Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease

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1 Neurobiology of Disease 16 (2004) Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease Ulrika Andersson, a David Smith, a Mylvaganam Jeyakumar, a Terry D. Butters, a Mario Cortina Borja, b Raymond A. Dwek, a and Frances M. Platt a, * a Department of Biochemistry, Glycobiology Institute, University of Oxford, Oxford OX1 3QU, UK b Centre for Paediatric Epidemiology and Biostatistics, Institute of Child Health, University College London, London WC1N 1EH, UK Received 6 August 2003; revised 31 March 2004; accepted 23 April 2004 Available online 7 June 2004 Sandhoff disease is a severe neurodegenerative glycosphingolipid (GSL) lysosomal storage disorder, currently without treatment options. One therapeutic approach under investigation is substrate reduction therapy (SRT). By partially inhibiting GSL biosynthesis, the impaired rate of GSL catabolism is balanced by a slower rate of influx of GSLs into the lysosome. In a previous study, we reported the beneficial effects of treating Sandhoff disease mice with the glucose analogue N- butyldeoxynojirimycin (NB-DNJ), a compound that inhibits the first step of GSL biosynthesis catalysed by the ceramide specific glucosyltransferase. NB-DNJ, however, exhibits adverse effects at high doses such as weight loss and GI tract distress (due to glucosidase inhibition). This might limit the therapeutic potential of NB-DNJ for treating diseases affecting the CNS where high dose therapy may be required to achieve therapeutic levels of the drug in the brain. In the present study, a more selective compound, the galactose analogue N-butyldeoxygalactonojirimycin (NB-DGJ), was evaluated in the Sandhoff disease mouse model. Treatment with NB-DGJ showed greater therapeutic efficacy than NB-DNJ with no detectable side effects. The ability to escalate the dose of NB-DGJ, leading to extended life expectancy and increased delay in symptom onset, demonstrates the greater therapeutic potential of NB-DGJ for the treatment of the human gangliosidoses. D 2004 Elsevier Inc. All rights reserved. Keywords: Sandhoff disease; Gangliosidosis; Substrate reduction therapy; Imino sugar Introduction Abbreviations: GSL, glycosphingolipid, NB-DNJ; secondary N-butyldeoxynojirimycin, NB-DGJ; tertiary N-butyldeoxygalactonojirimycin, SRT, substrate reduction therapy, BMT, bone marrow transplantation, CNS, central nervous system. * Corresponding author. Department of Biochemistry, Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Fax: address: fran@glycob.ox.ac.uk (F.M. Platt). Available online on ScienceDirect ( The GM2 gangliosidoses are a group of GSL lysosomal storage disorders including Tay Sachs disease, Sandhoff disease and GM2 activator deficiency. They result from mutations in genes encoding the hexosaminidase a-subunit, h-subunit and GM2 activator protein, respectively. In humans, they are characterised by progressive neurodegeneration in response to high levels of lysosomal storage of GM2 and related GSLs in neurons of the CNS (Gravel et al., 2001). There are currently no therapies for these diseases. Substrate reduction therapy (SRT) is a pharmacological approach that aims to partially inhibit the ceramide-specific glucosyltransferase that catalyses the first committed step in GSL biosynthesis to balance the rate of GSL synthesis with the reduced rate of GSL catabolism (Butters et al., 2000; Radin, 1996).The approach is generic because synthesis of all glucosylceramide (GlcCer) based GSLs is reduced and all diseases involving storage of GlcCer-based GSLs (Gaucher types 1, 2 and 3, Fabry, GM1 gangliosidosis, Tay-Sachs and Sandhoff disease) could potentially be treated with a single drug (Dwek et al., 2002). The imino sugar NB-DNJ has recently been shown to be an effective therapy in clinical trials in Type 1 Gaucher disease (Cox et al., 2000) and was approved for clinical use in Europe for the treatment of type 1 Gaucher disease late in As this drug has the potential to be effective in related diseases with CNS involvement, the drug has been evaluated in several mouse models of GSL storage diseases. Prevention of storage was shown in an asymptomatic mouse model of Tay Sachs disease (Platt et al., 1997a) and a symptomatic Sandhoff disease mouse model (Jeyakumar et al., 1999). In the latter study, reduced brain and peripheral storage, delayed onset of symptoms and increased life expectancy were achieved following NB-DNJ treatment (Jeyakumar et al., 1999). However, the use of NB-DNJ at high doses in mice is associated with adverse effects such as body weight reduction, lymphoid organ atrophy and GI tract distress (due to disaccharidase inhibition) (Platt et al., 1997b), which might offset the beneficial effects of SRT and limit dose escalation. High-dose therapy may be required for treating GSL storage diseases with brain involvement as only approximately 10% of the serum level of the drug penetrates the CNS (Platt et al., 1997a). In a previous study, we have shown the potential of the galactose analogue NB-DGJ as a second- generation compound for GSL storage disease therapy(andersson et al., 2000). Its greater selectivity and lack of side effects in healthy /$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi: /j.nbd

