Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.
These authors contributed equally: Michael C. Babcock and Christina R. Mikulka.
Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are severe, dysmyelinating or demyelinating diseases of the peripheral and central nervous systems (PNS and CNS)[
Graph: Figure 1 Synthesis and catabolism of galactosylceramide and sulfatide. CGT catalyzes the synthesis of GalCer by adding a galactose molecule to ceramide (A). Ceramide with a hydroxy group at the 2 position (dashed circle) is a more efficient CGT substrate than non-hydroxyceramide. CST catalyzes the sulfation of GalCer to generate both non-hydroxy and 2-hydroxy-sulfatide (B). GalCer and sulfatide are degraded in the lysosome by the enzymes GALC and ARSA, which are deficient in KD and MLD, respectively (C,D). GALC and ARSA deficiency results in lysosomal accumulation of these glycolipids and their subsequent deacylation into the toxic byproducts psychosine and lysosulfatide by acid ceramidase (E). Previous substrate reduction approaches using L-cycloserine targets a reaction upstream from ceramide synthesis (F).
UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45), encoded by the UGT8 gene, catalyzes the addition of galactose from UDP-galactose to ceramide to generate GalCer (Fig. 1A). Ceramide galactosyltransferase can utilize either 2-hydroxylated or non-hydroxylated ceramides as a substrate; however, 2-hydroxylated ceramides have been shown to be more efficiently galactosylated compared to non-hydroxylated forms[
The discovery of small molecules that inhibit the synthesis of galactosylceramide-based glycolipids that accumulate in KD and MLD is an area of active research[
We have discovered a novel, potent, selective, and brain-penetrant small molecule inhibitor of CGT (S202) and determined its potential as an SRT to treat KD and MLD. When administered to mice, S202 significantly decreased psychosine accumulation and increased life span of GALC-deficient Twitcher mice without overt toxicity. Similar doses of S202 reduced lysosulfatide levels in ARSA-deficient MLD mice. However, a closer examination of wild-type mice treated with S202 revealed negative impacts of CGT inhibition on myelin rich tissues including brain, spine, and peripheral nerves. In total, S202 or its analogs provide a critical step forward toward the development of a safe and efficacious SRT for KD and MLD.
A novel CGT inhibitor was identified by screening a small, focused library of bioactive lipids for inhibitors of CGT catalytic activity. Human CGT was obtained from lysates of CHO cells expressing human UGT8, the gene encoding CGT. A single compound was identified from this screen that weakly inhibited CGT activity (Fig. 2A). This molecule had previously been described as a cannabinoid receptor (CB1) inverse agonist[
Graph: Figure 2 Identification of a novel potent and brain penetrant CGT inhibitor. S221 was identified in a screen of a focused bioactive lipid library (A). S221 was determined to be an oxidation product of (CAY10508), which had acquired a hydroxyl group (in red) on the central ring. Through a medicinal chemistry effort S202 was discovered as a more potent CGT inhibitor (B) showing a 400-fold increased potency in a CGT enzyme assay (C) and single digit nanomolar potency in a CGT cellular assay (D) (n = 2 per concentration). Mouse pharmacokinetic (PK) and brain concentration profile graph (n = 3, E) and PK parameter table (F) for S202 after a single IP injection show favorable in vivo half-life and brain penetration properties.
Following identification of the initial low potency CGT inhibitor S221, a series of structural analogs with increased potency were synthesized[
Ceramide galatosyltransferase mRNA expression peaks in mice during periods of major myelin synthesis, corresponding roughly to postnatal days (PND) 10–30 for the CNS and PND 6–15 for the PNS[
Graph: Figure 3 Tolerability and biochemical changes in young mice treated with S202. Weight profiles of neonatal mice (n = 4–19) treated with daily IP S202 (mg/kg/day) from PND3 to PND40 (A) and occurrence of animal deaths and development of abnormal gait (B). LC–MS quantification of brain non-hydroxy-GalCer (C), 2-hydroxy-GalCer (D), non-hydroxy-sulfatide (E), and 2-hydroxy-sulfatide (F) from mice treated with S202. Sciatic nerve non-hydroxy-GalCer (G), 2-hydroxy-GalCer (H), non-hydroxy-sulfatide (I), and 2-hydroxy-sulfatide (J) from mice treated with S202. Refer to the Materials and Methods section for significance levels.
