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A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease
Molecular Genetics and Metabolism 91 (2007) 259–267 www.elsevier.com/locate/ymgme
A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease Kerry Anne McEachern a, John Fung a, Svetlana Komarnitsky a, Craig S. Siegel a, Wei-Lien Chuang a, Elizabeth Hutto a, James A. Shayman b, Gregory A. Grabowski c, Johannes M.F.G. Aerts d, Seng H. Cheng a,*, Diane P. Copeland a, John Marshall a a
Genzyme Corporation, 31 New York Avenue, Framingham, MA 01701-9322, USA b University of Michigan, Ann Arbor, MI 48103, USA c Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH 45229, USA d University of Amsterdam, Amsterdam, The Netherlands
Received 26 January 2007; received in revised form 3 April 2007; accepted 4 April 2007 Available online 16 May 2007
Abstract 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 (IC50 24 nM) 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. 2007 Elsevier Inc. All rights reserved. Keywords: Lysosomal storage disorders; Gaucher disease; Glucocerebrosidase; Substrate inhibition therapy; Glycosphingolipids; Glucosylceramide; Gangliosides
Introduction Gaucher disease is the most common of the glycosphingolipidoses, a group of rare inborn errors of metabolism that result from the deficiency of catabolic enzymes involved in the degradation of a variety of glycolipids [1,2]. Gaucher disease is caused by mutations in the gene encoding glucocerebrosidase with consequent abnormal lysosomal accumulation of glucosylceramide in histiocytes. This leads to characteristic and often severe pathology including hepatosplenomegaly, anemia, thrombocytopenia, and bone disease [3]. *
Corresponding author. Fax: +1 508 872 4091. E-mail address: [email protected] (S.H. Cheng).
1096-7192/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2007.04.001
Most patients with Gaucher disease type 1 are presently effectively managed by enzyme replacement therapy (ERT) using a glycan-modified, recombinant human glucocerebrosidase [3,4]. Systemic administration of the recombinant enzyme reduces the burden of accumulated glucosylceramide in affected cells, corrects the pathology in the visceral organs and improves the clinical sequelae. However, treatment is for a lifetime, requiring patients to have regular and frequent intravenous infusions of enzyme, with attendant impact on their quality of life [4]. Systemic administration of glucocerebrosidase also does not address the CNS disease in the neuronopathic forms of Gaucher disease (types 2 and 3) [5,6]. Bone marrow transplantation has been evaluated for these Gaucher patients, but requires HLA-matched donors
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and results have been mixed. Gene therapy shows promise for long-term efficacy, but gene therapy for systemic disorders has yet to be proven in humans [7,8]. Substrate inhibition therapy, also sometimes referred to as substrate reduction or deprivation therapy, represents an alternative, therapeutic strategy for Gaucher disease [8–10]. This strategy seeks to abate the accumulation of glucosylceramide through inhibition of glucosylceramide synthase, the enzyme that catalyzes the synthesis of the offending substrate [10]. As glucosylceramide synthesis is the rate-limiting first step in the glycosphingolipid biosynthetic pathway, inhibiting this synthase also has the potential for treating several other glucosylceramide-based glycosphingolipidoses such as Fabry, Sandhoff and Tay-Sachs disease [11]. However, substrate inhibition therapy is predicted to be most effective for treating type 1 Gaucher disease as the residual enzymatic activity in these subjects is proportionately higher than in the other glycosphingolipidoses. Therefore, in subjects who retain sufficient amounts of residual glucocerebrosidase activity, substrate inhibition therapy might not only arrest further accumulation but also allow a decrease in overall substrate levels [10–12]. Substrate inhibition therapy with the iminosugar, Nbutyl-deoxynojirimycin (NB-DNJ) was recently approved for treating patients with Gaucher disease type 1 for whom ERT is not a therapeutic option (e.g., allergy, hypersensitivity, or poor venous access) [13]. However, NB-DNJ exhibits inhibition of other glucosidases and intestinal glycosidases that may account, at least in part, for the observed gastrointestinal irritation including diarrhea, as well as peripheral neuropathy [14–16]. To address these potential limitations, we evaluated the therapeutic activity of other small molecule inhibitors of glucosylceramide synthase, particularly those based around 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) [17–22]. Until recently, the evaluation of experimental therapies for Gaucher disease has been hampered by the lack of an appropriate animal model. Attempts using gene knockout technology generated mice that were either non-viable or that lacked significant tissue accumulation of glucosylceramide or the appearance of Gaucher cells [23,24]. However, a recent attempt to generate a murine model of Gaucher disease harboring the D409V mutation in the mouse GC locus was successful [25]. This heterozygous mouse, gbaD409V/null, exhibits a normal life span, approximately 5–10% of normal GC activity in visceral tissues, and by 4 months of age, measurable accumulation of glucosylceramide-engorged macrophages (Gaucher cells) in the liver, spleen and lung. We report here our findings on the activity of an analog of PDMP (Genz-112638) at effecting substrate inhibition therapy in the gbaD409V/null mouse. We show that daily oral administration of the drug was well tolerated and effective in abating the lysosomal accumulation of glucosylceramide and the formation of Gaucher cells.
Materials and methods Biochemical characterization of Genz-112638 Genz-112638 is a synthetic small molecule designed to inhibit the enzymatic activity of glucosylceramide synthase [20]. It is a structural homologue of D-threo-ethylendioxyphenyl-2-palmitoylamino-3-pyrrilidino-propanol formulated as a tartrate salt. The chemical structure of Genz-112638 and the procedures for its synthesis are described elsewhere [26]. The inhibitory activity of Genz-112638 was determined indirectly by measuring its effect on the cell surface levels of the gangliosides GM1 and GM3 on either K562 or B16/F10 cells. The human erythroleukemic K562 and murine melanoma B16/F10 cells were obtained from the American Type Culture Collection (ATCC) and were cultured under standard conditions. GM1 levels on the K562 cells were determined by incubating the cells with increasing amounts of Genz-112638 (0.6–1000 nM) for 72 h after which the cells were harvested and stained using 10 lg of recombinant cholera toxin-FITC (Sigma, St. Louis, MO) in 100 ll phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) for 30 min on ice. Cells were washed, resuspended in PBS containing 0.5% BSA and the fluorescence quantitated using either a FACS Vantage or a FACS Aria flow cytometer. Non-viable or compromised cells were eliminated from the analysis by dye exclusion with propidium iodide (Sigma. St. Louis, MO). Fluorescence was quantitated for inter-assay variability using beads with known Molecules of Equivalent Soluble Fluorophores (MESF) (Bangs Laboratories, Fisher, IN). GM3 levels on the B16/F10 cells were measured by culturing the cells in the presence Genz-112638 (0.6–1000 nM) for 72 h after which the cells were harvested and resuspended in 50 ll of a 10 lg/ml stock of anti-GM3 monoclonal antibody (CosmoBio, Tokyo, Japan). After incubating for 30 min on ice, the cells were washed with PBS containing 0.5% BSA, incubated with goat-antimouse IgM (H+L)-FITC (Jackson, West Grove, PA), re-washed and resuspended in PBS containing 0.5% BSA, and analyzed as for GM1 above. The specificity of Genz-112638 was evaluated by testing its ability to inhibit a number of enzymes. The intestinal glycosidase enzymes were assayed in rat tissue homogenates [27], and the glycogen debranching enzyme was assayed in a cell free assay as described [28]. Non-lysosomal glucosylceramidase and lysosomal glucocerebrosidase were assayed in intact human cells using C6-NBD-glucosylceramide as substrate [29]. Conduritol b epoxide (a specific inhibitor of lysosomal glucocerebrosidase) was used to differentiate lysosomal versus the non-lysosomal activity. Glucocerebrosidase activity was also measured by fluorescence-activated cell sorting (FACS). K562 cells were cultured with increasing amounts of Genz-112638 in the presence of 1 lM 5-(pentafluorobenzoylamino)-fluorescein di-b-D-glucopyranoside (PFB-FDGlu, Molecular Probes/Invitrogen, Carlsbad, CA) for 30–60 min. Cells were immediately chilled on ice and the fluorescence quantitated as above.