2 U. Andersson et al. / Neurobiology of Disease 16 (2004) mice suggests that it may allow the escalation of dose. The present study therefore evaluated NB-DGJ in a mouse model of Sandhoff disease. Therapeutic efficacy was demonstrated without any detectable side effects. Dose escalation of NB-DGJ was possible with a further extension in life expectancy and a further delay in symptom onset, as compared to NB-DNJ treatment. Materials and methods Animals and compounds The Sandhoff disease mouse model was generously provided by Dr. Richard Proia (National Institute of Health, Bethesda, MD 20892; Sango et al., 1995). The mice were housed under standard nonsterile conditions and bred as homozygous males crossed with heterozygous females. The mice were provided with water ad libitum and before drug administration were fed pelleted chow (expended Rat and Mouse Chow 1, SDS Ltd., Witham, Essex, U.K.). NB-DNJ and NB-DGJ were gifts from Oxford Glyco- Sciences (OGS, Abingdon, U.K.). Sandhoff mice genotyping: b-hexosaminidase assay Mouse tail tips were surgically removed from anaesthetised mice, homogenised in 0.5 ml H 2 O using an Ultraturax T25 probe, spun in a microfuge ( rpm) for 20 s and the supernatant was retained for assay. The h-hexosaminidase activity of the tail sample was determined by incubating 10 Al of the supernatant enzyme source with 50 Al of 3 mm 4-MUGlcNAc in 100 mm citrate/200 mm sodium phosphate buffer, ph 4.5, for 15 min at 37jC, the reaction stopped with 1 ml NaOH Glycine (0.3 M, ph 10.5), and the released 4-MU measured at 365 k ex /460 k em nm (Perkin Elmer fluorimeter). The activity was standardised to protein concentration (Bio-Rad protein assay). Feeding mice with imino sugars Female Sandhoff mice were fed powdered diet (Rat and Mouse Chow 1, SDS Ltd.) with or without NB-DNJ or NB- DGJ as previously described (Andersson et al., 2000). From 6 weeks of age, the mice were maintained on 1200 or 2400 mg kg 1 day 1 either for 6 weeks or until they reached a defined humane end point, which was applied when the mice became moribund and unable to right themselves when laid on their side. As controls, heterozygous (Hexb +/ ) littermates were used. These mice are asymptomatic and have a normal life expectancy. Imino sugar determination Mice were sacrificed by CO 2 asphyxiation, blood collected by cardiac puncture, then immediately perfused through the left cardiac ventricle with 0.9% heparinised saline. Concentrations of NB-DNJ or NB-DGJ in mouse serum and/or brain samples were analysed by HPCE chromatography (Dionex BioLC) as previously described (Mellor et al., 2000). Compound concentration in brain was calculated by volume-standardisation against tissue weight (1 mg = 1 Al) assuming the density of brain equals the density of water. Glycosphingolipid analysis of brain and liver by TLC Gangliosides from mouse liver and brain were extracted and analysed as previously described (Andersson et al., 2000). Briefly, liver and brain samples were homogenised in water and lyophilised. Dried homogenates were extracted twice with chloroform/methanol (2:1, v/v), once overnight at 4jC and once for 3 h at room temperature, pooled and dried under nitrogen. Liver extracts were resuspended in 500 Al chloroform/methanol (1:1, v/v), base-treated by adding 83 Al of 0.35 M NaOH in methanol for 90 min at room temperature, and partitioned by adding 83 Al water/methanol (9:1, v/v), Al water and 416 Al chloroform. The upper phase containing the gangliosides was separated from the lower phase after mixing and low-speed centrifugation, and the lower phase was washed twice with Folch upper phase (chloroform/methanol/0.74% KCl, 3:48:47, v/v). Upper phases were combined, dried to half their original volume under nitrogen, dialysed against water to desalt, lyophilised and resuspended in chloroform/methanol (2:1, v/v). Gangliosides dissolved in chloroform/methanol were separated by HPTLC (in chloroform/methanol/0.22% dcacl 2, 60:35:8, v/v), loading an equivalent dry weight of 3 mg liver or 0.4 mg brain per sample. The TLC plates (HPTLC aluminium sheets, silica gel 60, Merck) were air-dried, sprayed with orcinol/ sulphuric acid [0.2% (w/v):1 M] and heat-treated (90jC for 10 min). The digital image of the HPTLC plate was captured using an AGFA Arcus II scanner (with Photoshop 6.0 software), and the intensity of the bands was quantified by scanning densitometry using NIH Image 1.61 software. Histology Brains were dissected and the right hemisphere placed in OCT (BDH) before freezing in 2-methylbutane in liquid nitrogen. Cryosections (10 Am) were cut and periodic-acid-schiff (PAS) stained at room temperature according to the manufacturer s instructions (Sigma). Immunohistochemistry for MHC class II was performed using MAb M5/114 (rat Ig; ATCC TIB120) as previously described (Jeyakumar et al., 2003). Horizontal bar crossing test Sandhoff disease mice were tested for their motor coordination and hind limb strength by performing a bar crossing test two to three times per week (Jeyakumar et al., 1999). Briefly, the mouse was held by its tail and allowed to grasp the centre of a suspended thin bar with only its forepaws, and after releasing the tail, the time to cross the bar was determined. The performance was scored where the higher the positive value the faster the mouse crossed the bar and the lower the negative value the faster the mouse fell from the bar (Jeyakumar et al., 1999). Open field test The spontaneous activity of each mouse was recorded for 2 min. The open field was a plastic box ( cm) with a 150-cm 2 floor grid pattern. Locomotion was recorded as the number of squares the mouse crossed on the floor grid (a cross was counted when the mouse entered a new square with both its front paws). Rearing was recorder as the number of

3 508 U. Andersson et al. / Neurobiology of Disease 16 (2004) times the mouse stood only on its hind legs (either upright without support or against the wall). Statistical analysis Conventional statistical methods were employed to calculate mean values and standard errors of the mean (SEM). Differences between groups of mice were tested for significance using twotailed Student s t test for unpaired observations. Results in the text and tables are presented as means F SEM. Survival data was analysed using the Weibull regression model (Meeker and Duke, 1981). The bar crossing scores were analysed with a nonlinear regression model, using a four-parameter logistic curve fit according to the equation below (Vonesh and Chinchilli, 1997): B A Y ¼ A þ 1 þ exp½ðage dþ=hš where A and B are the lower and upper asymptotes, respectively, d is the age at which the median time is reached and h is the scale parameter that determines how fast the curve declines (the smaller the h, the faster the decline). For open field data, a quadratic function was used to describe changes in vertical/ horizontal movements with respect to age in the four treatment groups during the presymptomatic period (<90 days). All the models were fitted in the context of linear mixed effects (Pinheiro and Bates, 2000). This allowed us to adequately consider the longitudinal nature of the data. Only random effects on the intercepts of the models were considered. Using the Box Cox family of transformations (Box and Cox, 1964), it was found that the optimal transformation for achieving normality and homogeneity of variance for the residuals was the square root of vertical and horizontal movements. Back transformation was performed to obtain the predicted values. Results Life expectancy of Sandhoff mice Homozygote female Sandhoff mice (Hexb / ) were treated orally with 1200 mg kg 1 day 1 of NB-DNJ (glucose analogue) or with 1200 or 2400 mg kg 1 day 1 of NB-DGJ (galactose analogue) from 6 weeks of age until they reached the humane end point and were sacrificed. It has previously been shown that 1200 mg kg 1 day 1 NB-DNJ is sufficient to reach the maximum therapeutic benefit achieved by this drug in this mouse model (Jeyakumar et al., 1999). At higher doses, no further increase in survival was achieved and weight loss became more severe. In the present study, treatment with 1200 mg kg 1 day 1 extended the survival from 124 F 1 days for untreated mice to 161 F 3 for NB- DNJ (30% increase compared to untreated, P < ) and 162 F Fig. 1. (a) Survival of Sandhoff mice treated with NB-DNJ or NB-DGJ. Mice were treated from 6 weeks of age and were killed at the humane end point (when the mice were unable to right themselves when placed on their side). The mice were either untreated (5, n = 13), given 1200 mg kg 1 day 1 of NB-DNJ (o, n =9)orNB-DGJ (., n = 12), or 2400 mg kg 1 day 1 of NB-DGJ (, n = 5). All treated groups lived significantly longer compared to the untreated group (P < ). The two 1200 mg kg 1 day 1 dose groups do not have significantly different life expectancies (P = 0.55). The 2400 mg kg 1 day 1 NB-DGJ mice show significantly longer life expectancy compared to 1200 mg kg 1 day 1 NB-DGJ (P < 0.001) and NB-DNJ (P < ). (b) Body weights of Sandhoff mice treated with NB-DNJ or NB-DGJ. (b) Comparison of body weights of Sandhoff mice treated with NB-DNJ or NB-DGJ [symbols are as in (a) plus heterozygote control mice (D, n = 5)]. The difference between NB-DNJ mice compared to untreated and NB-DGJ mice is statistically significant (P < 0.05) from day 57 until the end point. (c) Comparison of body weights at different doses of NB-DGJ. Untreated and 1200 mg kg 1 day 1 are as in (a) plus 2400 mg kg 1 day 1 NB-DGJ (). There were no statistical differences between the groups (P > 0.1).