GalCer and sulfatide levels in S202-treated mice were monitored by LC–MS/MS. Lower doses (0.015 to 0.5 mg/kg) of S202 greatly decreased non-hydroxy-GalCer levels in the brains of wild-type mice without reducing 2-hydroxy-GalCer (Fig. 3C,D). Remarkably, lower doses of S202 were found to consistently increase 2-hydroxy-GalCer levels. However, higher doses of S202 (> 1.5 mg/kg), reduced synthesis of both non-hydroxy-GalCer and 2-hydroxy-GalCer. Like their respective GalCer precursors, treatment with lower doses of S202 selectively reduced non-hydroxy-sulfatide and correspondingly increased 2-hydroxy-sulfatide, while higher doses reduced total sulfatide levels (Fig. 3E,F). While these data represent the sum response of all acyl chain lengths, a similar response was seen when individual acyl chains were monitored (see Supplementary Fig. S1 online). Preferential reduction in non-hydroxy-GalCer and non-hydroxy-sulfatides relative to the hydroxylated versions were also seen in the sciatic nerve. However, the effect at a given dose appeared greater in the sciatic nerve relative to the brain. For example, a dose of 0.5 mg/kg increased 2-hydroxy GalCer and 2-hydroxy sulfatide in the brain while partially reducing these molecules in the sciatic nerve (Fig. 3G–J).
To evaluate the therapeutic potential of CGT inhibition in a relevant disease model, we treated Twitcher mice with S202. Mice received IP injections of S202 three times per week starting on PND3 and disease progression was monitored by the onset of clinical signs and survival. The median lifespan of untreated or vehicle treated Twitcher mice is ~ 40 days[
Graph: Figure 4 CGT inhibition extends survival and reduces psychosine levels in a KD mouse model. Survival curves of Twitcher mice treated with S202 injected IP three times per week starting on PDN3 (A) and median days of survival (B). Untreated wild-type (UT WT) mice survived while vehicle treated Twitcher mice (vehicle) had a median survival of 43 days. S202-treated Twitcher mice survived longer except at the highest dose tested (1.5 mg/kg). Effect of S202 treatment on non-hydroxy-GalCer (C) and 2-hydroxy-GalCer in brain homogenates from mice treated from PND3-PND28 (D). Psychosine reduction in these brains correlates with reduction of non-hydroxy-GalCer (E) (n = 5–8, C–E). Quantification of psychosine generated by recombinant acid ceramidase incubated with either purified 2-hydroxy-GalCer or non-hydroxy-GalCer substrates (n = 2 per condition, F).
A separate cohort of Twitcher mice was dosed and sacrificed at PND28 to evaluate the biochemical and histological effects of CGT inhibition. As observed in wild-type animals, S202 produced a dose-dependent reduction in non-hydroxy-GalCer without reducing 2-hydroxy-GalCer in the brain (Fig. 4C,D). Surprisingly, psychosine levels were reduced to the same extent as the non-hydroxy-GalCer, even though 2-hydroxy-GalCer was not reduced (Fig. 4E).
To investigate the mechanism for this correlation between psychosine and non-hydroxy-GalCer but not 2-hydroxy-GalCer, we examined the ability of recombinant acid ceramidase to deacylate non-hydroxy-GalCer and 2-hydroxy-GalCer in vitro. We recently showed that psychosine is produced through deacylation of GalCer by acid ceramidase, rather than addition of galactose to sphingosine[
The effects of CGT inhibition on brain pathology were examined by immunofluorescent staining for inflammatory markers and myelin basic protein in PND28 Twitcher mice. Treatment with 0.15 mg/kg S202 IP three times per week showed a trend toward reduction in the levels of CD68-positive macrophages (Fig. 5A,B) and a marker of astrocytosis (GFAP, Fig. 5C,D) in Twitcher mouse brain sections. A significant reduction was seen in a marker of myelin degeneration (MBP, Fig. 5E,F). Cytokine analysis was also performed on brain lysates from the same animals treated with S202. Treatment reduced the levels of the macrophage markers MCP-1 and GROA in brain homogenates (Fig. 5G,H). Twitcher mice had disorganized myelin with a relatively large number of intensely staining puncta throughout the white matter tracts of the cerebellum (Fig. 5E). This led to an overall increase in myelin staining compared to wild-type mice (Fig. 5F). The myelin in S202-treated animals remained disorganized but had an apparent decrease in the number of intensely staining puncta.
