Báo cáo khoa học: The pharmacological chaperone isofagomine increases the activity of the Gaucher disease L444P mutant form of b-glucosidase docx

The pharmacological chaperone isofagomine increases the activity of the Gaucher disease L444P mutant form of b-glucosidase Richie Khanna1, Elfrida R Benjamin1, Lee Pellegrino1, Adriane Schilling1, Brigitte A Rigat2, Rebecca Soska1, Hadis Nafar3, Brian E Ranes1, Jessie Feng1, Yi Lun1, Allan C Powe1, David J Palling1, Brandon A Wustman3, Raphael Schiffmann4, Don J Mahuran2, David J Lockhart3 and Kenneth J Valenzano1 Amicus Therapeutics, Cranbury, NJ, USA Genetics and Genome Biology Program, Research Institute, The Hospital for Sick Children, Toronto, Canada Amicus Therapeutics, La Jolla, CA, USA Baylor Research Institute, Dallas, TX, USA Keywords Gaucher disease; isofagomine; L444P b-glucocerebrosidase; lysosomal storage disorder; pharmacological chaperone Correspondence R Khanna, Amicus Therapeutics, Cedar Brook Drive, Cranbury, NJ 08512, USA Fax: +1 609 662 2002 Tel: +1 609 662 2018 E-mail: rkhanna@amicustherapeutics.com (Received 30 November 2009, revised 11 January 2010, accepted 20 January 2010) doi:10.1111/j.1742-4658.2010.07588.x Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid b-glucosidase (GCase) We have shown previously that the small molecule pharmacological chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity In this study, we investigated the effect of IFG on L444P GCase Incubation of Gaucher patient-derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5- and 1.3-fold increases in L444P GCase activity, respectively, as measured in cell lysates The effect in fibroblasts was increased approximately 2-fold using glycoprotein-enrichment, GCase-immunocapture, or by incubating cells overnight in IFG-free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels Importantly, this reduction was seen only following three-day incubation in IFG-free media, underscoring the importance of IFG removal to restore lysosomal GCase activity In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2- to 5-fold) in GCase activity in disease-relevant tissues, including brain Additionally, eight-week IFG administration significantly lowered plasma chitin III and IgG levels, and 24-week administration significantly reduced spleen and liver weights Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non-neuronopathic Gaucher disease Structured digital abstract l MINT-7555323, MINT-7555338, MINT-7555398: LAMP1 (uniprotkb: P11279) and GCase (uniprotkb:P04062) colocalize (MI:0403) by fluorescence microscopy (MI:0416) Abbreviations CBE, conduritol-B-epoxide; CNS, central nervous system; ConA, concanavalin A; CSF, cerebrospinal fluid; ERT, enzyme replacement therapy; a-Gal A, a-galactosidase A GC, glucosylceramide; GCase, acid b-glucosidase; IFG, isofagomine; LAMP-1, lysosome-associated membrane protein 1; LCL, lymphoblastoid cell line; MI, McIlvaine; 4-MUG, 4-methylumbeliferryl-b-glucoside; NB-DNJ, N-butyl-1-deoxynojirimycin; SRT, substrate reduction therapy 1618 FEBS Journal 277 (2010) 1618–1638 ª 2010 Amicus Therapeutics Journal compilation ª 2010 FEBS R Khanna et al Effect of isofagomine on L444P acid b-glucosidase Introduction Gaucher disease is caused by inherited mutations in the gene (GBA) that encodes acid b-glucosidase (EC 3.2.1.45; GCase), the lysosomal enzyme responsible for the metabolism of glucosylceramide (GC) into ceramide and glucose [1] Mutations in GCase result in reduced cellular enzyme activity and progressive accumulation of GC, mainly within macrophages (Gaucher cells), leading to clinical manifestations including anemia, thrombocytopenia, hepatosplenomegaly, bone lesions and, in some cases, central nervous system (CNS) impairment [2,3] Patients with Gaucher disease without CNS involvement are classified as type I, whereas those with CNS involvement are classified as type II or type III [4,5] The two most prevalent missense mutant forms of GCase reported in patients with Gaucher disease are N370S and L444P [5] Patients homozygous or heterozygous for N370S GCase typically present with a non-neuronopathic form of Gaucher disease, whereas those homozygous for L444P GCase usually display a more severe neuronopathic form More than 70% of patients with Gaucher disease within the Ashkenazi Jewish population carry at least one N370S allele, and 38% of non-Jewish patients with Gaucher disease carry the L444P allele [5–7] Currently, enzyme replacement therapy (ERT) and small-molecule substrate reduction therapy (SRT) are the only approved treatment options for patients with the non-neuronopathic form of Gaucher disease [8–12] ERT, based on the intravenous administration of recombinant GCase, is the most effective treatment for type I and the visceral manifestations of types II and III disease