrosidase in the two brothers, the processing of its N-glycans in cell lysates was monitored. Lysates were subjected to endo-H digestion, a specific endoglycosidase that can distinguish between high mannose (more than 4 mannose residues) and a mature N-glycan complex (Maley et al. Ibid and Trimble and Tarentino Ibid).

Results presented in Figures 1C and 1D, showed that there was a significant difference in the endo-H cleavage pattern between cells derived from the brothers and that of normal cells and also between the two affected brothers. While in normal cells about 80 % of glucocerebrosidase was endo-H resistant, indicating that most of the proteins have already crossed the mid-Golgi (probably mature lysosomal), 66 % of glucocerebrosidase in cells of the mildly affected brother and only 32 % in cells of the severely affected brother were endo-H resistant, suggesting that different fractions of glucocerebrosidase in the cells of the two brothers were unprocessed and did not reach the mid Golgi apparatus. Since it has already been demonstrated that mutant glucocerebrosidase variants retain in the ER and undergo variable degrees of Endoplasmic Reticulum Associated Protein

Degradation (ERAD), the degree of ERAD in the brothers was assayed using the proteasome inhibitors MG-132 (Rock et al. (1994) Cell 78:761-771) and ALLN (Sasaki et al. (1990) J Enzyme lnhib 3: 195-201).

For the assay, cells were grown in the presence of the proteasome inhibitors ALLN and MG-132 and their lysates were subjected to Western blot analysis with p53 as a control, since it undergoes proteasomal degradation and can be stabilized by proteasome inhibitors (Maki et al. (1996) Cancer Res 56: 2649-2654). Figures 1 E and 1 F summarize results of the proteasome inhibition assay which indicated that, while normal glucocerebrosidase was not affected by proteasome inhibitors, mutant glucocerebrosidase accumulated in both affected brothers, indicating their stabilization in the presence of proteasome inhibitors. There was a higher stabilization in the severely affected brother (about 2.4 fold increase in glucocerebrosidase levels) compared to the mildly affected one (about 1.5 fold increase in glucocerebrosidase levels), indicating that the difference in glucocerebrosidase protein level in the two brothers is due, at least in part, to different degrees of proteasomal degradation. Results from this example, together with those of example 2, indicated that in the severely affected brother the ERAD of glucocerebrosidase was more extensive. The extensive ERAD of glucocerebrosidase presented a plausible explanation for the more severe disease phenotype.

EXAMPLE 4 Decreased stability of mutant glucocerebrosidase variants in the brothers

In order to directly test glucocerebrosidase stability, normal cells and cells from both affected brothers were chased with cycloheximide, with or without proteasome inhibitors.

Cycloheximide was used to block de-novo synthesis of proteins and, thus, allows detecting the stability of a tested expressed protein (Obrig et al. (1971) J Biol Chem 246: 74-18). The results (Figures 2A-F) clearly demonstrated that, while the half-life of the normal glucocerebrosidase was about the expected 60 hours (Erickson et al. (1985) J Biol Chem 260(26): 14319-14324), and was not significantly affected by proteasome inhibitors, the half-life of mutant glucocerebrosidase was 28 hours in the mildly affected brother and changed to 53 hours in the presence of proteasome inhibitors. In the severely affected brother the half-life of mutant glucocerebrosidase was 11.5 hours and changed to 20 hours in the presence of proteasome inhibitors.

These results clearly demonstrated that in both brothers there was a decrease in glucocerebrosidase stability due to proteasomal degradation. The stabilization, due to proteasome inhibition, was more significant in the severely affected brother, suggesting that his proteasomal degradation was more extensive. EXAMPLE 5 Intracellular localization of glucocerebrosidase in cells from the brothers

In order to test whether glucocerebrosidase is retained in the ER of the two affected brothers, indirect immunoflourescence was performed. Figures 3A-0 show that in normal cells glucocerebrosidase accumulated in punctate lysosomal structures, as presented by colocalization with the lysosomal marker lysotracker. In normal cells, only a negligible fraction of glucocerebrosidase was colocalized with calnexin, the

ER marker.

On the other hand, cells from both brothers demonstrated diverse levels of colocalization with calnexin. In cells from the severely affected brother there was almost complete colocalization of glucocerebrosidase with calnexin, indicating that most of the protein was retained in the ER and did not reach the lysosomes. In cells from the mildly affected brother, part of the protein showed a reticular accumulation in the calnexin positive ER, while it also appeared in punctate lysosomal structures. These results are consistent with those of Examples 3 and 4, which suggested that glucocerebrosidase is being degraded. Moreover, results from Example 5 suggested that the degradation process was mediated by a factor or factors in the ER.