Graph: Figure 5 CGT inhibition improves histological and immunological markers in Twitcher mice. Brain sections from Twitcher (Twi) mice treated with 0.15 mg/kg S202 by IP injection three times per week from PND3 to PND28 were immunologically stained for CD68, GFAP and MBP (A,C,E) and quantified (B,D,F). Percent immunoreactivity represents the area of staining within the region analyzed (cerebellar white matter, 100%). Cytokine levels from brain homogenates harvested from the same mice were analyzed and MCP-1 and GROA were reduced upon S202 treatment (G,H). N = 3–4 (A–H).
To determine the effects of CGT inhibition at an age that is roughly equivalent to the myelination status of a 1 to 2-year-old human, Twitcher mice were treated with varying doses of S202 IP three times per week starting at PND15[
Graph: Figure 6 Older Twitcher mice better tolerate S202 but higher doses are required to increase survival. Survival curves of Twitcher mice treated with S202 starting on PND15 (A) and median days of survival (B). Treating Twitcher mice starting from PND15 with S202 dose dependently reduced non-hydroxy-GalCer (C), 2-hydroxy-GalCer (D), and psychosine (E) (n = 3–4).
Lysosulfatide accumulation in MLD is analogous to psychosine accumulation in KD (Fig. 1E). The ARSA-deficient mouse model of MLD has a very mild disease course; however, MLD mice do accumulate sulfatide and its cytotoxic metabolite, lysosulfatide[
Graph: Figure 7 S202 treatment produced similar effects on sulfatides in an MLD mouse model. ARSA knockout mice were treated with S202 from PND3 to PND28 (n = 4, A–C) or PND15 to PND40 (n = 4, D–F). Effect of S202 treatment on non-hydroxy-sulfatide (A,D) and 2-hydroxy-sulfatide in brain homogenates (B,E). Like psychosine in Twitcher mice, lysosulfatide reduction in brain correlated with reduction of non-hydroxy-sulfatide (C,F).
Due to the essential role of GalCer and sulfatides in myelin formation, we characterized the longer-term (8 week) tolerability to S202 treatment. Wild-type mice were treated IP with S202 three times per week at doses lsimilar to those that extended median Twitcher survival to ~ 60 days when treatment started on PND15 (2.3 mg/kg and 5.4 mg/kg). Generally, treatment with these dose levels were well tolerated with no mortality, change in weight, or grossly abnormal clinical signs. Clinical chemistry and hematology analysis revealed no significant effects on systemic health (see Supplementary Table S2 online). These mice also lacked histopathological changes in the major peripheral organs, with the notable exception of the testes and brain.
The testes of treated mice showed histopathological changes that were identical to those described in CGT deficient mice (see Supplementary Fig. S2 online)[
While seemingly well tolerated, some mice treated chronically with higher (2.3 and 5.4 mg/kg) doses of S202 developed a mild gait abnormality and impaired performance on the wire hang test (Fig. 8A). We hypothesized that these behavioral deficits resulted from CGT inhibition affecting PNS function. Therefore, nerve conduction velocity (NCV) was measured in the sciatic nerve of treated and untreated animals. There was a significant and dose-dependent reduction in NCV with treatment (Fig. 8B). Light microscopic examination of sciatic nerve did not reveal obvious structural abnormalities. However, quantitative g-ratio analysis of toluidine blue stained sections revealed a trend towards increased large diameter axons with decreased myelin thickness (Fig. 8C).
Graph: Figure 8 Chronic CGT inhibition affects PNS and CNS function and integrity. Wild-type mice were treated with S202 by IP injection three times per week for 8 weeks starting on PND15. The ability of mice to hang from a wire mesh was evaluated weekly starting on week 2 of treatment (A). Sciatic NCVs were recorded from mice that had been treated with S202 (B). G-ratio analysis of sciatic nerve (C). H&E stained brain sections from S202 revealed vacuolation in S202 treated animals at either dose (E arrow) that was not seen in vehicle treated animals (D). Severity of vacuolation was scored by a trained pathologist on a scale of 1–4 in white matter (F) or gray matter (G) in brain and spinal cord. (n = 6–12).