ERT generally leads to reduced spleen and liver weights, as well as increased platelet counts and hemoglobin levels [13–16] However, the CNS manifestations of types II and III Gaucher disease not respond well to ERT because of the inability of exogenous enzyme to cross the blood–brain barrier [17] SRT drugs have the potential for better CNS penetration and some neurological benefit as the therapeutic agent is a small molecule, such as N-butyl-1-deoxynojirimycin (NB-DNJ, miglustat, ZavescaÒ), which acts as a weak inhibitor of glucosyl-transferase, thus reducing the synthesis of GC Miglustat has been approved for use in patients with mild to moderate type I Gaucher disease [9,11,12], and is currently being evaluated in patients with neuronopathic Gaucher disease, although a recent report has shown no significant benefit for the neurological manifestations of type III patients [18] Furthermore, many patients treated with miglustat have experienced side-effects, including diarrhea, weight loss, tremor and peripheral neuropathy [19] More recently, pharmacological chaperone therapy has been proposed as a potential treatment for Gaucher disease [20–23] Small-molecule pharmacological chaperones are designed to selectively bind and stabilize mutant GCase, thereby facilitating correct folding and trafficking to lysosomes, and increasing total cellular GCase activity [24,25] In addition, pharmacological chaperones have the potential to cross the blood–brain barrier and to be orally available A number of iminosugar-based pharmacological chaperones have been shown to increase the cellular activity of various mutant forms of GCase in cell lines derived from patients with Gaucher disease [26–34] The iminosugar isofagomine (IFG) has been shown to stabilize and promote lysosomal trafficking of N370S GCase [24,25] To date, however, the effects of pharmacological chaperones on L444P GCase in vitro have varied, with some reports showing small increases in enzyme activity [33] and others showing no response at all [28,31,34] Importantly, IFG has not been evaluated extensively in vitro against L444P GCase and, in vivo, the testing of IFG has been hampered by the lack of a suitable Gaucher mouse model Initial attempts to create mice with an L444P GCase point mutation resulted in a perinatal lethal phenotype [35] However, rescue of lethality was achieved using a genetically modified background (GC synthase heterozygosity), optimized breeding schemes and improved husbandry [36] Phenotypically, L444P GCase mice not exhibit the severe features generally associated with the L444P mutation in humans, such as GC accumulation, Gaucher cells, gross hepatosplenomegaly or neurological symptoms However, they manifest an attenuated, Gaucher-related phenotype characterized by reduced GCase activity in disease-relevant tissues, such as liver, spleen, lung and brain, moderate increases in spleen and liver weights, and elevated plasma chitin III and IgG levels [36] Given that other mouse models generated for Gaucher disease not carry the L444P mutation [37,38] and that L444P GCase mice were readily available, viable and easy to breed, we chose this mouse model to test the effects of the pharmacological chaperone IFG on L444P GCase in vivo In this study, we report the effects of IFG on human L444P GCase activity and GC levels in cell lines derived from patients with Gaucher disease and on murine L444P GCase activity in mice Five-day incubation of lymphoblastoid cell lines (LCLs), derived FEBS Journal 277 (2010) 1618–1638 ª 2010 Amicus Therapeutics Journal compilation ª 2010 FEBS 1619 Effect of isofagomine on L444P acid b-glucosidase from patients with Gaucher disease, with IFG led to approximately 3.5-fold increases in L444P GCase activity, as measured in cell lysates; the magnitude was much smaller in patient-derived fibroblasts (up to 1.3-fold) The measured effect of IFG on L444P GCase activity in fibroblasts could be increased approximately two-fold after glycoprotein or GCase enrichment using concanavalin A (ConA) and immunocapture, respectively, prior to assay, or by incubating the cultured cells in IFG-free medium for 24 h prior to direct assay of the cell lysates IFG incubation also increased the lysosomal trafficking of L444P GCase in fibroblasts and reduced the GC levels in situ in L444P GCase fibroblasts and LCLs Oral administration of IFG to L444P mice for weeks resulted in selective and significant increases in GCase activity (two- to five-fold) in liver, spleen, lung and brain, and, after 24 weeks, resulted in significant increases in GCase activity (up to two-fold) in mineralized bone and bone marrow Furthermore, oral administration of IFG for weeks lowered significantly plasma chitin III and IgG levels and, after 24 weeks, reduced spleen and liver weights significantly