Animal studies Procedures involving animals were reviewed and approved by an Institutional animal care and use committee (IACUC) following Association for assessment and accreditation of laboratory animal care (AAALAC), State and Federal guidelines. The Gaucher gbaD409V/null mice [25] were allowed to mature according to study requirements. No difference in phenotype or response to Genz-112638 has been found between males and females, so both sexes were used in the studies. Genz-112638 delivery was by a single daily oral gavage at a volume of 10 ml/kg. Animals were acclimated to oral gavaging with a similar volume of water for one week prior to initiation of treatment. Genz-112638 was dissolved in Water For Injection (WFI; VWR, West Chester, PA) and administered in a dose escalation from 75 mg/kg/day to 150 mg/kg/day over the course of nine days, with three days at each dose and increments of 25 mg/kg/day. Mice were weighed three times per week to monitor the potential impact of the drug on their overall health. Animals were killed by carbon dioxide inhalation
K.A. McEachern et al. / Molecular Genetics and Metabolism 91 (2007) 259–267 and their tissues harvested immediately. Half of each tissue was snap frozen on dry ice and stored at 80 C until ready for further processing. The other half was collected for histological analysis.
Quantitation of tissue glucosylceramide levels by high performance thin layer chromatography High performance thin layer chromatography (HP-TLC) analyses were as described [22,30,31]. Briefly, a total lipid fraction was obtained by homogenizing tissue in cold PBS, extracting with 2:1 (v/v) chloroform:methanol, and sonicating in a water bath sonicator. Samples were centrifuged to separate the phases and the supernatant was recovered. The pellets were re-sonicated in chloroform:methanol:saline, centrifuged and the resulting second supernatant was collected and combined with the first. A 1:1 (v/v) chloroform:saline mixture was added to the combined supernatants, vortexed, and centrifuged. After discarding the upper aqueous layer, methanol:saline was added, vortexed and re-centrifuged. The organic phase was taken and dried under nitrogen, dissolved in 2:1 (v/v) chloroform:methanol at 1 ml per 0.1 g original tissue weight and stored at 20 C. A portion of the lipid extract was used to measure total phosphate [32], i.e., the phospholipid content, to use as an internal standard. The remainder underwent alkaline methanolysis to remove phospholipids that migrate with glucosylceramide on the HP-TLC plate. Aliquots of the extracts containing equivalent amounts of the total phosphate were spotted onto a HP-TLC plate along with known glucosylceramide standards (Matreya inc., Pleasant Gap, PA). The lipids were resolved and visualized with 3% cupric acetate monohydrate (w/v), 15% phosphoric acid (v/v) followed by baking for 10 min at 150 C. The lipid bands were scanned on a densitometer (GS-700, Bio-Rad, Hercules, CA) and analyzed by Quantity One software (Bio-Rad).
Quantitation of tissue glucosylceramide levels by mass spectrometry Glucosylceramide was quantified by mass spectrometry as described [33,34]. Tissue was homogenized in 2:1 (v/v) chloroform:methanol and incubated at 37 C. Samples were centrifuged and the supernatants were extracted with 0.2 volumes of water overnight. The samples were centrifuged again, the aqueous phase was discarded, and the organic phase dried down to a film under nitrogen. For electrospray ionization mass spectrometry (ESI/MS) analysis, tissue samples were reconstituted to the equivalent of 50 ng original tissue weight in 1 ml chloroform/methanol (2:1, v/v) and vortexed for 5 min. Aliquots of each sample (40 ll) were delivered to Waters total recovery vials and 50 ll of a 10 lg/ml d3-C16-GL-1 internal standard (Matreya, Inc., Pleasant Gap, PA) was added. Samples were dried under nitrogen and reconstituted with 200 ll of 1:4 DMSO:methanol. ESI/MS analysis of glucosylceramides of different carbon chain lengths was performed on a Waters alliance HPLC (Separation Module 2695) coupled to a Micromass Quattro Micro system equipped with an electrospray ion source. Twenty microliter lipid extract samples were injected on a C8 column (4 ml · 3 mm i.d; Phenomenex, Torrance, CA) at 45 C and eluted with a gradient of 50–100% acetonitrile (2 mM ammonium acetate, 0.1% formic acid) at 0.5 ml/min. The first 0.5 min are held at 50% organic and then quickly switched to 100% for the final 3.5 min. The source temperature was held constant at 150 C and nitrogen was used as the desolvation gas at a flow rate of 670 L/h. The capillary voltage was maintained at 3.80 KV with a cone voltage of 23 V, while the dwell time for each ion species was 100 ms. Spectra were acquired by the MRM mode to monitor eight dominant isoforms (C16:0, C18:0, C20:0, C22:1, C22:0, C22:1-OH, C24:1, and C24:0). Quantitation of glucosylceramide is based on the sum of these eight isoforms to the internal standard, with a calibration curve range from 0.1 to 10 lg/mL.
Histology For histological analysis, tissues were fixed in zinc formalin (Electron Microscopy Sciences, Hatfield, PA) at room temperature for 24 h, then stored in PBS at 4 C until ready for further processing. All samples were
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dehydrated in ascending concentrations of alcohol, cleared in xylenes and infiltrated and embedded in Surgipath R paraffin (Surgipath, Richmond, IL). Five micron sections were cut using a rotary microtome and dried in a 60 C oven prior to staining. Sections were deparaffinized in xylenes, and rehydrated in descending concentrations of alcohol followed by a water wash. After a 1 min rinse in 3% acetic acid, slides were stained for 40 min in 1% Alcian Blue 8GX (Electron Microscopy Sciences) in 3% acetic acid pH 2.0. After rinsing in water and oxidizing in 1% periodic acid for 1 min, slides were stained with Schiff’s reagent (Surgipath) for 12 min. After washing for 5 min in hot water, the slides were dehydrated in alcohol and cleared in xylenes prior to mounting with SHUR/Mount coverglass mounting medium (TBS, Durham, NC). Gaucher cells identified morphologically in the liver were quantified using a manual cell count per 10 high power fields (HPFs, 400·).