The impact of chronic S202 treatment on the CNS was evaluated by both H&E and luxol fast blue/PAS (myelin) staining of the brain and spinal cord, revealing widespread vacuolation (Fig. 8D,E). In the brain, gray matter vacuolation occurred in specific subsites, but was generally most extensive in the cerebral cortex, basal ganglia, and thalamus, with less involvement in the hippocampus, pons/medulla, and cerebellum. In all affected spinal cord segments, grey matter vacuolation was randomly scattered throughout the central area and ventral and dorsal horns. White matter vacuolation in brain/spinal cord was sporadically distributed in various subsites and characterized by variable size and sharply demarcated extracellular vacuoles scattered through the neuropil. Scoring vacuolation severity by a U.S. board certified pathologist revealed that white matter vacuolation was greater in the brain and upper spinal regions while grey matter vacuolation was more evenly distributed in all brain and spine regions (Fig. 8F,G).
The effects of longer-term CGT inhibition on GalCer levels were evaluated in the same wild-type mice. The selective reduction of non-hydroxy-GalCer and relative sparing of 2-hydroxy-GalCer levels observed in the animals receiving short-term S202 was observed in chronically treated animals (see Supplementary Fig. S3 online). We hypothesized that inhibition of CGT might also alter ceramide levels, which could negatively impact chronically treated animals. While there was only a small increase in non-hydroxy-ceramide in the animals injected with S202 (see Supplementary Fig. S3 online), there was a dramatic increase in 2-hydroxy-ceramide in S202-treated mice (see Supplementary Fig. S3 online).
In Twitcher mice treated with S202 starting at PND3, much lower doses (> 30 fold less) were needed to provide the same survival benefit observed when treatment was started at PND15. Based on these findings, we speculated that the optimal dose in younger mice was due to a balance between sufficient CGT inhibition and long-term tolerability and that this low dose level may not produce the vacuoles observed at higher dose levels. To test this hypothesis, wild-type mice were treated from PND3 to PND70 with 0.15 mg/kg S202 and brain, spine, and sciatic nerve were examined for signs of vacuolation. Surprisingly, even at this low dose, vacuolation was observed in a similar pattern, but was much less severe to that seen at higher doses (see Supplementary Fig. S4 online).
This study describes the identification and characterization of S202, a potent and selective inhibitor of CGT with favorable drug-like properties. While having single digit nanomolar potency toward CGT, this inhibitor only weakly inhibited the CB1 receptor (IC
Treating wild-type mice with lower doses of S202 selectively reduced non-hydroxy-GalCer synthesis with a corresponding increase in 2-hydroxy-GalCer. While the nature of this selective reduction is unknown, 2-hydroxy-Cer is a significantly more efficient substrate for CGT than non-hydroxy-Cer, possibility explaining the dose-dependent reduction of one product over the other[
The ability to selectively reduce non-hydroxy-GalCer could have important therapeutic implications for KD. The reduction in non-hydroxy-GalCer correlated with the reduction in psychosine in the Twitcher mice, even while 2-hydroxy-GalCer levels were unchanged or even elevated. We also found that recombinant acid ceramidase more efficiently catabolizes non-hydroxy-GalCer to produce psychosine compared to 2-hydroxy-GalCer. Collectively, these data suggest non-hydroxy-GalCer is the primary source of psychosine and are consistent with the proportional reduction of non-hydroxy-GalCer and psychosine following CGT inhibition in vivo.
Treating neonatal Twitcher mice with S202 significantly increased survival. However, PND3 is an age in mice that precedes both CNS and PNS myelination[
When treatment was started on PND15, the percentage of non-hydroxy-GalCer and psychosine reduction in brain was lower compared to PND3 treatment start. Similar effects were seen for non-hydroxylated sulfatide and lysosulfatide (Fig. 7). This diminished response is likely due to preexisting non-hydroxy-GalCer, non-hydroxylated sulfatide, and/or their deacylated metabolites (psychosine and lysosulfatide) prior to treatment start on PND15. In addition, the higher doses used in PND15 mice resulted in reduction of total GalCer levels, including non-hydroxy-GalCer, psychosine, and 2-hydroxy-GalCer. These results suggest that reduction of total GalCer, not just non-hydroxy-GalCer and psychosine, contribute to survival benefit when treatment starts later in development.