Collectively, these data indicate that IFG increases L444P GCase activity both in vitro and in vivo, and may warrant clinical evaluation for patients with both neuronopathic and nonneuronopathic Gaucher disease Results IFG increases L444P GCase activity in cells derived from patients with Gaucher disease Primary skin fibroblasts and LCLs derived from patients with Gaucher disease homozygous for either N370S or L444P GCase were used to investigate the effects of the pharmacological chaperone IFG As reported previously, the incubation of N370S GCase fibroblasts for days with IFG tartrate resulted in a statistically significant and concentration-dependent increase in GCase activity, as measured directly in cell lysates using the fluorogenic substrate 4-methylumbeliferryl-b-glucoside (4-MUG) [± conduritolB-epoxide (CBE)] [24,25,31–33,39] (Fig 1A) In contrast, incubation of L444P GCase fibroblasts under the same conditions resulted in small, but reproducible, increases in GCase activity (Fig 1B, left panel), again as reported previously [33] This effect was seen in L444P fibroblast cell lines derived from four different patients with Gaucher disease, with maximal increases from 1.2- to 1.3-fold above baseline (Table 1) Importantly, incubation of L444P GCase LCLs for days with IFG tartrate resulted in robust increases in 1620 R Khanna et al GCase activity, as measured in lysed cells (Fig 1B, right panel) Again, similar responses were seen in L444P LCL cell lines derived from five different patients with Gaucher disease, with maximal increases from 2.5- to 3.5-fold above baseline (Table 1) The effects of IFG were also seen directly on GCase protein levels, as assessed by western blotting Here, IFG incubation increased the mature, lysosomal 69-kDa form of GCase in fibroblasts (Fig 1B, left panel inset), and both the immature, Golgi 59-kDa and mature 69-kDa forms of GCase in LCLs (Fig 1B, right panel inset) These data indicate that IFG increases the total quantity of L444P GCase that is capable of trafficking through the Golgi and to lysosomes [25] As patient-derived L444P GCase cell lines have very low GCase levels (Table 1), we developed methods to enrich GCase and simultaneously remove IFG, thereby increasing the sensitivity of the GCase measurements To this end, the lysed cell assay protocol was modified to include either glycoprotein enrichment using ConA precipitation or GCase immunocapture, followed by extensive washing of the pellets to remove bound IFG from immobilized GCase GCase activity was then measured in the absence or presence of CBE using 4-MUG [24,25,31–33,39] Under these conditions, 5-day incubation of patient-derived fibroblasts with IFG tartrate increased significantly L444P GCase activity by approximately 2.0-fold (Fig 1C) This effect was seen in fibroblast cell lines derived from four different patients with Gaucher disease, with maximal increases from 1.8- to 2.2-fold (Table 2) Finally, three different L444P GCase fibroblast cell lines incubated with IFG for days, followed by a 1-day incubation in growth medium only (washout), showed maximal increases in GCase activity from 1.6- to 1.7-fold, as measured directly in cell lysates (Fig 1D; Table 2) Collectively, these data indicate that IFG can increase L444P GCase activity and protein levels in vitro, although the effect is more pronounced in LCLs than fibroblasts derived from patients with Gaucher disease In addition, the measured response in fibroblast lysates is larger after removal of IFG, which can otherwise inhibit the enzyme activity if carried into the assay IFG increases the lysosomal pool of L444P GCase Indirect immunofluorescence staining and confocal microscopy imaging were used to determine whether IFG increases the trafficking of L444P GCase to lysosomes Fibroblasts derived from healthy volunteers FEBS Journal 277 (2010) 1618–1638 ª 2010 Amicus Therapeutics Journal compilation ª 2010 FEBS R Khanna et al N370S fibroblasts Lysed-cell assay 3.0 *** 2.5 ** 2.0 L444P fibroblasts and LCLs Lysed-cell assay B Relative GCase activity Relative GCase activity A Effect of isofagomine on L444P acid b-glucosidase * 1.5 1.0 0.5 0.0 *** ** * * 20 [IFG tartrate] (µM) 60 L444P fibroblasts GCase enrichment C 2.5 D * * 2.0 20 60 20 60 LCLs Fibroblasts [IFG tartrate] (µM) * * 1.5 1.0 0.5 Relative GCase activity Relative GCase activity 2.5 L444P fibroblasts Lysed-cell assay with and without washout * 2.0 * 1.5 * 1.0 0.5 0.0 0.0 20 60 20 60 Con A Immunocapture [IFG tartrate] (µM) 20 60 20 60 + washout –washout [IFG tartrate] (µM) Fig IFG increases N370S and L444P GCase activity in cells derived from patients with Gaucher disease (A) N370S fibroblasts (DMN89.45) were incubated with the indicated concentrations of IFG tartrate for days and GCase activity was measured directly in lysed