EXAMPLE 6 Effect of cholesterol reduction on processing of mutant glucocerebrosidase variants

In order to test whether there is a difference in the intracellular cholesterol levels between the two affected brothers, and whether the intracellular cholesterol level affects glucocerebrosidase processing and trafficking both total and free cholesterol were measured in cells that originated from the two affected brothers as well as in normal cells.

Figures 4A-B clearly show that, while there was no significant difference in the intracellular level of cholesterol between normal cells and cells that originated from the mildly affected brother, cells from the severely affected brother demonstrated about 65-70 % increase in both total and free cholesterol. Moreover, growing the cells of the severely affected brother in LPDS resulted in a approximately 30 % reduction in both total and free cholesterol, which reached almost normal levels, while it did not significantly alter cholesterol levels in normal cells or in cells from the mildly affected brother (Figures 4A-B). Free intracellular cholesterol was also tested by staining with Filipin, an intrinsically fluorescent, sterol-binding, antibiotic. Figure 4C shows significantly higher levels of free cholesterol in cells from the severely affected brother in comparison to normal cells and it was markedly reduced following LPDS treatment, which did not effect normal cells. In order to follow glucocerebrosidase processing and trafficking, glucocerebrosidase levels as well as its endo-H sensitivity and intracellular localization were tested. As is clear from Figures 4D-F the depletion of cholesterol did not significantly alter glucocerebrosidase amount or its endo-H sensitivity in normal cells or in cells that were derived from the mildly affected brother. In sharp contrast, in cells that were derived from the severely affected brother, there was a two-fold increase in glucocerebrosidase levels as well as an increase in the endo-H resistant fraction (an increase from about 30 % to about 60 %).

Furthermore, Figure 4G shows that the intracellular localization of glucocerebrosidase in the severely affected brother was altered from reticular to more punctate structures relative to normal cells, namely from the ER to endosomes/lysosomes.

In order to assay the effect of cholesterol on glucocerebrosidase activity, normal cells and cells from the two brothers were grown in the presence of 20 % FCS or 10 % LPDS. Seven days later, lysates were prepared and the in-vitro glucocerebrosidase activity was tested toward 4-MUG. Fold glucocerebrosidase activity was calculated by dividing glucocerebrosidase activity in LPDS treated cells by that of the same cells without treatment. The results represented the mean ± SEM measured in 3 experiments with 2 repetitions for each. As is clear from the results (Table 2, hereinbelow), reduction of cholesterol by treatment with LPDS led to an approximately 2.4 fold increase in glucocerebrosidase activity in the severely affected brother, as measured in vitro toward the artificial substrate 4-MUG, while it increased by only about 20 % the glucocerebrosidase activity in cells that were derived from the mildly affected brother, and barely had an effect on normal cells.

Table 2: Fold increase of glucocerebrosidase activity as a result of cholesterol reduction

Ce// line Fold increase of in-vitro glucocerebrosidase activity

Normal 1.1 ± 0.08

Mildly affected brother 1.2 ± 0.38

Severely affected brother 2.4 ± 0.49

EXAMPLE 7

Effect of mevastatin on glucocerebrosidase in the two GD brothers

In order to test whether the decrease in intracellular cholesterol level can be achieved by mevastatin, cells that originated from both brothers were treated with 20 (g/ml of mevastatin; Sigma, Israel) for 72 hours, after which endo-H sensitivity of glucocerebrosidase and enzymatic activity were tested. Mevastatin is an inhibitor of HMG CoA reductase, used as a drug to lower cholesterol level in patients that suffer from hypercholesterolemia [Endo A. et al., FEBS Lett. (1976) 72: 323-326; Yamamoto A. et al., Atherosclerosis (1980) 35: 259-266].

As presented in Figures 5A-C, tissue culture fibroblasts that originated from the severely affected brother demonstrated almost two-fold increase in endo-H resistant fraction of glucocerebrosidase as well as a two fold increase in its total amount following treatment with mevastatin, while normal cells and cells of the mildly affected brother were not significantly affected by the treatment.