Substrate reduction therapy targeting major lipid components of myelin membranes presents several challenges. These glycolipids are essential for healthy myelin formation and chronic inhibition of their synthesis could potentially destabilize myelin. Furthermore, once synthesized and incorporated into normal myelin, these molecules have half-lives approaching 1 year or more in humans[
Although generally well-tolerated, chronic administration of higher doses of S202 to wild-type mice resulted in dose-dependent physiological (NCV), behavioral (wire hang), and histological (vacuolation) changes. While vacuolation occurred in both grey and white matter of the spine and brain, the severity in white matter was greater in regions undergoing myelination during the treatment period and less in regions that had previously undergone significant myelination. This supports the hypothesis that tissue myelinated at treatment initiation is more resistant to adverse effects of CGT inhibition. In contrast, vacuolation observed in grey matter did not coincide with the timing of myelination and was relatively uniform in distal spine regions and brain, possibly indicating a novel role for CGT in non-myelinating cells such as astrocytes, microglia, neurons, or endothelial cells. The severity, distribution, and nature of vacuolation observed in our study is similar to CGT deficient mice as well as mice with reduced GalCer levels due to a deficiency in the Cers2 gene[
Although treatment of Twitcher mice with S202 resulted in significantly increased life span and decreased neuroinflammation, the improvements were modest. These results are consistent with numerous previous studies showing monotherapies result in minor therapeutic benefit in Twitcher mice. However, studies have also found dramatic and synergistic effects by combining disparate approaches that target different pathogenic mechanisms. For example, while SRT using L-cycloserine alone produces an incremental increase in survival, benefits are significantly enhanced when combined with therapies like bone marrow transplant or gene therapy. A rational combination approach might also require lower doses of a CGT inhibitor, thereby decreasing the dose-dependent toxicity observed with S202. Thus, the true potential of inhibiting CGT to treat patients with infantile KD might be observed when used in combination with other therapies. Also, juvenile and adult-onset forms of KD might respond better to CGT inhibition compared to infantile forms not only because they progress more slowly but because they also retain residual enzyme activity that can slowly metabolize lysosomal GalCer.
Ceramide galactosyltransferase inhibition reduces non-hydroxy-sulfatide and 2-hydroxy-sulfatide and could also be used to treat MLD. MLD is a less severe lipid storage disease potentially due to the lower abundance of sulfatide in myelin compared to GalCer. Because MLD is slower progressing, CGT inhibition might produce greater clinical benefits. Also, a large proportion of MLD patients are juvenile and adult-onset and could significantly benefit from lower-dose CGT inhibition. Recently, autologous stem cell gene therapy (SCGT) has resulted in significant clinical improvements in presymptomatic MLD patients[
In conclusion, we have identified a novel CGT inhibitor with favorable drug-like properties that will be a valuable tool for exploring the role that GalCer and sulfatides play in normal development and disease. This inhibitor will be useful in exploring how galactosylceramides contribute to the biology of myelin, testes, and other non-myelinating cells. The ability to selectively inhibit the synthesis of non-hydroxylated galactosylceramides should be a useful attribute to enable the further understanding of the roles of the different hydroxylated species. Given that CGT inhibitors are actively being pursued as potential SRT therapy for KD and MLD[
These studies were carried out in compliance with the ARRIVE guidelines. For these unblinded, descriptive, and novel exploratory research studies, samples sizes were chosen arbitrarily and adjusted as needed. Most normal mouse, disease model, and tolerability studies were repeated independently, at least twice, at different research facilities, both academic and contract research organizations, without prior knowledge of expected outcomes. Several analogs of S202 were also tested and produced consistent findings for survival, selective biochemical changes, and tolerability. Most endpoints were determined prospectively. No data or outliers were excluded from the analysis unless noted in the methods. All methods were carried out in accordance with relevant guidelines and regulations.
Human CGT was obtained from a cell lysate from CHO cells expressing UGT8 (made by BioMarin) prepared in M-PER with proteinase inhibitors (Sigma). Compounds, serially diluted in DMSO and Tween 80, were added to 4 µg/well of CHO/CGT cell lysate to make 20 µL final reaction volume containing 0.5% DMSO and 0.01% Tween 80 in the reaction buffer consisting of 10 µM NDB-(2R-OH)-C6 ceramide (made by BioMarin), 17.5 µM UDP-Galactose (Sigma), 35 µM of dioleoylphosphatidylcholine (DOPC), 5 mM MgCl
Modified from Larsen et al.[
A 50 µL reaction consisting of 4 µg MPER lysate prepared from CHO cells stably expressing GAL3ST1 cDNA construct (made by BioMarin), 3 µM N-dodecanoyl-NBD-galactosylceramide or 10 µM PAPS (Sigma) added as substrate (in DMSO/Tween 80), reaction buffer (100 mM Tris/HCl (pH 7.0), 2.5 mM ATP, 20 mM MgCl
100 µL assay buffer (50 mM Tris buffer pH 7.5 containing 25 mM KCl, 0.5 mM EDTA, 10 µM NBD-C6 ceramide, 100 µM DOPC, and 0.01% Tween 80), 60 µg MDCK cell lysate, and serial diluted compounds were incubated at 37 ℃ for 1 h. The reaction was terminated and prepared for LC–MS using the same protocol as the GCS assay, however, a NBD-C6-sphingomyelin (Avanti) reference standard was used to quantify the sphingomyelin synthesis.