cells as described in Materials and methods In the experiment shown, a concentration-dependent increase of approximately 2.5-fold was seen in GCase activity The increase in GCase activity was found to be significant for a linear trend (one-way ANOVA), indicating a concentration-dependent effect (B) L444P fibroblasts (GM07968) and LCLs (GS0505) were incubated with the indicated concentrations of IFG tartrate for days and GCase activity was measured directly in lysed cells In the experiments shown, a small, but reproducible, 1.3-fold increase in GCase activity was seen in fibroblast lysates (left panel), and a 3.5-fold increase was seen in LCL lysates (right panel) The increase in GCase activity measured in LCLs was found to be significant for a linear trend (one-way ANOVA) Summary data from the fibroblast and LCL cell lines shown here, as well as others, are presented in Table Insets: GCase protein levels were increased in Gaucher fibroblasts and LCLs after a 5-day incubation with IFG, as measured directly by western blotting (50 lg total protein per lane) Blots were probed with rabbit polyclonal anti-human GCase serum (upper panels) and mouse monoclonal anti-b-actin IgG (lower panels) (loading control) The data shown are representative of three independent experiments (C) Gaucher fibroblasts homozygous for L444P GCase (GM07968) were incubated for days with the indicated concentrations of IFG tartrate Cell lysates were then subjected to either glycoprotein or GCase enrichment using ConA and immunocapture, respectively, as described in Materials and methods GCase activity was measured on the precipitated beads In the experiments shown, concentration-dependent increases (approximately two-fold) were seen in GCase activity The increases were found to be significant for a linear trend (one-way ANOVA) (D) Gaucher fibroblasts homozygous for L444P GCase (GM07968) were incubated for days with the indicated concentrations of IFG tartrate, followed by a 24-h washout (medium only) GCase activity was measured directly in lysed cells In the experiments shown, an approximately 1.7-fold increase was seen in L444P GCase activity after a 24-h washout This increase was found to be significant for a linear trend (one-way ANOVA) In all panels, the data were normalized to baseline (untreated) values and are representative of three or six independent experiments, as indicated in Tables and 2, with each point the mean ± SEM of triplicate determinations Statistically significant differences from untreated were determined using a two-tailed, unpaired, Student’s t-test with *P < 0.05, **P < 0.01 and ***P < 0.001 (wild-type) or Gaucher patients homozygous for the N370S or L444P mutant forms of GCase were incubated for 14 days with or without 100 lm IFG tartrate Fixed cells were incubated with primary antibodies against GCase and the lysosomal marker LAMP-1 (lysosome-associated membrane protein 1), FEBS Journal 277 (2010) 1618–1638 ª 2010 Amicus Therapeutics Journal compilation ª 2010 FEBS 1621 Effect of isofagomine on L444P acid b-glucosidase R Khanna et al Table Effect of IFG tartrate on N370S and L444P GCase activity in lysates from fibroblasts and LCLs derived from patients with Gaucher disease GCase activity in cell lysates was determined after a 5-day incubation of Gaucher fibroblasts or LCLs with the indicated concentrations of IFG tartrate The GCase activities in fibroblasts derived from three different healthy volunteers (CRL1509, CRL2076 and CRL2097) were 25 ± 2, 30 ± 0.9 and 16 ± 1.0 nmolỈ(mg protein))1Ỉh)1, respectively The average GCase activity in LCLs derived from two different healthy volunteers (GM02184 and GM03201) was 15 ± nmolỈ(mg protein))1Ỉh)1 All cell lines were homozygous for the specified GCase mutations The data for each cell line have been normalized to the GCase activity in untreated cells, and are presented as the mean ± SEM F, fibroblast; L, lymphoblastoid cell line GCase activity IFG tartrate – lM (% increase) Cell ID Mutation Cell type Baseline (nmolỈmg)1Ỉh)1) DMN89.45 GM07968 GM00877 GM10915 GM08760 GS0501 GS0505 GS0502 GS0503 GS0504 N370S L444P L444P L444P L444P L444P L444P L444P L444P L444P F F F F F L L L L L 4.0 0.2 1.0 3.0 2.3 3.1 0.4 0.8 0.7 0.7 ± ± ± ± ± ± ± ± ± ± 35 15 25 25 20 227 220 120 141 129 0.3 0.03 0.1 0.04 0.1 0.2 0.1 0.2 0.1 0.2 20 ± ± ± ± ± ± ± ± ± ± 10 5 10 23 18 14 14 28 60 95 30 30 30 20 251 232 150 146 152 ± ± ± ± ± ± ± ± ± ± 10 4 10 15 27 15 24 34 115 17 17 16 15 146 124 134 124 129 n ± ± ± ± ± ± ± ± ± ± 7 28 13 19 13 32 3 3 3 6 Table Effect of IFG tartrate on L444P GCase activity in lysates from fibroblasts derived from patients with Gaucher disease after IFG removal GCase activity was measured in fibroblasts derived from patients with Gaucher disease homozygous for L444P GCase after a 5-day incubation with the