4 U. Andersson et al. / Neurobiology of Disease 16 (2004) Table 1 Statistical analysis of bar crossing data Age of decline (days) Untreated 100 F 1 NB-DNJ (1200) 128 F 2 NB-DGJ (1200) 125 F 0.7 NB-DGJ (2400) 134 F 0.3 P values NB-DNJ (1200) NB-DGJ (1200) NB-DGJ (2400) Untreated <0.001 <0.001 <0.001 NB-DNJ (1200) NB-DGJ (1200) <0.001 The bar crossing data (Fig. 2a) was fitted with a nonlinear regression model according to the equation defined in Materials and methods. The age of decline (d) is defined when the median time of the decline is reached. Behavioural analysis of Sandhoff mice The horizontal bar crossing test was performed to measure the progressive decline in hind limb strength and coordination. The heterozygote controls crossed the bar in less than the 120-s duration of the test (average crossing time 8 s = score of 112) (Fig. 2a). The early symptomatic phase in the untreated Hexb / mice began at 12 weeks of age and was characterised by a fine tremor that was not prevented by imino sugar treatment (in agreement with previous studies; Jeyakumar et al., 1999). Untreated mice rapidly developed difficulties in crossing the bar from approximately 90 days of age, and by 100 days all fell from the apparatus before crossing (Table 1). NB-DNJ and NB-DGJ at 2 for NB-DGJ-treated mice (31% increase compared to untreated, P < ) (Fig. 1a). When increasing the NB-DGJ dose to 2400 mg kg 1 day 1, the survival was increased to 185 F 3 days (49% increase compared to untreated, P < ) with the longest surviving mice reaching the humane end point at 196 days of age. The life expectancy of the high dose NB-DGJ group (2400 mg kg 1 day 1 ) was also significantly increased when compared to the 1200 mg kg 1 day 1 dose groups (P < compared to NB-DNJ treated, and P < compared to NB-DGJ treated). The body weights of untreated homozygotes, 1200 mg kg 1 day 1 NB-DGJ-treated homozygotes, and heterozygote controls were almost identical until the mice were approximately 100 days old (Fig. 1b). At that point, untreated homozygous mice became symptomatic and lost weight. At approximately 145 days, the NB-DGJ-treated homozygote mice started loosing weight as a result of the disease. NB-DNJ-treated mice weighed considerably less than any of the other three groups throughout their life span (P < 0.05 at all time points from 57 days of age). Treating the mice with the higher NB-DGJ dose (2400 mg kg 1 day 1 ) resulted in a small, nonsignificant, increase in body weight as compared to untreated and NB-DGJ treated at 1200 mg kg 1 day 1 (P > 0.1 until symptom onset of untreated mice, Fig. 1c). Fig. 2. Bar crossing (a) and open field test (b, c). The Sandhoff mice were assessed for their ability to cross a horizontal bar. Values above zero indicate ability to cross the bar; the higher the score, the more rapid the crossing [score = 120 crossing time (s)]. Negative scores indicate falling from the bar; the lower the score, the less time before falling [score = falling time (s) 120]. The mice were heterozygote controls (D, n =5)or homozygote Sandhoff mice untreated (5, n = 13), treated with 1200 mg kg 1 day 1 NB-DNJ (o, n =9)orNB-DGJ (., n = 12), or treated with 2400 mg kg 1 day 1 NB-DGJ (, n = 5). P values are presented in Table 1. Exploration and mobility were determined in the open field test with the number of times the mice crossed a line moving from one square to another (see text for method) defined as horizontal movement, and the number of times the mice reared as vertical movement. (b) Horizontal movement expressed as lines crossed per minute for heterozygotes (average F SEM shown as a dotted area), homozygotes untreated (5, n = 13), treated with 1200 mg kg 1 day 1 NB-DNJ (o, n =9)orNB-DGJ (., n = 12), or treated with 2400 mg kg 1 day 1 NB-DGJ (, n = 5). 5 heterozygotes were tested 23 times over the time period contributing to the calculated average and standard error. (c) Vertical movement expressed as rears per minute, symbols as in (a).