Recombinant human glucosylceramidase (R&D Systems) activity was monitored using the artificial fluorescent substrate 4-methylumbelliferyl-β-d-glucopyranoside (Sigma) using the assay condition on the enzyme product datasheet. Recombinant human galactosylceramidase (R&D Systems) activity was monitored using the artificial fluorescent substrate 4-methylumbelliferyl-β-d-galactopyranoside (Sigma) using the assay conditions described on the enzyme product datasheet.
A postnuclear supernatant (PNS) was prepared from HEK293 cells transiently expressing a GBA2 cDNA construct. 5 µg of PNS was added to pH 6 McIlvaine's Buffer (0.2 M Na
The ARSA assay was modified from a previously reported method[
UGT1A activity was evaluated at Eurofins Panlabs Taiwan, Ltd. ITEM 196000.
CB1R activity was evaluated at Eurofin Cerep, Le Bois l'Evêque, France ITEM G012.
Maximum obtainable terminal blood collection was taken via cardiac puncture and split. 200 µL into clot-activating tubes for serum processing and the following chemistry panel: AST, ALT, CKMB and electrolytes. 220 µL stored on wet ice and used for CBC.
3 × 10
25 µM of 2-hydroxy-GalCer or non-hydroxy-GalCer (Matreya) were incubated with 7 µL of purified acid ceramidase in a reaction containing 15 µL of 0.2 M citrate phosphate buffer (pH 4.5), 2.25 µL of 2 M NaCl, 1.5 µL of 10 mg/mL BSA, and 0.3 µL 10% IGEPAL CA630. The reaction was incubated at 37 °C for 18 h without agitation and then stopped by adding 60 µL of acidified methanol. Psychosine formation was determined by LC–MS. Briefly, samples were extracted in acetonitrile containing d5-glucosysphingosine (Avanti) IS and analyzed using a Shimadzu HPLC/Autosampler with Halo-Hilic (150 mm, 5 µm) and an API-4000 Qtrap Mass Spectrometer (ESI positive, multiple reaction monitoring [MRM] scan). Mobile phase A was 0.5% FA, 5 mM NH
All procedures were carried out in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols and the BioMarin Animal Resource Committee (ARC). Twitcher mouse studies were conducted using a protocol approved by the Washington University School of Medicine IACUC. PND3 wild-type mouse studies and ARSA -/- studies were conducted using a protocol that was approved by the Shanghai ChemPartner Co. IACUC. PND15 wild-type mice tolerability studies were conducted using a protocol approved by the Jackson labs IACUC. For all animal studies S202 was formulated in 5% dimethylacetamide (Sigma-Aldrich), 10% Solutol HS15 (Sigma-Aldrich), 85% 10 mM citrate buffer (pH 4.5).
C57BL/6 mice were given a single 20 mg/kg IP injection of S202 and plasma pharmacokinetics and brain tissue levels were monitored (n = 3 per timepoint). Blood was collected in K
Wild-type C57BL/6 mice were given daily IP injections of S202 from PND3 to PND40 and weight and clinical signs were monitored daily. Animals were euthanized by exsanguinations under deep anesthesia, the brain and sciatic nerve were removed, rinsed in cold saline, dried, and snap frozen for lipid quantification.
Mice heterozygous for the Twitcher mutation on the congenic C57BL/6 background were originally purchased from The Jackson Laboratories (stock #000845). Homozygous GALC −/− animals were generated from heterozygous mating. Genotypes were determined on PND1 by PCR as previously described[
ARSA mice were obtained from Volkmar Gieselmann, University of Bonn. ARSA −/− mice were generated from heterozygous pairing and confirmed by PCR genotyping. Treatment IP with S202 began on either PND3 or PND15 and continued until PND28 or PND40 respectively. Animals were euthanized by exsanguination under deep anesthesia and brain and sciatic nerve removed, rinsed in cold saline, dried, and snap frozen for lipid quantification.