indicated concentrations of IFG tartrate Prior to assay, glycoproteins or GCase were enriched from cell lysates using either ConA or immunocapture, respectively, or cells were incubated for 24 h in medium only (IFG washout), as described in Materials and methods The data presented are the mean ± SEM from three independent experiments ND, not determined GCase activity (% increase) IFG tartrate (lM) 20 Cell ID 64 ± 13 99 ± 21 25 ± ND Glycoprotein enrichment GM07968 GM00877 GM10915 GM08760 60 88 ± 19 113 ± 39 55 ± ND 20 Immunocapture 115 ± 33 100 ± 20 100 ± ND 33 27 55 38 ± ± ± ± 16 12 followed by labeling with secondary antibodies conjugated with different fluorophores Strong punctate signals for GCase and LAMP-1 were recorded in wildtype fibroblasts in the absence or presence of IFG; the degree of colocalization of the GCase and LAMP-1 signals was increased after IFG incubation (Fig 2A) By comparison, the overall GCase signal in untreated N370S fibroblasts was weaker However, N370S GCase levels were increased significantly and showed increased colocalization with LAMP-1 after incubation with IFG (Fig 2B), as reported previously [39] The GCase signal was even weaker with a more diffuse pattern in the three L444P GCase fibroblast lines investigated (Fig 2C–E), with lines 00877 and 10915 showing some 1622 60 55 41 84 62 20 60 55 ± 13 51 ± 45 ± ND 74 ± 61 ± 12 55 ± ND IFG washout ± ± ± ± 17 10 19 115 39 73 72 ± ± ± ± 46 19 40 ± 12 33 ± 25 ± ND low-level colocalization with LAMP-1 prior to IFG incubation Importantly, IFG increased the overall GCase signal (more intense punctate signals) in all three L444P GCase cell lines, resulting in clear colocalization with LAMP-1 Collectively, these data indicate that IFG can increase the lysosomal content of L444P GCase in cells derived from patients with Gaucher disease IFG reduces GC levels in L444P GCase fibroblasts and LCLs We next determined whether increased L444P GCase levels and lysosomal trafficking resulted in increased FEBS Journal 277 (2010) 1618–1638 ª 2010 Amicus Therapeutics Journal compilation ª 2010 FEBS R Khanna et al GCase A B WT Effect of isofagomine on L444P acid b-glucosidase LAMP1 Merge GCase LAMP1 Merge WT + IFG N307S N370S + IFG L444P (07968) L444P (07968) + IFG D L444P (00877) L444P (00877) + IFG E L444P (10915) + IFG C L444P (10915) Fig IFG increases the lysosomal pool of L444P GCase in fibroblasts derived from patients with Gaucher disease Fibroblasts derived from healthy volunteers (WT; CRL2097) and patients with Gaucher disease homozygous for the N370S (DMN89.45) or L444P (GM07968, GM00877, GM10915) mutant forms of GCase were incubated in the absence or presence of 100 lM IFG tartrate for 14 days GCase (green) and the lysosomal marker LAMP-1 (red) were visualized by confocal microscopy after indirect immunofluorescence staining, as described in Materials and methods In the merged images, yellow denotes the colocalization of the two proteins, indicative of their lysosomal localization Nuclei are stained with 4¢,6-diamidino-2-phenylindole (blue) IFG treatment increased the lysosomal pool of GCase in wild-type as well as N370S and L444P GCase fibroblasts (as shown by the increased amount of yellow in the merged images) Representative cells are shown to demonstrate the degree of colocalized GCase and LAMP-1 Magnification, ·63 FEBS Journal 277 (2010) 1618–1638 ª 2010 Amicus Therapeutics Journal compilation ª 2010 FEBS 1623 Normal L444P # ** * 10 on 10 on NJ NB -D on/3 off Untreated IFG Untreated 10 on 10 on NJ -D on/3 off ** NB IFG Untreated *** Untreated 20 18 16 14 12 10 R Khanna et al LCL GC Llvels [μg·(mg protein)–1] Fibroblast GC levels [μg·(mg protein)–1] Effect of isofagomine on L444P acid b-glucosidase Fig IFG reduces GC levels in Gaucher fibroblasts and LCLs Fibroblasts (GM07968, left) and LCLs (GS0505, right) homozygous for L444P GCase were incubated in the absence or presence of 30 lM IFG for days, followed by a 3-day washout (‘7 on ⁄ off’) Parallel cultures of these cell lines were incubated for 10 days with 30 lM IFG or 500 lM NB-DNJ (‘10 on’) GC levels were then measured as described in Materials and methods, as well as in normal control fibroblasts (CRL2076) or LCLs (WT0003) The data are expressed as the mean ± SEM from three flasks for each condition tested Statistically significant differences from untreated GC levels were determined using a two-tailed, unpaired, Student’s t-test with *P < 0.05, **P < 0.01 and ***P < 0.001, or #P < 0.05 for untreated versus ‘10 on’ Similar results were seen in two other L444P GCase cell lines (GM10915 fibroblasts and GS0501 lymphoblasts; see Table 3) substrate turnover in situ (Fig 3; Table 3) GC levels in Gaucher fibroblasts and LCLs were measured after a 7-day incubation in the absence or presence of 30 lm IFG tartrate, followed by a 3-day