Results Activity of Genz-112638 at inhibiting glycosphingolipid synthesis in vitro Two assays were used to quantify the inhibitory activity of Genz-112638 for glucosylceramide synthase. Since glucosylceramide is the first and rate-limiting step in the biosynthesis of glycosphingolipids, a flow cytometry assay that measured cell surface levels of GM1 and GM3 was used to indirectly assess the activity of the inhibitor in intact cells. Incubating K562 or B16/F10 cells for 72 h with increasing amounts of Genz-112638 (0.6–1000 nM) resulted in a dose-dependent reduction of cell surface levels of both GM1 and GM3. The mean IC50 value for inhibiting the cell surface presentation of GM1 in K562 cells was 24 nM (range 14–34 nM) (Table 1) and that for GM3 in B16/F10 cells was 29 nM (range 12–48 nM). No overt cellular toxicity was noted in either cell line even when tested at the highest dose. An alternative assay that measured intracellular levels of glucosylceramide was also used. In this assay, K562 cells were incubated with increasing amounts of Genz-112638 (0–1000 nM) for 3 days after which the cells were lysed and total cellular glucosylceramide levels determined using HP-TLC. The mean IC50 value for inhibiting glucosylceramide synthesis was 40 nM. This value was similar to those obtained above for GM1 and GM3 and suggests that measurements of these cell surface glycolipids are good surrogates of the activity of Genz-112638 for glucosylceramide synthase.
Table 1 Biochemical activities of Genz-112638 in vitro Substrate inhibition potency (in vitro IC50)
0.024 lM
Enzyme specificities, IC50: a-Glucosidase I and II Lysosomal glucocerebrosidase (GBA1) Non-lysosomal glucosylceramidase (GBA2) Glycogen debranching enzyme
>2500 lM >2500 lM 1600 lM >2500 lM
Enzyme specificities, Ki: Sucrase inhibition Maltase inhibition
No inhib. to 10 lM No inhib. to 10 lM
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Specificity of substrate synthesis inhibition by Genz-112638 Previous studies of an imino sugar-based inhibitor (NBDNJ) of glucosylceramide synthase showed inhibition of other enzymes including the a-glucosidases I and II located in endoplasmic reticulum, and the digestive saccharases (maltase, lactase and sucrase) [35]. The specificity of Genz-112638 was evaluated in a series of in vitro cell-based and cell-free assays. No detectable inhibition of intestinal glycosidases (lactase, maltase, sucrase), a-glucosidase I and II, and the cytosolic debranching enzyme (a-1,6-glucosidase), was found at concentrations up to 2500 lM (Table 1). The non-lysosomal glucosylceramidase was weakly inhibited with an IC50 of 1600 lM. There was no inhibition of lysosomal glucocerebrosidase, the enzyme that is deficient in Gaucher disease, up to the highest concentration of 2500 lM (Table 1). Hence, a differential of approximately 40,000 in the concentration was required to inhibit glucosylceramide synthase compared to any of the other enzymes tested. This suggests that Genz-112638 should display improved specificity in human clinical studies, at least when compared to NB-DNJ. Effect of administering of Genz-112638 to D409V/null mice The effect of administering Genz-112638 to D409V/null mice was assessed. Approximately 7-month-old mice were administered 150 mg/kg/day Genz-112638 (a dose shown in preliminary studies to be effective at inhibiting glucosylceramide synthase) by oral gavage for 10 weeks. This treatment had no notable effects on the well-being or feeding habits of the mice. Measurements of their body weight throughout the study showed no significant deviation from those of untreated mice (Fig. 1) suggesting that Genz112638 was well tolerated at a dose shown to be effective at inhibiting the synthase.
Fig. 1. Effect of treatment with Genz-112638 on body weight. The body weights of D409V/null mice treated with 150 mg/kg/day Genz-112638 by oral gavage for 10 weeks were measured. Mice were weighed 3 times per week. Data are presented as means ± standard error of the mean (SEM) (n = 8).
Efficacy of Genz-112638 at treating young, pre-symptomatic Gaucher mice Genz-112638 was evaluated for abatement of the lysosomal accumulation of glucosylceramide and the appearance of Gaucher cells in young (10-week old) D409V/null mouse. These young Gaucher mice exhibit low levels of GL-1 in the affected tissues. Ten-week-old animals were administered either 75 or 150 mg/kg/day of Genz-112638 by oral gavage for 10 weeks. Measurement of glucosylceramide levels showed a dose-dependent reduction when compared to age-matched vehicle-treated controls. In the cohort that had been treated with 150 mg/kg/day, glucosylceramide levels were 60, 40 and 75% of those in the controls, in the liver, lung and spleen, respectively (Fig. 2). The statistically significantly lower levels of glucosylceramide observed in the liver and lung of treated D409V/null mice indicated that Genz-112638 was effective at reducing the accumulation of this glycosphingolipid in these tissues. Histopathological evaluation of the livers of untreated D409V/null mice at the end of the study (20 weeks of age) showed the presence of Gaucher cells throughout the liver (Fig. 3B). Mice treated with 150 mg/kg/day of Genz-112638 for 10 weeks showed only the occasional presence of Gaucher cells that were also invariably smaller in size (Fig. 3C). Quantitation of these cells in a number of different sections confirmed that the frequency of Gaucher cells were significantly lower in the Genz-112638-treated mice (Fig. 3D). Together, these biochemical and histological findings suggested that daily oral administration of Genz-112638 to pre-symptomatic Gaucher mice was effective at decreasing the accumulation of glucosylceramide in the affected tissues and the consequent formation of Gaucher cells in the liver.
Fig. 2. Efficacy of Genz-112638 in young D409V/null mice. Genz-112638 was administered to 10-week-old D409V/null mice daily by oral gavage at a dose of 75 or 150 mg/kg for 10 weeks. Glucosylceramide levels in liver, lung and spleen were evaluated at the end of the study by HP-TLC. Data are presented as a percentage of GL-1 in untreated age-matched control mice. Dashed lines indicate glucosylceramide levels observed in normal wild type mice. *p < 0.05; **p < 0.01 relative to untreated control (twotailed, unpaired t-test). Data are represented as means ± standard error of the mean (SEM) (n = 5 for 75 mg/kg; n = 6 for 150 mg/kg).
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Fig. 3. Histopathological evaluation of liver sections from young D409V/null mice administered Genz-112638. Tissues from young D409V/null mice treated with 150 mg/kg Genz-112638 for 10 weeks in the study described in Fig. 3 were processed for histological evaluation. Gaucher cells (indicated by arrows) were observed using an Alcian Blue/periodic acid Schiff stain. Representative liver sections from wild-type (A), untreated D409V/null (B) and Genz-112638-treated D409V/null mice (C) are shown. Quantitative Gaucher cell counts were performed on the liver sections (D). *p < 0.05 relative to untreated control (two-tailed, unpaired t-test). Data are expressed as means ± standard error of the mean (SEM) (n = 10 fields).