Treatment IP with S202 began on PND15-16 and continued three times/week until PND71. Animals were euthanized with CO
Beginning on the 2nd week of treatment mice were trained weekly for two weeks. Mice are placed on a wire grid, which is shaken to increase wire grabbing, and then immediately turned over. Mice are timed for their ability to hang from the inverted grid for 1 min. Three trials are performed, up to 1 min each, with a 30 s recovery period between trials. After training, mice were tested weekly and times recorded. The test was done with the grid held 10–12 in. over a box of clean shavings.
Whole brain was homogenized in 3 volumes H
Previously diluted brain homogenate was serially diluted (10 µL into 90 µL and then 20 µL into 80 µL) in ACN/H
Galactosylsphingosine (psychosine), and galactosylceramide were measured by tandem mass spectrometry as previously described[
Sulfatides were quantified using the methods described above. For lysosulfatide, 30 µL of brain homogenates were added to 200 µL ACN with IS (N-acetylsulfatide, Matreya), vortexed for 10 min, and centrifuged at 5800 rpm for 10 min. 3 µL of supernatant was injected for LC–MS/MS analysis. UPLC–MS/MS methods were the same used for sulfatides described above except lysosulfatide (Matreya) was used as a reference standard.
The right midsagittal brain hemisphere was harvested, wet weights recorded, and then homogenized in 1 mL and sciatic nerve in 300 µL of H
Five sections from a one in four series of sagittal brain sections from each mouse were stained on slides using a modified immunofluorescence protocol for the following antibodies—astrocytes (rabbit anti-GFAP, 1:1000, DAKO Z0334), microglia (rat anti-mouse CD68, 1:400, Bio-Rad MCA1957), and myelin basic protein (MBP) (rat anti-MBP, 1:500, Merck Millipore MAB386). Briefly, 40 µm sagittal sections were mounted on Superfrost Plus slides (Fisher Scientific), air-dried for 30 min, and blocked in 15% normal goat serum solution (Vector Labs, S-1000) in 2% TBS-T (1 × Tris Buffered Saline, pH 7.6 with 2% Triton-X100, Fisher Scientific) for 1 h. Slides were then incubated in primary antibody in 10% serum solution in 2% TBS-T for 2 h. Slides were washed three times in 1×TBS and incubated in fluorescent Alexa-Fluor labelled IgG secondary antibodies (Alexa-Fluor goat anti-rabbit 488, Invitrogen A-11008 and goat anti-rat 546, Invitrogen A-11081) in 10% serum solution in 2% TBS-T for 2 h, washed three times in 1×TBS, and incubated in a 1 × solution of TrueBlack lipofuscin autofluorescence quencher (Biotium, Fremont, CA, #23,007) in 70% ethanol for 2 min before rinsing in 1×TBS. Slides were coverslipped in fluoromount-G mounting medium with DAPI (Southern Biotech). To analyze the glial activation (GFAP-positive astrocytes + CD68-positive microglia) as well as MBP staining of punctate bodies in the cerebellar white matter, a semiautomated thresholding image analysis method was used with Image-Pro Premier software (Media Cybernetics). Briefly, this involved the collection of slide-scanned images at 5 × magnification for a one in four series of 3 sections per animal followed by demarcation of all regions (in this case the white matter of the cerebellum) of interest while maintaining the lamp intensity, video camera setup, and calibration constant throughout image capturing. Images were subsequently analyzed using Image-Pro Premier using an appropriate threshold that selected the foreground immunoreactivity above background. This threshold was then applied as a constant to all subsequent images analyzed per batch of animals and reagent used to determine the specific area of immunoreactivity for each antigen. Image densitometry for the average pixel luminance data was also gathered for MBP staining of the cerebellar white matter due to the higher density of staining. Slide-scanned images at 5 × magnification were collected for a one in forty-eight series of sections per animal followed by collecting the mean luminance data across all pixels from the regions of interest delineated using Image-Pro Premier.