5 510 U. Andersson et al. / Neurobiology of Disease 16 (2004) mg kg 1 day 1 delayed the onset of the late symptomatic phase showing a close to normal performance on the bar until just after 100 days of age and by 135 days they all fell from the bar. The mice receiving the higher NB-DGJ dose (2400 mg kg 1 day 1 ) showed an even greater delay in symptom onset and all crossed the bar without difficulties until approximately 135 days of age. A nonlinear regression analysis was used to determine the medium time for the functional decline (d in Materials and methods). The age for the progressive decline in performance was significantly delayed in the 2400 mg kg 1 day 1 NB-DGJ group compared to all the other groups (P < 0.02). The two lowdose groups had similar age of decline, which were both significantly delayed as compared to untreated mice (P < 0.001). The overall symptomatic period was extended in all drug-treated groups, in the lower dose groups however more so than the high NB-DGJ dose group. The moribund end stage period was, as a proportion of life expectancy, similar for all the groups. Locomotion and rearing activity were measured using the open field test. Overall, mice treated with either dose of NB-DGJ were more active than untreated or NB-DNJ-treated mice (Figs. 2b,c). Both for horizontal movement and rearing, the general trend of activity before symptom onset was NB-DGJ (2400 mg kg 1 day 1 )>NB-DGJ (1200) > untreated > NB-DNJ. For horizontal movement during the presymptomatic period, the treatment groups were all significantly different (P < 0.05), apart from the untreated group compared to 1200 mg kg 1 day 1 NB-DGJ. For vertical movement (also during the presymptomatic period), the untreated and the NB-DNJ-treated groups were significantly different to the two NB-DGJ-treated groups (P < 0.05). Only NB-DGJ-treated groups (during the presymptomatic phase) were as active as heterozygote controls (but not significantly more active than the heterozygotes, i.e., not hyperactive). In agreement with the performance scored in the bar crossing test, a decline in activity/ rearing was first seen in the untreated group, then in the two lowdose groups (NB-DNJ and NB-DGJ), and finally in the high-dose NB-DGJ group. Brain GSL concentration in Sandhoff mice treated for 6 weeks Gangliosides GA2 and GM2 concentrations were analysed by histological and biochemical methods in brain from Sandhoff mice killed at 12 weeks of age after 6 weeks of drug treatment (1200 mg kg 1 day 1 of NB-DNJ or NB-DGJ). The histochemical analysis (Figs. 3a i) showed a decrease in GSL storage in imino sugartreated mice as compared to untreated controls, particularly in the hippocampal and the thalamic regions. This reduction was confirmed by TLC of a brain homogenate containing basal ganglia, hypothalamus, thalamus, midbrain, pons and medulla (Table 2), in which NB-DGJ-treated mice showed a 12 F 4% decrease in GA2 (P < 0.05) and 10 F 5% in GM2 (P = 0.09), and NB-DNJ-treated mice showed a 7 F 2% decrease in GA2 ( P = 0.15) and 6 F 2% in GM2 (P = 0.15). In addition to reducing neuronal storage, imino sugars delayed or reduced the onset of CNS inflammation in Sandhoff mice as assessed by MHC class II immunostaining (Figs. 3j l). Inhibitor levels in serum and brain were also assessed after 6 weeks of 1200 mg kg 1 day 1 NB-DNJ or 1200 or 2400 mg kg 1 day 1 NB-DGJ treatment (Fig. 4). In 1200 mg kg 1 day 1 NB- DNJ, the serum drug levels ranged from 13 to 55 AM with a weighted mean value of 29.9 F 2.1 and 28.4 F 1.5 AM for wildtype and Sandhoff mice, respectively. NB-DNJ levels in brain varied from 5 to 9 AM with a weighted mean value of 5.8 F 0.5M and 7.3 F 1.4 AM for wild-type and Sandhoff mice respectively. In the NB-DGJ-treated group, a dose-dependent increase was noted in both serum and brain. From 1200 to 2400 mg kg 1 day 1 NB-DGJ, the weighted mean values increased from 31.5 F 2.7 to 55.5 F 5.9 AM and 8.4 F 0.1 to 15.4 F 0.3 AM for serum and brain, respectively. Ratio of serum: brain was 4:1 for both NB-DNJ and NB-DGJ in Sandhoff mice. The measured brain NB-DNJ levels from Sandhoff mice was approximately 25% (P = 0.03) higher that that from the wild-type littermate mice, probably due to a compromised blood brain barrier (Jeyakumar et al., 2003). Brain GSL concentrations in Sandhoff mice at end point Liver and brain from Sandhoff mice killed at the humane end point were analysed for GSL concentration. TLC analysis of brain GSL showed that all homozygote Sandhoff mice displayed similar GSL patterns at their respective end points with elevated concentrations of GSLs GA2 and GM2 as compared to the heterozygote controls (Fig. 5a). Between the different treatment groups, there were, however, slight differences in this elevation. NB-DGJtreated mice showed a small increase in both GA2 and GM2 compared to untreated mice at their respective end points (age 124, 162 and 185 days, respectively, for untreated, lower dose NB-DGJ and higher dose NB-DGJ). The lower NB-DGJ dose (1200 mg kg 1 day 1 ) was associated with a 10 F 2% increase in GA2 and 26 F 0.8% for GM2 (P = 0.07 and 0.02, respectively), whereas the mice on the higher dose (2400 mg kg 1 day 1 )at their end point showed a 16 F 5% increase in GA2 and 31 F 9% in GM2 (P = 