washout period to minimize potential GCase inhibition by IFG in situ (‘7 on ⁄ off’) For comparison, the effects of a continuous 10-day incubation (‘10 on’) with either 30 lm IFG or the GC synthase inhibitor NB-DNJ (500 lm) [40] were also assessed Baseline GC levels in the Gaucher fibroblast cell lines 07968 and 10915 were elevated 7.1 ± 2.2and 1.9 ± 0.6-fold, respectively, compared with the normal fibroblast cell line CRL2076 Similarly, baseline GC levels in the Gaucher LCL cell lines GS0501 and GS0505 were elevated 3.4 ± 0.8- and 3.4 ± 0.5fold, respectively, compared with the normal LCL cell line WT0003 Importantly, all tested L444P GCase fibroblasts and LCLs incubated with IFG in the ‘7 on ⁄ off’ regimen showed significant decreases in GC levels In contrast, IFG incubation in the ‘10 on’ regimen did not reduce GC levels in any cell line tested As expected, 10-day incubation with NB-DNJ decreased GC levels significantly in these cell lines These data indicate that the IFG-mediated increases in cellular and lysosomal GCase can lead to a reduction in GC levels in L444P GCase fibroblasts and LCLs, provided that IFG is sufficiently washed out from the cells for several days IFG is orally available and shows broad tissue distribution Two different salt forms of IFG, IFG hydrochloride (IFG HCl) and IFG tartrate, were used for the in vivo studies We first determined the tissue distribution and rate of clearance of IFG in plasma, liver, spleen and brain of male Sprague–Dawley rats Animals were administered a single oral dose (by gavage) of IFG tartrate (600 mgỈkg)1, equivalent to 300 mgỈkg)1 free base), Table Effect of IFG and NB-DNJ on GC levels in cells derived from patients with Gaucher disease homozygous for the L444P mutation GC levels in Gaucher fibroblasts and LCLs homozygous for L444P GCase were determined after a 7-day incubation in the absence or presence of 30 lM IFG, followed by a 3-day washout (‘7 on ⁄ off’) For comparison, cells were also incubated for 10 days (‘10 on’) with 30 lM IFG or 500 lM NB-DNJ The data for each cell line were normalized to the GC levels in untreated cells, and are expressed as the mean ± SEM from three flasks for each condition tested Differences in GC levels between treated and untreated cells were determined using a two-tailed, unpaired Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001) Although incubation with 30 lM IFG for 10 days did not reduce GC levels significantly in any cell line tested, significant increases were seen in fibroblast cell lines 07968 and 10915 (16% and 35%, respectively; P < 0.05 compared with untreated) GC levels in fibroblasts and LCLs derived from healthy volunteers (CRL2076 and WT0003, respectively) were 1.2 ± 0.02 and 0.85 ± 0.2 lgỈ(mg protein))1, respectively (see Fig 3) F, fibroblast; L, lymphoblastoid cell line; –, no decrease GC levels Compound ⁄ regimen (% decrease) Cell ID Cell type Baseline [lgỈ(mg protein)–1] GM07968 GM10915 GS0501 GS0505 F F L L 14 2.5 2.2 2.7 1624 ± ± ± ± 0.5 0.1 0.3 0.2 IFG ‘7 on ⁄ off’ IFG ‘10 on’ NB-DNJ ‘10 on’ 23 32 50 26 – – – – 66 76 77 75 ± ± ± ± 3** 4** 2* 1* ± ± ± ± 1*** 1*** 1** 1*** FEBS Journal 277 (2010) 1618–1638 ª 2010 Amicus Therapeutics Journal compilation ª 2010 FEBS R Khanna et al Effect of isofagomine on L444P acid b-glucosidase [IFG] (ng·mL–1 or ng·g–1) 10000 Plasma Liver Spleen Brain 1000 100 10 12 24 Time (h) 36 48 Fig Tissue distribution pharmacokinetics of IFG Eight-week-old male Sprague–Dawley rats were fasted overnight prior to the administration of IFG tartrate (600 mgỈkg)1, equivalent to 300 mgỈkg)1 free base) by oral gavage Tissue and blood samples were drawn as a function of time IFG levels were assessed by LC-MS ⁄ MS in plasma and tissue homogenates as described in Materials and methods Each point represents the mean ± SEM from three rats and plasma and tissue concentrations of IFG were measured by LC-MS ⁄ MS as a function of time after administration (Fig 4) Maximal IFG levels were attained within h after administration IFG was detected in all peripheral tissues tested, with peak levels of 3.9 ± 1.0 lgỈmL)1, 17.7 ± 3.3 lgỈg)1 and 1.2 ± 0.1 lgỈg)1 in plasma, liver and spleen, respectively (approximately 26, 120 and lm, assuming that g of tissue is equivalent to mL of volume) Twentyfour hours post-administration, concentrations fell to 0.007 ± 0.002 lgỈmL)1 and 0.04 ± 0.01 lgỈg)1 in plasma and spleen, respectively, with levels in the liver below the limit of quantification (0.025 lgỈg)1) By 48 h, the IFG concentrations in plasma and spleen were less than 0.003 lgỈmL)1 and 0.01 lgỈg)1, respectively IFG penetration into the brain was slower and reached lower levels than in other tissues, with a maximal concentration of 0.25 ± 0.09 lgỈg)1 (approximately 1.7 lm) at h; 48 h post-administration, brain levels were less than 