Efficacy of Genz-112638 in treating older Gaucher mice with pre-existing pathology The efficacy of Genz-112638 at arresting or reversing disease progression in older, symptomatic Gaucher mice was also evaluated. Seven-month old D409V/null mice were administered 150 mg/kg/day Genz-112638 by oral gavage for 10 weeks. Analyses of glucosylceramide levels in the liver, lung and spleen of treated mice at 5 and 10 weeks post-treatment showed they had not increased beyond those observed at the start of the study (Fig. 4). After 10 weeks of treatment, glucosylceramide levels were determined to be 60% lower in liver (Fig. 4A), 50% lower in lung (Fig. 4B) and 40% lower in spleen (Fig. 4C) than in vehicle-treated mice. These results showed that Genz112638 was effective at inhibiting the further accumulation of glucosylceramide in mice with an existing burden of storage pathology. Histopathological analysis of tissue sections showed a reduced number of Gaucher cells in the liver of treated
D409V/null mice when compared to untreated controls (Figs. 5B and C). Quantitation of the number of Gaucher cells corroborated the biochemical findings (Fig. 5D); treated D409V/null mice displayed Gaucher cell counts that were not significantly different from those at the beginning of treatment at both the 5- and 10-week time points. Gaucher cell numbers at both these time points were significantly lower than those of untreated D409V/null mice. Together, these data demonstrate that Genz-112638 effectively inhibited further glucosylceramide accumulation and Gaucher cell development in animals with pre-existing pathology. Discussion Gaucher disease is the first lysosomal storage disorder for which an ERT was successfully developed to treat the visceral manifestations of the disease [4,36,37]. However, in a small proportion of patients, ERT is less than a favorable therapeutic option (e.g., those hypersensitive to the
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Fig. 4. Effect of Genz-112638 treatment in mature D409V/null mice. 7month-old Gaucher mice were administered 150 mg/kg Genz-112638 daily by oral gavage for up to 10 weeks. Glucosylceramide levels were evaluated in liver (A), lung (B) and spleen (C) at baseline and after 5 and 10 weeks of treatment. *p < 0.05; **p < 0.01; ***p < 0.001 relative to age-matched untreated control (two-tailed, unpaired t-test). Data shown correspond to means ± standard error of the mean (SEM) (n = 8).
enzyme or having poor venous access) [38], or is not effective (neuronopathic Gaucher disease types 2 and 3) [39]. Alternative therapies have been investigated, including bone marrow transplantation [40,41], substrate inhibition therapy [42], genetically modified autologous stem cell therapy [43] and systemic gene therapy [33]. Currently, stem cell and gene therapy rely on as yet unproven technology for treating lysosomal storage disorders, and bone marrow transplantation has associated donor and morbidity issues. However, substrate inhibition therapy has the potential to provide benefit where there is a remaining clinical need for Gaucher patients.
The hypothesis behind substrate inhibition therapy is that a reduction in the rate of substrate synthesis will result in an attenuation of disease progression. Here, the efficacy of this approach is demonstrated for the first time in a mouse model of Gaucher disease. NB-DNJ is an approved drug for substrate inhibition therapy of type 1 Gaucher disease patients who cannot be treated by intravenous infusions of recombinant glucocerebrosidase. However, the NB-DNJ treatment regimen is associated with some side effects in patients [42], which may be partially due to the relative promiscuity of the drug for other enzymes [35], especially glycosidases. Genz-112638 is a molecule that was designed to mimic the transition state between the combination of UDP-glucose and ceramide and the resulting glucosylceramide [20]. As expected, it demonstrated a higher degree of specificity for the enzyme glucosylceramide synthase resulting in directed competitive inhibition. There was also no measurable inhibition of glucocerebrosidase activity at the effective dose, which is an important feature when treating Gaucher disease type 1 patients, the majority of whom retain residual glucocerebrosidase activity. At the effective dose of 150 mg/kg/day, there were no observable gastro-intestinal issues and there was no difference in body weights between the treated and control untreated groups. Serum concentrations at and above the IC50 (24–40 nM) were readily attainable with oral doses that were below the maximum tolerated level. Genz112638 also was readily metabolized and cleared; both parent compound and metabolites effectively cleared within 24 h as shown in single and repeat oral dose ADME studies with 14C-radiolabelled compound in rats and dogs (data not shown). Using a non-optimized dosing regimen of a single daily oral gavage successfully prevented glucosylceramide accumulation in both young, pre-symptomatic mice and in older Gaucher mice that already exhibited storage pathology. The young, 10-week old mice, although harboring elevated glucosylceramide levels relative to wildtype controls, had not yet developed the characteristic engorged tissue macrophages, termed Gaucher cells. Treatment with 150 mg/kg/day of Genz-112638 halted all measurable disease progression and inhibited the development of Gaucher cells. In older mice exhibiting a higher level of lysosomal glucosylceramide and number of Gaucher cells, there was no further increase in the levels of the glycosphingolipid or in the number of storage cells after either 5 weeks or 10 weeks of treatment. As the major source of glucosylceramide in Gaucher cells is reported to be extracellular in origin these results implied that Genz-112638 inhibition of glucosylceramide synthase was systemic. The observation that Genz-112638 was effective in preventing further accumulation of glucosylceramide suggests a therapeutic strategy that could further enhance the treatment of Gaucher disease. A combination therapy could be envisaged wherein patients would be debulked of accumulated substrate using ERT followed by a maintenance treat-
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Fig. 5. Histopathological assessment of liver sections from older Gaucher mice following treatment with Genz-112638. Tissues from D409V/null mice in the study described in Fig. 5 were processed for histological evaluation. An Alcian Blue/periodic acid Schiff stain was used to visualize Gaucher cells (indicated by arrows). Representative sections from the livers of wild type (A), untreated D409V/null (B) and Genz-112638 treated D409V/null mice (C) at the 10-week time point are shown. Gaucher cells were quantified in these liver sections (D). *p < 0.05; **p < 0.01 relative to age-matched untreated control (two-tailed, unpaired t-test). Data are presented as means ± standard error of the mean (SEM) (n = 10 fields).
ment with substrate inhibition therapy. An important observation for a potential combination therapy was that in a cell free assay, glucocerebrosidase was not inhibited by Genz-112638 at concentrations up to 2500 lM. Such a combination therapy would provide for less frequent enzyme infusions that in turn should improve the quality of care for the patients. A further projection could be made about the utility of substrate inhibition therapy based on the observation that the synthesis of glucosylceramide is the first step in the glycosphingolipid biosynthetic pathway. By reducing glucosylceramide synthesis, there should be an effect on the levels of more complex glycosphingolipids [27,30] that are associated with other lysosomal storage disorders. Hence, substrate inhibition therapy using Genz-112638 may be effective for not only Gaucher disease but also other diseases such as Fabry [21]. In summary, the data presented here demonstrated that Genz-112638 is an active and specific inhibitor of glucosyl-
ceramide synthase exhibiting no overt adverse effects in a mouse model of Gaucher disease. It successfully prevented disease progression in both pre-symptomatic and older diseased Gaucher mice by inhibiting glucosylceramide accumulation and Gaucher cell formation. These findings suggest that Genz-112638 may represent yet another therapeutic option for both pediatric and adult Gaucher type 1 disease and potentially other glycosphingolipid storage disorders. Acknowledgments The authors thank Dr. Ron Scheule for his help with experimental design, discussion and critical reading of this manuscript, Leah Curtin and the Department of Comparative Medicine at Genzyme for animal welfare and technical assistance, Jennifer Johnson and Sue Ryan for histology support and Joshua Pacheco for mass spectroscopy analysis.