0.06 and 0.03, respectively) (Fig. 5b). Comparison of end point GSL concentrations to predicted levels GA2 and GM2 concentrations were determined by TLC at several time points throughout the life span of untreated Sandhoff mice (Fig. 6a). The GSL accumulation was found to be linear, and through regression analysis was calculated to be (expressed in Ag GSL per mg dry tissue): [GM2] = 0.12 age (days) (error coefficient r = 0.989) and [GA2] = 0.21 age (days) (error coefficient r = 0.983). In Fig. 6b, these equations are plotted (solid line) and extrapolated (dashed line) to calculate the predicted storage levels for ages beyond the natural life span of the Sandhoff mouse. The GA2 and GM2 levels determined in the present study plotted in Fig. 6b show that imino sugar treatment results in brain GSL accumulation below the predicted values at their respective end stages. This, together with the brain GSL reduction seen in the younger mice (Figs. 3a i; Table 2), indicates that although the treated mice show slightly increased brain storage at their respective end stages as compared to untreated mice, as predicted substrate reduction therapy with NB-DNJ or NB-DGJ is associated with reduced GSL accumulation (i.e., it has reduced the rate of storage). Liver GSL concentrations in Sandhoff mice at end point In liver, the GSL accumulation at the respective end stages was very different from that in brain. Here, NB-DGJ-treated mice displayed the lowest GSL levels (Fig. 7). The GA2 concentrations (Ag/mg dry tissue) compared to untreated homozygotes were 67 F 19% (P = 0.2), 56 F 9% (P = 0.02) and 42 F 6% (P = 0.003) for NB- DNJ (1200 mg kg 1 day 1 ), NB-DGJ (1200 mg kg 1 day 1 ) and

6 U. Andersson et al. / Neurobiology of Disease 16 (2004) Fig. 3. PAS staining (a i) and MHC class II immunoreactivity (j l) of brain from Sandhoff mice after 6 weeks of treatment. Sandhoff mice were treated with 1200 mg kg 1 day 1 of NB-DGJ or NB-DNJ for 6 weeks. Storage GSLs were visualised in frozen brain sections by PAS staining. Panels a c and d f show regions of the hippocampus. Panels g i show a representative region of the thalamus. Panels j l show a representative region of the brain stem immunostained for MHC class II. Panels a, d, g and j represent untreated mice, b, e, h and k NB-DGJ-treated mice, and c, f, i and l NB-DNJ-treated mice. Representative sections were chosen from each group. n =4. The scale bar represents 50 Am. NB-DGJ (2400 mg kg 1 day 1 ) treated, respectively. For GM2, they were 44 F 9% (P = 0.04), 39 F 3% (P = 0.02) and 36 F 6% (P = 0.02) for NB-DNJ (1200 mg kg 1 day 1 ), NB-DGJ (1200 mg kg 1 day 1 ) and NB-DGJ (2400 mg kg 1 day 1 ) treated, respectively. Discussion Previous studies of substrate reduction therapy (SRT) using NB- DNJ to treat Sandhoff mice showed increased life expectancy and delay in symptom onset (Jeyakumar et al., 1999). The adverse effects seen with high dose NB-DNJ treatment in mice (i.e., body weight decrease, lymphoid organ shrinkage, GI tract distress) limited dose escalation. Increasing the NB-DNJ dose above 1200 mg kg 1 day 1 did not improve the therapeutic outcome in Sandhoff mice (Jeyakumar et al., 1999). Using mainly male mice, life expectancies for Sandhoff mice were 175 days with 1200 mg kg 1 day 1 (treatment started at 4 weeks of age), 170 days with 2400 mg kg 1 day 1 (starting at 6 weeks), and 169 days with 4800 mg kg 1 day 1 (starting at 3 weeks). The higher life expectancy of 175 days at 1200 mg kg 1 day 1 of NB-DNJ as compared to the 161 days achieved in this study (females) is due to the slightly longer life expectancy of male Sandhoff mice (Andersson and Platt, unpublished) in addition to a slightly different definition of the humane end point. In the present study, due to animal welfare, mice were sacrificed as soon as the mice were unable to right themselves when

7 512 U. Andersson et al. / Neurobiology of Disease 16 (2004) Table 2 HPTLC analysis of brain GA2 and GM2 from Sandhoff mice after 6 weeks treatment UT NB-DGJ NB-DNJ GA2 100 F F 4.1* 93 F 2.0 GM2 100 F F F 1.8 n =4. Six-week-old Sandhoff disease mice were treated with 1200 mg kg 1 day 1 of NB-DNJ or NB-DGJ for 6 weeks. The mice were sacrificed, and GA2 and GM2 concentrations were determined by HPTLC in a homogenate containing basal ganglia, hypothalamus, thalamus, midbrain, pons and medulla. The values are expressed as a percentage of ganglioside levels in untreated mice. * P < 0.05 as compared to untreated. laid on their side, whereas in the earlier study the humane end point was applied when mice were almost completely moribund and were unable to feed. The results of the present study showed that NB-DGJ treatment of Sandhoff mice, in keeping with previous studies in healthy mice (Andersson et al., 2000), caused no overt adverse effects and the life expectancy increased with increased dose. At 1200 mg kg 1 day 1, the mice on NB-DNJ and NB-DGJ had similar life expectancy and similar decline in behavioural performance. The NB- DGJ mice, however, had higher body weights and were more active in the open field test showing the improved functional benefits of NB-DGJ treatment. When the NB-DGJ dose was further increased to 2400 mg kg 1 day 1, the Sandhoff mice lived longer than the lower dose