EXAMPLE 8

Effect of Mevastatin on Glucocerebrosidase in other GD patients

In order to ascertain whether the results of Example 7 were generally applicable, the effect of mevastatin was tested on cells that were derived from two other patients, homozygous for the N370S mutation (Tsuji et al. (1985) Proc Natl Acad Sci U S A 7:2349-2352). This is a mild mutation typically associated with a mild form of type 1 Gaucher disease. However, one patient presented a severe type 1 Gaucher disease while the other one was very mild. Cells of both patients were treated with mevastatin (20 mg/ml for 72 hours) after which the total amount of glucocerebrosidase, its endo-H resistant fraction as well as its activity were measured. Results summarized in Figures 6A-C clearly indicated that lowering intracellular cholesterol level by treatment with mevastatin increased the total amount of glucocerebrosidase, its endo-H resistant fraction (Figure 6A-B) and its activity (results not shown) in cells of the severely affected patient, while it had no significant effect on cells of the mildly affected patient. These results confirmed the general applicability of the principles demonstrated in Example 7. Results presented in Examples 7 and 8 clearly indicated that in patients whose ER associated degradation of glucocerebrosidase is more excessive due to high levels of intracellular cholesterol, treatment with an HMG CoA reductase inhibitor leads to a significant increase in enzymatic activity. This, in turn, should lead to improvement in phenotypic expression of the disease.

EXAMPLE 9 Effect of Mevastatin on Glucocerebrosidase in other GD patients

Next, the effect of cholesterol trafficking on GCD activity was measured. Cells were treated with 12 μg/ml of the amphiphatic amine U18666A (Sigma, Israel; Sparrow et al., (1999)

Neurochem Res 24, 69-77), which impairs normal cholesterol trafficking, and its effect on glucocerebrosidase activity was tested. As shown in Figures 7A-C, treatment with U18666A led to an increase in the endo-H sensitive fraction of mutant as well as normal glucocerebrosidase, and led to a decrease in enzymatic activity. Taken together, these results suggest that abnormal intracellular cholesterol trafficking leads to a decrease in glucocerebrosidase enzymatic activity of mutant and even normal glucocerebrosidase due to its retention in the ER.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, and patent applications and sequences identified by their GenBank accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, or patent application or sequence identified by its GenBank accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:

1. Use of a cholesterol synthesis inhibitor for the manufacture of a medicament identified for treating a sphingolipid storage disease (SLSD) in a subject in need thereof.

2. A method of treating a sphingolipid storage disease (SLSD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a cholesterol synthesis inhibitor, thereby inhibiting the sphingolipid storage disease in the subject.

3. A method of treating a sphingolipid storage disease (SLSD) in a subject in need thereof, the method comprising:

(a) determining in the subject at least one of an activity level of an enzyme associated with the SLSD and an intracellular cholesterol level; and

(b) treating the subject with a cholesterol synthesis inhibitor according to the result of step (a), thereby treating the sphingolipid storage disease.

4. The use or methods of any of claims 1 , 2 or 3, wherein the sphingolipid storage disease (SLSD) comprises Gaucher's disease.

5. The use or methods of any of claims 1 , 2 or 3, wherein the sphingolipid storage disease (SLSD) is selected from the group consisting of Gaucher's Disease, Niemann-Pick Disease, Krabbe's Disease (Globoid leukodystrophy), Metachromatic leukodystrophy, Ceramide lactoside lipidosis, Fabry's Disease, Tay-Sachs Disease, Sandhoff s Disease and Landing's Disease.

6. The use or methods of any of claims 1 , 2 or 3, wherein the cholesterol synthesis inhibitor comprises an inhibitor of HMG CoA-reductase.

7. The use or methods of claim 6, wherein the inhibitor of HMG CoA-reductase comprises a statin.

8. The use or methods of claim 7, wherein said statin comprises mevastatin.

9. The use or methods of claim 7, wherein said statin is selected from the group consisting of mevastatin, lovastatin, pravastatin, fluvastatin, simvastatin, rosuvastatin, cerivastatin and atorvastatin.

10. The use or methods of any of claims 1 , 2 or 3, wherein the subject has Gaucher's disease and a glucocerebrosidase enzymatic activity at least below 30 % of that of a normal subject free of said Gaucher's disease.

11. The use or methods of any of claims 1 , 2 or 3, wherein the subject has at least 30 % higher cholesterol level than a normal subject not having the sphingolipid storage disease.

12. The use or methods of any of claims 1 , 2 or 3, further comprising administering to the subject a therapeutically effective amount of an agent capable of treating the sphingolipid storage disease.