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A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease Kerry Anne McEachern a, John Fung a, Svetlana Komarnitsky a, Craig S. Siegel a, Wei-Lien Chuang a, Elizabeth Hutto a, James A. Shayman b, Gregory A. Grabowski c, Johannes M.F.G. Aerts d, Seng H. Cheng a,*, Diane P. Copeland a, John Marshall a a
Genzyme Corporation, 31 New York Avenue, Framingham, MA 01701-9322, USA b University of Michigan, Ann Arbor, MI 48103, USA c Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH 45229, USA d University of Amsterdam, Amsterdam, The Netherlands
Received 26 January 2007; received in revised form 3 April 2007; accepted 4 April 2007 Available online 16 May 2007
Abstract 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 (IC50 24 nM) 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. 2007 Elsevier Inc. All rights reserved. Keywords: Lysosomal storage disorders; Gaucher disease; Glucocerebrosidase; Substrate inhibition therapy; Glycosphingolipids; Glucosylceramide; Gangliosides
Introduction Gaucher disease is the most common of the glycosphingolipidoses, a group of rare inborn errors of metabolism that result from the deficiency of catabolic enzymes involved in the degradation of a variety of glycolipids [1,2]. Gaucher disease is caused by mutations in the gene encoding glucocerebrosidase with consequent abnormal lysosomal accumulation of glucosylceramide in histiocytes. This leads to characteristic and often severe pathology including hepatosplenomegaly, anemia, thrombocytopenia, and bone disease [3]. *
Corresponding author. Fax: +1 508 872 4091. E-mail address: [email protected] (S.H. Cheng).
1096-7192/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2007.04.001
Most patients with Gaucher disease type 1 are presently effectively managed by enzyme replacement therapy (ERT) using a glycan-modified, recombinant human glucocerebrosidase [3,4]. Systemic administration of the recombinant enzyme reduces the burden of accumulated glucosylceramide in affected cells, corrects the pathology in the visceral organs and improves the clinical sequelae. However, treatment is for a lifetime, requiring patients to have regular and frequent intravenous infusions of enzyme, with attendant impact on their quality of life [4]. Systemic administration of glucocerebrosidase also does not address the CNS disease in the neuronopathic forms of Gaucher disease (types 2 and 3) [5,6]. Bone marrow transplantation has been evaluated for these Gaucher patients, but requires HLA-matched donors
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and results have been mixed. Gene therapy shows promise for long-term efficacy, but gene therapy for systemic disorders has yet to be proven in humans [7,8]. Substrate inhibition therapy, also sometimes referred to as substrate reduction or deprivation therapy, represents an alternative, therapeutic strategy for Gaucher disease [8–10]. This strategy seeks to abate the accumulation of glucosylceramide through inhibition of glucosylceramide synthase, the enzyme that catalyzes the synthesis of the offending substrate [10]. As glucosylceramide synthesis is the rate-limiting first step in the glycosphingolipid biosynthetic pathway, inhibiting this synthase also has the potential for treating several other glucosylceramide-based glycosphingolipidoses such as Fabry, Sandhoff and Tay-Sachs disease [11]. However, substrate inhibition therapy is predicted to be most effective for treating type 1 Gaucher disease as the residual enzymatic activity in these subjects is proportionately higher than in the other glycosphingolipidoses. Therefore, in subjects who retain sufficient amounts of residual glucocerebrosidase activity, substrate inhibition therapy might not only arrest further accumulation but also allow a decrease in overall substrate levels [10–12]. Substrate inhibition therapy with the iminosugar, Nbutyl-deoxynojirimycin (NB-DNJ) was recently approved for treating patients with Gaucher disease type 1 for whom ERT is not a therapeutic option (e.g., allergy, hypersensitivity, or poor venous access) [13]. However, NB-DNJ exhibits inhibition of other glucosidases and intestinal glycosidases that may account, at least in part, for the observed gastrointestinal irritation including diarrhea, as well as peripheral neuropathy [14–16]. To address these potential limitations, we evaluated the therapeutic activity of other small molecule inhibitors of glucosylceramide synthase, particularly those based around 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) [17–22]. Until recently, the evaluation of experimental therapies for Gaucher disease has been hampered by the lack of an appropriate animal model. Attempts using gene knockout technology generated mice that were either non-viable or that lacked significant tissue accumulation of glucosylceramide or the appearance of Gaucher cells [23,24]. However, a recent attempt to generate a murine model of Gaucher disease harboring the D409V mutation in the mouse GC locus was successful [25]. This heterozygous mouse, gbaD409V/null, exhibits a normal life span, approximately 5–10% of normal GC activity in visceral tissues, and by 4 months of age, measurable accumulation of glucosylceramide-engorged macrophages (Gaucher cells) in the liver, spleen and lung. We report here our findings on the activity of an analog of PDMP (Genz-112638) at effecting substrate inhibition therapy in the gbaD409V/null mouse. We show that daily oral administration of the drug was well tolerated and effective in abating the lysosomal accumulation of glucosylceramide and the formation of Gaucher cells.
Materials and methods Biochemical characterization of Genz-112638 Genz-112638 is a synthetic small molecule designed to inhibit the enzymatic activity of glucosylceramide synthase [20]. It is a structural homologue of D-threo-ethylendioxyphenyl-2-palmitoylamino-3-pyrrilidino-propanol formulated as a tartrate salt. The chemical structure of Genz-112638 and the procedures for its synthesis are described elsewhere [26]. The inhibitory activity of Genz-112638 was determined indirectly by measuring its effect on the cell surface levels of the gangliosides GM1 and GM3 on either K562 or B16/F10 cells. The human erythroleukemic K562 and murine melanoma B16/F10 cells were obtained from the American Type Culture Collection (ATCC) and were cultured under standard conditions. GM1 levels on the K562 cells were determined by incubating the cells with increasing amounts of Genz-112638 (0.6–1000 nM) for 72 h after which the cells were harvested and stained using 10 lg of recombinant cholera toxin-FITC (Sigma, St. Louis, MO) in 100 ll phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) for 30 min on ice. Cells were washed, resuspended in PBS containing 0.5% BSA and the fluorescence quantitated using either a FACS Vantage or a FACS Aria flow cytometer. Non-viable or compromised cells were eliminated from the analysis by dye exclusion with propidium iodide (Sigma. St. Louis, MO). Fluorescence was quantitated for inter-assay variability using beads with known Molecules of Equivalent Soluble Fluorophores (MESF) (Bangs Laboratories, Fisher, IN). GM3 levels on the B16/F10 cells were measured by culturing the cells in the presence Genz-112638 (0.6–1000 nM) for 72 h after which the cells were harvested and resuspended in 50 ll of a 10 lg/ml stock of anti-GM3 monoclonal antibody (CosmoBio, Tokyo, Japan). After incubating for 30 min on ice, the cells were washed with PBS containing 0.5% BSA, incubated with goat-antimouse IgM (H+L)-FITC (Jackson, West Grove, PA), re-washed and resuspended in PBS containing 0.5% BSA, and analyzed as for GM1 above. The specificity of Genz-112638 was evaluated by testing its ability to inhibit a number of enzymes. The intestinal glycosidase enzymes were assayed in rat tissue homogenates [27], and the glycogen debranching enzyme was assayed in a cell free assay as described [28]. Non-lysosomal glucosylceramidase and lysosomal glucocerebrosidase were assayed in intact human cells using C6-NBD-glucosylceramide as substrate [29]. Conduritol b epoxide (a specific inhibitor of lysosomal glucocerebrosidase) was used to differentiate lysosomal versus the non-lysosomal activity. Glucocerebrosidase activity was also measured by fluorescence-activated cell sorting (FACS). K562 cells were cultured with increasing amounts of Genz-112638 in the presence of 1 lM 5-(pentafluorobenzoylamino)-fluorescein di-b-D-glucopyranoside (PFB-FDGlu, Molecular Probes/Invitrogen, Carlsbad, CA) for 30–60 min. Cells were immediately chilled on ice and the fluorescence quantitated as above.