group, and their decline in motor coordination, strength, and activity were further delayed. The low activity in the open field test by NB-DNJ mice is probably a cause of the general stress these mice are under because of lack of growth (reduced skeletal growth and body mass) and GI tract distension (particularly of the caecum, due to disaccharidase inhibition) found in mice on high dose NB-DNJ (Andersson and Platt, unpublished observation). The NB-DGJ fed mice were significantly more active than both NB-DNJ-treated and untreated mice and had activities near or slightly above that of heterozygote (asymptomatic) mice. NB-DGJtreated mice were either healthier with greater motor function and/ or had a higher motivation to explore. However, results from an exploration test, the hole board test (Lister, 1987), showed no differences in their motivation to explore between the different groups of mice (data not shown). This indicates that differences in the open field are not motivation-based, but are due to difference in motor strength and adverse side effects of NB-DNJ. Of some concern when treating the Sandhoff disease mice in a previous study with NB-DNJ was the prolonged end stage of the disease (Jeyakumar et al., 1999). In the present study, the symptomatic period was prolonged with NB-DNJ treatment but the end stage was not. As with the difference between the two studies in life expectancy, this could be explained either because a different gender was used in the present study (females), or more likely by the slightly earlier defined humane end point applied in this study as compared to Jeyakumar et al. (1999). Although both NB- DNJ and NB-DGJ at 1200 mg kg 1 day 1 prolonged the symptomatic phase, NB-DGJ fed mice at 2400 mg kg 1 day 1, with further prolonged life expectancy, displayed a steep decline in motor coordination and muscle strength with a symptomatic period shorter than for the lower doses. As a percentage of life expectancy, the symptomatic period in 2400 mg kg 1 day 1 NB-DGJ-treated mice was similar to that of untreated mice. The basis of SRT suggests that the therapeutic effect is due to a decrease in GSL influx into lysosomes and therefore a reduced rate of accumulation of storage material. The only known activity shared by NB-DNJ and NB-DGJ is inhibition of GSL biosynthesis. The present study showing therapeutic benefits of both NB-DNJ and NB-DGJ therefore supports the hypothesis of substrate reduction as the underlying mechanism and excludes other activities of NB-DNJ such as glucosidase inhibition and body weight loss (caloric restriction). In support of this, data in the present study from Sandhoff mice treated for 6 weeks demonstrated a reduction in brain GA2 and GM2 in NB-DNJ and NB-DGJ-treated mice. This was true to an even greater extent in liver. The liver in common with other peripheral organs is exposed to higher levels of inhibitor, whereas only approximately 19 25% of the serum level is reflected in the brain. The percentage of serum level achieved in brain is higher than values measured in single-dose pharmacokinetics studies (Platt et al., 1997a), presumably reflecting the steady state concentration or the fact that in those studies CSF was compared with serum not brain tissue. The present study also shows, in agreement with previous reports, that NB-DNJ-treated mice with their prolonged life expectancy have similar concentrations of brain gangliosides at their end Fig. 4. Serum and brain imino sugar levels after 6 weeks of treatment in wild-type and Sandhoff mice. Data are weighted mean F SEM (n = 6 7 per group). Brain imino sugar concentration was determined by volume standardisation against tissue weight (1 mg = 1 Al) assuming the density of brain equals the density of water.

8 U. Andersson et al. / Neurobiology of Disease 16 (2004) Fig. 5. Ganglioside analysis of brain from Sandhoff mice at their respective end points. Sandhoff mice were treated with imino sugars from 6 weeks of age until the humane end point was reached and the mice were sacrificed. The groups included homozygote Sandhoff mice treated with control diet (average age 124 days), with 1200 mg kg 1 day 1 NB-DNJ (age 161 days), with 1200 mg kg 1 day 1 NB-DGJ (age 162 days), or with 2400 mg kg 1 day 1 NB-DGJ (age 185 days), or control heterozygote mice (normal life span, killed at same age as homozygotes). (a) Brain glycolipids separated by HPTLC. The extracts loaded were standardised to 0.4 mg dry brain weight. (b) Quantification of GA2 and GM2 bands by scanning densitometry (n =3).