0.01 lgỈg)1 The total brain exposure was approximately 20% of the plasma exposure The terminal half-lives of IFG were estimated to be 4.4, 2.6, 4.6 and h in plasma, liver, spleen and brain, respectively Collectively, these results indicate that IFG is orally available and has a wide tissue distribution profile, including the CNS IFG selectively increases L444P GCase activity in mouse tissues To investigate the effect of IFG on L444P GCase in vivo, 2-month-old male L444P GCase mice were administered IFG HCl (3, 10 or 30 mgỈkg)1Ỉday)1, equivalent to 2.5, 8.2 and 25 mgỈkg)1 free base, respectively) ad libitum in drinking water for weeks Mice were then euthanized and the GCase activity was measured in liver homogenates A statistically significant and dose-dependent increase in L444P GCase activity (approximately four-fold) was seen (Fig 5A), with a maximal increase at a daily dose of 10 mgỈkg)1Ỉday)1 In a follow-up study, 2-month-old male L444P GCase mice were administered IFG tartrate (20 mgỈkg)1Ỉday)1, equivalent to 10 mgỈkg)1 free base) ad libitum in drinking water for weeks (Fig 5B) Again, a statistically significant increase (approximately four-fold) in L444P GCase activity was seen in the liver In addition, L444P GCase activity was also elevated in the spleen, lung and brain, with increases of approximately four-, five- and two-fold, respectively (Fig 5B) In separate studies, oral administration (ad libitum) of IFG tartrate (20 mgỈkg)1 Ỉday)1) to 6-month-old L444P GCase mice for 24 weeks resulted in a significant increase in L444P GCase activity (up to two-fold) in mineralized bone and bone marrow (Fig 5B, inset) Oral administration of IFG tartrate increased tissue L444P GCase activity to 15–40% of that measured in the respective tissues of age-matched, untreated, wildtype C57BL ⁄ mice (Fig 5B) IFG administration did not affect the tissue activity of four other lysosomal hydrolases, including a-galactosidase A (a-Gal A), acid a-glucosidase, b-glucuronidase and b-galactosidase, in L444P GCase mice (data not shown), indicating that the increase in L444P GCase activity in vivo is selective Furthermore, the 69-kDa form of L444P GCase was increased three-, two-, four- and 1.2-fold in liver, spleen, lung and brain homogenates, respectively, of L444P GCase mice administered 20 mgỈkg)1Ỉday)1 of IFG tartrate for weeks, as measured by western blotting (Fig 5C) Finally, the increased activity of murine L444P GCase in liver tissue correlated with increased quantities of GCase protein in lysosomal fractions isolated from liver homogenates of mice administered IFG tartrate (20 mgỈkg)1Ỉday)1) for 24 weeks (Fig S1, see Supporting Information) To determine whether the effect of IFG tartrate on L444P GCase could be reproduced in cells derived from the L444P GCase mice, primary macrophage cultures were established from liver Five-day ex vivo incubation with increasing concentrations of IFG tartrate resulted in a ± 0.3-fold increase in L444P GCase activity, as measured in lysates from the cultured macrophages (Fig 5D) Collectively, these data indicate that IFG can selectively increase murine L444P GCase activity and lysosomal levels both in vitro and in vivo FEBS Journal 277 (2010) 1618–1638 ª 2010 Amicus Therapeutics Journal compilation ª 2010 FEBS 1625 12 B Liver ** 10 GCase activity [nmol·(mg protein)–1·h–1] GCase activity [nmol·(mg protein)–1·h–1] A R Khanna et al * * 23 L444P L444P+IFG C57BL/6 GCase activity [nmol·(mg protein)–1·h–1] Effect of isofagomine on L444P acid b-glucosidase 50 30 Bone 10 * * Mineralized 10 Marrow *** *** ** * Lung Brain 0 10 IFG HCl (mg kg–1) C 30 Liver GCase 69 kDa β-actin IFG tartrate – + Liver – + Spleen – + Lung – + Brain Relative GCase activity D 2.0 Spleen ** Liver macrophages * 1.5 1.0 0.5 0.0 20 IFG tartrate (µM) 60 Fig IFG increases tissue L444P GCase activity in vivo (A) Two-month-old male L444P GCase mice were administered IFG HCl (3, 10 or 30 mgỈkg)1Ỉday)1, equivalent to 2.5, 8.2 and 25 mgỈkg)1 free base, respectively) ad libitum in drinking water for weeks GCase activity in liver lysates was measured as described in Materials and methods Significant increases in GCase activity were seen at all three doses Each bar represents the mean ± SEM GCase activity from four mice per group analyzed in triplicate The treatment was also found to be significant for a linear trend (one-way ANOVA), indicating a dose-dependent effect (B) Two-month-old male L444P GCase mice were administered IFG tartrate (20 mgỈkg)1Ỉday)1, equivalent to 10 mgỈkg)1 free base) ad libitum for weeks GCase activity was measured in tissue lysates as described in Materials and methods Significant increases in GCase activity were seen in liver (four-fold), spleen (four-fold), lung (five-fold) and brain (two-fold) Tissue GCase activity from untreated wild-type