Animal studies Procedures involving animals were reviewed and approved by an Institutional animal care and use committee (IACUC) following Association for assessment and accreditation of laboratory animal care (AAALAC), State and Federal guidelines. The Gaucher gbaD409V/null mice [25] were allowed to mature according to study requirements. No difference in phenotype or response to Genz-112638 has been found between males and females, so both sexes were used in the studies. Genz-112638 delivery was by a single daily oral gavage at a volume of 10 ml/kg. Animals were acclimated to oral gavaging with a similar volume of water for one week prior to initiation of treatment. Genz-112638 was dissolved in Water For Injection (WFI; VWR, West Chester, PA) and administered in a dose escalation from 75 mg/kg/day to 150 mg/kg/day over the course of nine days, with three days at each dose and increments of 25 mg/kg/day. Mice were weighed three times per week to monitor the potential impact of the drug on their overall health. Animals were killed by carbon dioxide inhalation
K.A. McEachern et al. / Molecular Genetics and Metabolism 91 (2007) 259–267 and their tissues harvested immediately. Half of each tissue was snap frozen on dry ice and stored at 80 C until ready for further processing. The other half was collected for histological analysis.
Quantitation of tissue glucosylceramide levels by high performance thin layer chromatography High performance thin layer chromatography (HP-TLC) analyses were as described [22,30,31]. Briefly, a total lipid fraction was obtained by homogenizing tissue in cold PBS, extracting with 2:1 (v/v) chloroform:methanol, and sonicating in a water bath sonicator. Samples were centrifuged to separate the phases and the supernatant was recovered. The pellets were re-sonicated in chloroform:methanol:saline, centrifuged and the resulting second supernatant was collected and combined with the first. A 1:1 (v/v) chloroform:saline mixture was added to the combined supernatants, vortexed, and centrifuged. After discarding the upper aqueous layer, methanol:saline was added, vortexed and re-centrifuged. The organic phase was taken and dried under nitrogen, dissolved in 2:1 (v/v) chloroform:methanol at 1 ml per 0.1 g original tissue weight and stored at 20 C. A portion of the lipid extract was used to measure total phosphate [32], i.e., the phospholipid content, to use as an internal standard. The remainder underwent alkaline methanolysis to remove phospholipids that migrate with glucosylceramide on the HP-TLC plate. Aliquots of the extracts containing equivalent amounts of the total phosphate were spotted onto a HP-TLC plate along with known glucosylceramide standards (Matreya inc., Pleasant Gap, PA). The lipids were resolved and visualized with 3% cupric acetate monohydrate (w/v), 15% phosphoric acid (v/v) followed by baking for 10 min at 150 C. The lipid bands were scanned on a densitometer (GS-700, Bio-Rad, Hercules, CA) and analyzed by Quantity One software (Bio-Rad).
Quantitation of tissue glucosylceramide levels by mass spectrometry Glucosylceramide was quantified by mass spectrometry as described [33,34]. Tissue was homogenized in 2:1 (v/v) chloroform:methanol and incubated at 37 C. Samples were centrifuged and the supernatants were extracted with 0.2 volumes of water overnight. The samples were centrifuged again, the aqueous phase was discarded, and the organic phase dried down to a film under nitrogen. For electrospray ionization mass spectrometry (ESI/MS) analysis, tissue samples were reconstituted to the equivalent of 50 ng original tissue weight in 1 ml chloroform/methanol (2:1, v/v) and vortexed for 5 min. Aliquots of each sample (40 ll) were delivered to Waters total recovery vials and 50 ll of a 10 lg/ml d3-C16-GL-1 internal standard (Matreya, Inc., Pleasant Gap, PA) was added. Samples were dried under nitrogen and reconstituted with 200 ll of 1:4 DMSO:methanol. ESI/MS analysis of glucosylceramides of different carbon chain lengths was performed on a Waters alliance HPLC (Separation Module 2695) coupled to a Micromass Quattro Micro system equipped with an electrospray ion source. Twenty microliter lipid extract samples were injected on a C8 column (4 ml · 3 mm i.d; Phenomenex, Torrance, CA) at 45 C and eluted with a gradient of 50–100% acetonitrile (2 mM ammonium acetate, 0.1% formic acid) at 0.5 ml/min. The first 0.5 min are held at 50% organic and then quickly switched to 100% for the final 3.5 min. The source temperature was held constant at 150 C and nitrogen was used as the desolvation gas at a flow rate of 670 L/h. The capillary voltage was maintained at 3.80 KV with a cone voltage of 23 V, while the dwell time for each ion species was 100 ms. Spectra were acquired by the MRM mode to monitor eight dominant isoforms (C16:0, C18:0, C20:0, C22:1, C22:0, C22:1-OH, C24:1, and C24:0). Quantitation of glucosylceramide is based on the sum of these eight isoforms to the internal standard, with a calibration curve range from 0.1 to 10 lg/mL.
Histology For histological analysis, tissues were fixed in zinc formalin (Electron Microscopy Sciences, Hatfield, PA) at room temperature for 24 h, then stored in PBS at 4 C until ready for further processing. All samples were
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dehydrated in ascending concentrations of alcohol, cleared in xylenes and infiltrated and embedded in Surgipath R paraffin (Surgipath, Richmond, IL). Five micron sections were cut using a rotary microtome and dried in a 60 C oven prior to staining. Sections were deparaffinized in xylenes, and rehydrated in descending concentrations of alcohol followed by a water wash. After a 1 min rinse in 3% acetic acid, slides were stained for 40 min in 1% Alcian Blue 8GX (Electron Microscopy Sciences) in 3% acetic acid pH 2.0. After rinsing in water and oxidizing in 1% periodic acid for 1 min, slides were stained with Schiff’s reagent (Surgipath) for 12 min. After washing for 5 min in hot water, the slides were dehydrated in alcohol and cleared in xylenes prior to mounting with SHUR/Mount coverglass mounting medium (TBS, Durham, NC). Gaucher cells identified morphologically in the liver were quantified using a manual cell count per 10 high power fields (HPFs, 400·).