*Statistically significant (P < 0.05). stage as do the untreated mice (Jeyakumar et al., 1999); that is, treatment has slowed the rate of storage as predicted by the mechanism of action of the drug. NB-DGJ-treated mice, however, in particular at higher dose when survival was maximal, show slightly elevated storage levels of GA2 and GM2 in brain at their end stage. The brain ganglioside levels were, however, not as high as would be expected in Sandhoff mice of that age with linear accumulation of storage GSLs. In particular, the accumulation of GA2 was reduced as compared to expected values, which agrees well with observations in mice that received bone marrow transplants (BMT) where the GA2 rather than GM2 levels show correlation with disease progression (Jeyakumar et al., 2001). The longer life expectancy and the delay in disease onset in these mice, with a higher degree of storage, imply that the level of brain ganglioside storage alone does not dictate disease progression. This has also been observed in BMT Sandhoff mice where the transplanted cells prolonged life expectancy despite a continued accumulation of brain GSLs (Jeyakumar et al., 2001; Norflus et al., 1998). In this case, it may be a delay in the inflammatory response to storage, which is important (Jeyakumar et al., 2003; Wada et al., 2000). Consistent with previous reports, reduced or delayed CNS inflammation was evident in Sandhoff mice treated with either NB- DNJ or NB-DGJ, as compared to the untreated age match controls. This suggests the level of neuronal storage can influence the kinetics of immune activation in the CNS. The liver ganglioside data in the present study suggests the importance of peripheral GSL depletion where the longest living mice (2400 NB-DGJ) showed the lowest liver ganglioside concentration when comparing mice at their respective end stages. The relationship between peripheral storage and CNS storage in disease progression is currently unknown. The difference in GSL depletion between brain and liver in the present study simply reflects greater accessibility of drug in the liver when compared to brain. The clinical relevance of the present findings might be of importance. Efficacy of NB-DNJ has been demonstrated in clinical trials in Gaucher type 1 patients (low oral dosing of approximately 4 mg kg 1 day 1 leading to a plasma level of 2 5 AM (Cox et al., 2000), equivalent to mg kg 1 day 1 in mice (Platt et al, unpublished data)). NB-DNJ has recently been licensed in Europe, Israel, and the USA for use in the treatment

9 514 U. Andersson et al. / Neurobiology of Disease 16 (2004) Fig. 6. Measured vs. predicted ganglioside levels in the Sandhoff mouse brain. (a) Brain ganglioside concentrations in untreated Sandhoff mice were determined by HPTLC. A linear relationship between age and accumulated gangliosides GA2 (o) and GM2 (5) was found over the natural life span. At least two animals were used for every point. (b) Linear regression and extrapolation of the values in panel a (solid line) give predicted ganglioside values for mice aged beyond the normal life span of the Sandhoff mouse (dashed line). Plotted in the figure are experimental GA2 and GM2 concentrations determined in the present study (Fig. 5) for untreated (5), treated with 1200 mg kg 1 day 1 NB-DNJ ( w )ornb-dgj (y), or treated with 2400 mg kg 1 day 1 NB-DGJ (). (Values for 1200 mg kg 1 day 1 NB-DGJ and NB-DNJ overlap in the GA2 graph resulting in a half solid half clear symbol.) The determined GSL concentrations as percentages of calculated values are tabulated. Fig. 7. Ganglioside analysis of liver from Sandhoff mice at their respective end points. Sandhoff mice were treated with imino sugars from 6 weeks of age until the humane end point was reached and the mice were sacrificed. The groups included homozygote Sandhoff mice treated with control diet (average age of 124 days), with 1200 mg kg 1 day 1 NB-DNJ (age 161 days), with 1200 mg kg 1 day 1 NB-DGJ (age 162 days) or with 2400 mg kg 1 day 1 NB-DGJ (age 185 days). Liver gangliosides (GA2 and GM2) were extracted, base treated, separated by HPTLC and quantified by scanning densitometry according to Materials and methods (n = 3). *Statistically significant ( P < 0.05).

10 U. Andersson et al. / Neurobiology of Disease 16 (2004) of type 1 Gaucher disease (for patients with mild to moderate disease unwilling or unable to receive enzyme replacement therapy). In patients with CNS involvement, it may be necessary to use higher dose therapy (relative to the Gaucher clinical trial) to maximise the level of compound in the CNS. With NB-DNJ, there is the risk of increasing side effects at high doses. Individuals with juvenile or adult onset gangliosidoses are predicted to benefit the most from SRT due to the presence of some residual enzyme activity in these individuals. We would predict that NB-DGJ would be the drug of choice for this group due to the likely tolerance of high-dose therapy without inducing weight loss or GI tract side effects. Weight loss is particularly undesirable in paediatric patients due to its negative effects on growth. Combination studies of NB-DNJ and BMT in the Sandhoff mice have shown synergy between the enzyme augmenting therapy (BMT) and the substrate-lowering drug (NB-DNJ) (Jeyakumar et al., 2001). It will be interesting to see if even greater synergy is achieved with high-dose NB-DGJ in combination with enzyme augmentation. In summary, NB-DGJ shows greater efficacy than NB-DNJ in a mouse model of Sandhoff disease because its dose can be increased beyond that of NB-DNJ. The lack of side effects in mice treated with NB-DGJ and the capacity to escalate dose suggest this compound merits evaluation as a potential therapy for the human GSL storage diseases with CNS involvement. Acknowledgments UA is supported by BBSRC and the British Council. FNP is a Lister Institute Research Fellow. MJ is supported by The Wellcome Trust. 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