C57BL ⁄ mice is also shown Each bar represents the mean ± SEM of GCase activity from four mice per group analyzed in triplicate Inset: six-month-old male L444P GCase mice were administered IFG tartrate (20 mgỈkg)1Ỉday)1, equivalent to 10 mgỈkg)1 free base) ad libitum for 24 weeks and GCase activity was measured in mineralized bone and bone marrow lysates as described in Materials and methods Significant increases in L444P GCase activity (up to two-fold) were seen with IFG administration Each bar represents the mean ± SEM of GCase activity from seven to eight mice per group analyzed in triplicate (C) GCase protein levels in the tissue samples (50 lg) used in (B) were measured directly by western blotting using rabbit polyclonal anti-mouse GCase serum and mouse monoclonal anti-b-actin IgG (loading control) antibodies as described in Materials and methods IFG tartrate administration increased GCase activity in liver (three-fold), spleen (two-fold), lung (four-fold) and brain (1.2-fold) Each lane represents one mouse from each group and is representative of two experiments with two different mice from each group (D) Primary cultures of mouse liver macrophages were derived from 2-month-old untreated male L444P GCase mice and incubated with IFG tartrate for days at the concentrations indicated, as described in Materials and methods In the experiment shown, a significant and concentration-dependent increase (approximately two-fold) in L444P GCase activity was seen in macrophage lysates The increase was also found to be significant for a linear trend (one-way ANOVA) The data shown were normalized to untreated values and are representative of three independent experiments, with each point the mean ± SEM of triplicate determinations In (A), (B) and (D), statistically significant differences from untreated were determined using a two-tailed, unpaired, Student’s t-test with *P < 0.05, **P < 0.01 and ***P < 0.001 Tissue L444P GCase activity is elevated for days after withdrawal of IFG To determine the duration of elevated L444P GCase after IFG withdrawal in vivo, 4-month-old, male L444P GCase mice were administered IFG tartrate (20 mgỈkg)1Ỉday)1, equivalent to 10 mgỈkg)1 free base) ad libitum in drinking water After a 4-week 1626 administration, IFG tartrate was removed and mice were provided access to drinking water only Groups of mice were then euthanized and tissue GCase activity was measured 0, 2, and days after IFG tartrate withdrawal (Fig 6) On the last day of IFG administration (day 0), L444P GCase activity was increased significantly in the liver, spleen, lung and brain The elevated tissue GCase activity was FEBS Journal 277 (2010) 1618–1638 ª 2010 Amicus Therapeutics Journal compilation ª 2010 FEBS Effect of isofagomine on L444P acid b-glucosidase GCase activity [nmol·(mg protein)–1·h–1] R Khanna et al ** * 1.5 Spleen *** ** * 0 GCase activity [nmol·(mg protein)–1·h–1] Liver *** *** 2.5 Lung ** Brain 2.0 1.5 1.0 * 0.5 * 1.0 * 0.5 0.0 0.0 Washout (days) Washout (days) Fig Time course for the decay of increased L444P GCase activity after IFG withdrawal Four-month-old male L444P GCase mice were administered drinking water (broken lines) or IFG tartrate [20 mgỈkg)1Ỉday)1, equivalent to 10 mgỈkg)1 free base (full line)] ad libitum in drinking water for weeks, followed by a washout period (drinking water only) for up to days Groups of mice were then euthanized on days 0, 2, or after IFG tartrate withdrawal and GCase activity was measured in tissue lysates Statistically significant increases above baseline were maintained in liver, spleen and lung GCase activity for up to days, and in brain for up to days Each data point represents the mean ± SEM of tissue GCase activity from six mice per group analyzed in triplicate Statistically significant differences from untreated were determined using a two-tailed, unpaired, Student’s t-test with *P < 0.05, **P < 0.01 and ***P < 0.001 Table Tissue levels of IFG Four-month-old L444P GCase mice were administered IFG tartrate (20 mgỈkg)1Ỉday)1, equivalent to 10 mgỈkg)1 free base) ad libitum in drinking water for weeks Mice were euthanized on the last day of dosing (day 0) or days after IFG tartrate withdrawal (day 2) For monkeys (cynomolgus), a single dose of IFG tartrate (1000 mgỈkg)1, equivalent to 500 mgỈkg)1 free base) was administered by oral gavage with CSF collected h post-administration IFG levels were quantified by LC-MS ⁄ MS and expressed as ngỈmL)1 (plasma and CSF) or ngỈg)1 (liver, spleen, lung and brain) Values represent the mean ± SEM for groups of six (L444P GCase mice) or 10 (monkeys) LOQ, limit of quantification Species IFG tartrate (mgỈkg)1) L444P GCase mice 20 Monkey 1000 [IFG] (ngỈg)1 or ngỈmL)1) Tissue Day Day LOQ (ngỈg)1 or ngỈmL)1) Plasma Liver Spleen Lung Brain CSF 71 ± 177 ± 17 70 ± 69 ± 10