Results Activity of Genz-112638 at inhibiting glycosphingolipid synthesis in vitro Two assays were used to quantify the inhibitory activity of Genz-112638 for glucosylceramide synthase. Since glucosylceramide is the first and rate-limiting step in the biosynthesis of glycosphingolipids, a flow cytometry assay that measured cell surface levels of GM1 and GM3 was used to indirectly assess the activity of the inhibitor in intact cells. Incubating K562 or B16/F10 cells for 72 h with increasing amounts of Genz-112638 (0.6–1000 nM) resulted in a dose-dependent reduction of cell surface levels of both GM1 and GM3. The mean IC50 value for inhibiting the cell surface presentation of GM1 in K562 cells was 24 nM (range 14–34 nM) (Table 1) and that for GM3 in B16/F10 cells was 29 nM (range 12–48 nM). No overt cellular toxicity was noted in either cell line even when tested at the highest dose. An alternative assay that measured intracellular levels of glucosylceramide was also used. In this assay, K562 cells were incubated with increasing amounts of Genz-112638 (0–1000 nM) for 3 days after which the cells were lysed and total cellular glucosylceramide levels determined using HP-TLC. The mean IC50 value for inhibiting glucosylceramide synthesis was 40 nM. This value was similar to those obtained above for GM1 and GM3 and suggests that measurements of these cell surface glycolipids are good surrogates of the activity of Genz-112638 for glucosylceramide synthase.
Table 1 Biochemical activities of Genz-112638 in vitro Substrate inhibition potency (in vitro IC50)
0.024 lM
Enzyme specificities, IC50: a-Glucosidase I and II Lysosomal glucocerebrosidase (GBA1) Non-lysosomal glucosylceramidase (GBA2) Glycogen debranching enzyme
>2500 lM >2500 lM 1600 lM >2500 lM
Enzyme specificities, Ki: Sucrase inhibition Maltase inhibition
No inhib. to 10 lM No inhib. to 10 lM
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Specificity of substrate synthesis inhibition by Genz-112638 Previous studies of an imino sugar-based inhibitor (NBDNJ) of glucosylceramide synthase showed inhibition of other enzymes including the a-glucosidases I and II located in endoplasmic reticulum, and the digestive saccharases (maltase, lactase and sucrase) [35]. The specificity of Genz-112638 was evaluated in a series of in vitro cell-based and cell-free assays. No detectable inhibition of intestinal glycosidases (lactase, maltase, sucrase), a-glucosidase I and II, and the cytosolic debranching enzyme (a-1,6-glucosidase), was found at concentrations up to 2500 lM (Table 1). The non-lysosomal glucosylceramidase was weakly inhibited with an IC50 of 1600 lM. There was no inhibition of lysosomal glucocerebrosidase, the enzyme that is deficient in Gaucher disease, up to the highest concentration of 2500 lM (Table 1). Hence, a differential of approximately 40,000 in the concentration was required to inhibit glucosylceramide synthase compared to any of the other enzymes tested. This suggests that Genz-112638 should display improved specificity in human clinical studies, at least when compared to NB-DNJ. Effect of administering of Genz-112638 to D409V/null mice The effect of administering Genz-112638 to D409V/null mice was assessed. Approximately 7-month-old mice were administered 150 mg/kg/day Genz-112638 (a dose shown in preliminary studies to be effective at inhibiting glucosylceramide synthase) by oral gavage for 10 weeks. This treatment had no notable effects on the well-being or feeding habits of the mice. Measurements of their body weight throughout the study showed no significant deviation from those of untreated mice (Fig. 1) suggesting that Genz112638 was well tolerated at a dose shown to be effective at inhibiting the synthase.
Fig. 1. Effect of treatment with Genz-112638 on body weight. The body weights of D409V/null mice treated with 150 mg/kg/day Genz-112638 by oral gavage for 10 weeks were measured. Mice were weighed 3 times per week. Data are presented as means ± standard error of the mean (SEM) (n = 8).
Efficacy of Genz-112638 at treating young, pre-symptomatic Gaucher mice Genz-112638 was evaluated for abatement of the lysosomal accumulation of glucosylceramide and the appearance of Gaucher cells in young (10-week old) D409V/null mouse. These young Gaucher mice exhibit low levels of GL-1 in the affected tissues. Ten-week-old animals were administered either 75 or 150 mg/kg/day of Genz-112638 by oral gavage for 10 weeks. Measurement of glucosylceramide levels showed a dose-dependent reduction when compared to age-matched vehicle-treated controls. In the cohort that had been treated with 150 mg/kg/day, glucosylceramide levels were 60, 40 and 75% of those in the controls, in the liver, lung and spleen, respectively (Fig. 2). The statistically significantly lower levels of glucosylceramide observed in the liver and lung of treated D409V/null mice indicated that Genz-112638 was effective at reducing the accumulation of this glycosphingolipid in these tissues. Histopathological evaluation of the livers of untreated D409V/null mice at the end of the study (20 weeks of age) showed the presence of Gaucher cells throughout the liver (Fig. 3B). Mice treated with 150 mg/kg/day of Genz-112638 for 10 weeks showed only the occasional presence of Gaucher cells that were also invariably smaller in size (Fig. 3C). Quantitation of these cells in a number of different sections confirmed that the frequency of Gaucher cells were significantly lower in the Genz-112638-treated mice (Fig. 3D). Together, these biochemical and histological findings suggested that daily oral administration of Genz-112638 to pre-symptomatic Gaucher mice was effective at decreasing the accumulation of glucosylceramide in the affected tissues and the consequent formation of Gaucher cells in the liver.
Fig. 2. Efficacy of Genz-112638 in young D409V/null mice. Genz-112638 was administered to 10-week-old D409V/null mice daily by oral gavage at a dose of 75 or 150 mg/kg for 10 weeks. Glucosylceramide levels in liver, lung and spleen were evaluated at the end of the study by HP-TLC. Data are presented as a percentage of GL-1 in untreated age-matched control mice. Dashed lines indicate glucosylceramide levels observed in normal wild type mice. *p < 0.05; **p < 0.01 relative to untreated control (twotailed, unpaired t-test). Data are represented as means ± standard error of the mean (SEM) (n = 5 for 75 mg/kg; n = 6 for 150 mg/kg).
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Fig. 3. Histopathological evaluation of liver sections from young D409V/null mice administered Genz-112638. Tissues from young D409V/null mice treated with 150 mg/kg Genz-112638 for 10 weeks in the study described in Fig. 3 were processed for histological evaluation. Gaucher cells (indicated by arrows) were observed using an Alcian Blue/periodic acid Schiff stain. Representative liver sections from wild-type (A), untreated D409V/null (B) and Genz-112638-treated D409V/null mice (C) are shown. Quantitative Gaucher cell counts were performed on the liver sections (D). *p < 0.05 relative to untreated control (two-tailed, unpaired t-test). Data are expressed as means ± standard error of the mean (SEM) (n = 10 fields).
Efficacy of Genz-112638 in treating older Gaucher mice with pre-existing pathology The efficacy of Genz-112638 at arresting or reversing disease progression in older, symptomatic Gaucher mice was also evaluated. Seven-month old D409V/null mice were administered 150 mg/kg/day Genz-112638 by oral gavage for 10 weeks. Analyses of glucosylceramide levels in the liver, lung and spleen of treated mice at 5 and 10 weeks post-treatment showed they had not increased beyond those observed at the start of the study (Fig. 4). After 10 weeks of treatment, glucosylceramide levels were determined to be 60% lower in liver (Fig. 4A), 50% lower in lung (Fig. 4B) and 40% lower in spleen (Fig. 4C) than in vehicle-treated mice. These results showed that Genz112638 was effective at inhibiting the further accumulation of glucosylceramide in mice with an existing burden of storage pathology. Histopathological analysis of tissue sections showed a reduced number of Gaucher cells in the liver of treated
D409V/null mice when compared to untreated controls (Figs. 5B and C). Quantitation of the number of Gaucher cells corroborated the biochemical findings (Fig. 5D); treated D409V/null mice displayed Gaucher cell counts that were not significantly different from those at the beginning of treatment at both the 5- and 10-week time points. Gaucher cell numbers at both these time points were significantly lower than those of untreated D409V/null mice. Together, these data demonstrate that Genz-112638 effectively inhibited further glucosylceramide accumulation and Gaucher cell development in animals with pre-existing pathology. Discussion Gaucher disease is the first lysosomal storage disorder for which an ERT was successfully developed to treat the visceral manifestations of the disease [4,36,37]. However, in a small proportion of patients, ERT is less than a favorable therapeutic option (e.g., those hypersensitive to the
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Fig. 4. Effect of Genz-112638 treatment in mature D409V/null mice. 7month-old Gaucher mice were administered 150 mg/kg Genz-112638 daily by oral gavage for up to 10 weeks. Glucosylceramide levels were evaluated in liver (A), lung (B) and spleen (C) at baseline and after 5 and 10 weeks of treatment. *p < 0.05; **p < 0.01; ***p < 0.001 relative to age-matched untreated control (two-tailed, unpaired t-test). Data shown correspond to means ± standard error of the mean (SEM) (n = 8).
enzyme or having poor venous access) [38], or is not effective (neuronopathic Gaucher disease types 2 and 3) [39]. Alternative therapies have been investigated, including bone marrow transplantation [40,41], substrate inhibition therapy [42], genetically modified autologous stem cell therapy [43] and systemic gene therapy [33]. Currently, stem cell and gene therapy rely on as yet unproven technology for treating lysosomal storage disorders, and bone marrow transplantation has associated donor and morbidity issues. However, substrate inhibition therapy has the potential to provide benefit where there is a remaining clinical need for Gaucher patients.
The hypothesis behind substrate inhibition therapy is that a reduction in the rate of substrate synthesis will result in an attenuation of disease progression. Here, the efficacy of this approach is demonstrated for the first time in a mouse model of Gaucher disease. NB-DNJ is an approved drug for substrate inhibition therapy of type 1 Gaucher disease patients who cannot be treated by intravenous infusions of recombinant glucocerebrosidase. However, the NB-DNJ treatment regimen is associated with some side effects in patients [42], which may be partially due to the relative promiscuity of the drug for other enzymes [35], especially glycosidases. Genz-112638 is a molecule that was designed to mimic the transition state between the combination of UDP-glucose and ceramide and the resulting glucosylceramide [20]. As expected, it demonstrated a higher degree of specificity for the enzyme glucosylceramide synthase resulting in directed competitive inhibition. There was also no measurable inhibition of glucocerebrosidase activity at the effective dose, which is an important feature when treating Gaucher disease type 1 patients, the majority of whom retain residual glucocerebrosidase activity. At the effective dose of 150 mg/kg/day, there were no observable gastro-intestinal issues and there was no difference in body weights between the treated and control untreated groups. Serum concentrations at and above the IC50 (24–40 nM) were readily attainable with oral doses that were below the maximum tolerated level. Genz112638 also was readily metabolized and cleared; both parent compound and metabolites effectively cleared within 24 h as shown in single and repeat oral dose ADME studies with 14C-radiolabelled compound in rats and dogs (data not shown). Using a non-optimized dosing regimen of a single daily oral gavage successfully prevented glucosylceramide accumulation in both young, pre-symptomatic mice and in older Gaucher mice that already exhibited storage pathology. The young, 10-week old mice, although harboring elevated glucosylceramide levels relative to wildtype controls, had not yet developed the characteristic engorged tissue macrophages, termed Gaucher cells. Treatment with 150 mg/kg/day of Genz-112638 halted all measurable disease progression and inhibited the development of Gaucher cells. In older mice exhibiting a higher level of lysosomal glucosylceramide and number of Gaucher cells, there was no further increase in the levels of the glycosphingolipid or in the number of storage cells after either 5 weeks or 10 weeks of treatment. As the major source of glucosylceramide in Gaucher cells is reported to be extracellular in origin these results implied that Genz-112638 inhibition of glucosylceramide synthase was systemic. The observation that Genz-112638 was effective in preventing further accumulation of glucosylceramide suggests a therapeutic strategy that could further enhance the treatment of Gaucher disease. A combination therapy could be envisaged wherein patients would be debulked of accumulated substrate using ERT followed by a maintenance treat-
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Fig. 5. Histopathological assessment of liver sections from older Gaucher mice following treatment with Genz-112638. Tissues from D409V/null mice in the study described in Fig. 5 were processed for histological evaluation. An Alcian Blue/periodic acid Schiff stain was used to visualize Gaucher cells (indicated by arrows). Representative sections from the livers of wild type (A), untreated D409V/null (B) and Genz-112638 treated D409V/null mice (C) at the 10-week time point are shown. Gaucher cells were quantified in these liver sections (D). *p < 0.05; **p < 0.01 relative to age-matched untreated control (two-tailed, unpaired t-test). Data are presented as means ± standard error of the mean (SEM) (n = 10 fields).
ment with substrate inhibition therapy. An important observation for a potential combination therapy was that in a cell free assay, glucocerebrosidase was not inhibited by Genz-112638 at concentrations up to 2500 lM. Such a combination therapy would provide for less frequent enzyme infusions that in turn should improve the quality of care for the patients. A further projection could be made about the utility of substrate inhibition therapy based on the observation that the synthesis of glucosylceramide is the first step in the glycosphingolipid biosynthetic pathway. By reducing glucosylceramide synthesis, there should be an effect on the levels of more complex glycosphingolipids [27,30] that are associated with other lysosomal storage disorders. Hence, substrate inhibition therapy using Genz-112638 may be effective for not only Gaucher disease but also other diseases such as Fabry [21]. In summary, the data presented here demonstrated that Genz-112638 is an active and specific inhibitor of glucosyl-
ceramide synthase exhibiting no overt adverse effects in a mouse model of Gaucher disease. It successfully prevented disease progression in both pre-symptomatic and older diseased Gaucher mice by inhibiting glucosylceramide accumulation and Gaucher cell formation. These findings suggest that Genz-112638 may represent yet another therapeutic option for both pediatric and adult Gaucher type 1 disease and potentially other glycosphingolipid storage disorders. Acknowledgments The authors thank Dr. Ron Scheule for his help with experimental design, discussion and critical reading of this manuscript, Leah Curtin and the Department of Comparative Medicine at Genzyme for animal welfare and technical assistance, Jennifer Johnson and Sue Ryan for histology support and Joshua Pacheco for mass spectroscopy analysis.
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