Cytokine/chemokine analyses were performed on the brains from the same animals used for immunofluorescent imaging. Briefly, one sagittal half of the brain was homogenized in 10 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 2 mM EDTA containing a protease inhibitor cocktail (Sigma, St. Louis, MO, cat# p8849). The lysates were adjusted to 10 mg/mL total protein with PBS, centrifuged at 10,000 g × 10 min 4 °C to remove particulates, then 25 µL per well in duplicate was added to 25 µL PBS with premixed magnetic beads (ThermoFisher/Procartaplex mouse simplex beads) capturing IL-10, MIP-1α, GRO-α, MCP-1, IP-10, IL-1β, IFNγ, IL-12p70, IL-4, IL-5, IL-6, and TNF-α. The plate was incubated overnight on a shaker at 4 °C with the final detection steps/analysis using the Luminex FlexMAP3D performed the next morning. A calibration curve was generated from the manufacturer-expressed-protein standard using a 5-parameter curve fitting software program (Milliplex Analyst 5.1, Verigene) and a two-fold dilution factor applied to the extrapolated pg/mL sample values.
Brains were collected after removal of the calvarium, weighted, and halved following the midline. The brain was sectioned in the mid-sagittal plane with half placed in 10% NBF. One sciatic nerve and other organs [heart, lung, liver, spleen, kidney, adrenal glands, stomach, small intestine, large intestine, spinal cord (cervical, thoracic, lumbar), skin, testes, and lymph nodes] were also collected and fixed in 10% NBF. Sections of sciatic nerve were also fixed in 2% PFA, 2% glutaraldehyde, embedded, sectioned and stained with Toluidine blue before scanning. Images were analyzed to determine the G-ratio in the dorsal funiculus.
Mice were anesthetized with isoflurane and placed on a thermostatically controlled heating pad to maintain body temperature. The recording electrode was inserted into the plantar muscles and a reference electrode was placed in the skin between toes. A pair of subcutaneous stimulating electrodes were placed distally on both sides of the ankle to stimulate the lateral plantar nerve. For proximal stimulation, the electrodes are moved to the level of the sciatic notch, to the depth of the sciatic nerve. A current pulse was delivered at low frequency and was gradually increased from zero until a maximal compound muscle action potential (CMAP) in the plantar muscles was evident on the oscilloscope. Six distally-produced CMAPs were recorded and stored. The procedure was repeated using the proximal electrode pair, and current was increased until a CMAP that matches that produced by distal stimulation was evident. Six proximally-produced CMAPs were recorded and stored. The distance between the sites of stimulation was measured. NCV was later calculated using the two latencies and conduction distance. The H wave was recorded during the recording of the distally stimulated CMAPs.
Significance from control group are represented as follows: * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. All error bars represent standard error. A two-way repeated measures ANOVA with Tukey correction for multiple comparisons was used for weight curves and wire hang. All glycolipid bar graphs, immunohistochemistry, and cytokine analyses used one-way ANOVA on log-transformed data with Tukey correction for multiple comparisons. Log-rank (Mantel-Cox) test with Bonferroni correction was used for survival curves. In vitro psychosine generation used an unpaired t-test. IC
We would like to acknowledge HD Bio for conducting the CGT enzyme and cellular assays and screening the bioactive lipid library. ChemPartner for conducting the synthesis of the CGT inhibitors, mouse PK studies, MLD mouse studies, and accompanying bioanalysis. Dr. Mike Gelb and Xinying Hong for running the ARSA assays. Dr. Gieselmann for providing the MLD mouse model. Jax Bar Harbor for conducting the wild-type mouse tolerability studies including NCV, G-ratios, clinical pathology, and wire hang. EPL for evaluating histopathological changes including scoring of the vacuolation. Dean and Teryn Suhr from the MLD Foundation for advice and support. This work was supported by BioMarin Pharmaceutical Inc. and the National Institutes of Health NIH R01 NS100779 (MSS).
M.C.B., C.R.M., B.W., Sa.C., J.C.W., Y.S., S.B., M.S.S., and B.E.C. conceptualized the studies, M.C.B. and C.R.M. performed formal analysis, Y.S., S.B., M.S.S., and B.E.C. acquired funding, M.C.B., C.R.M., Su.C., Y.X., and K.W. performed experimental investigation, M.C.B., C.R.M., B.W., Sa.C., Y.X., K.W., B.K.Y., X.J., Q.C., J.C.W. developed methodology, M.C.B., B.W., Sa.C., Su.C., Y.X., Y.F., Q.C., J.C.W., Y.S., and M.S.S. supported project administration, B.W., A.G., B.K.Y., M.L., X.J., Q.C., and M.S.S. provided resources, M.C.B., B.W., Q.C., J.C.W., Y.S., S.B., M.S.S., and B.E.C. provided supervision of the studies, M.C.B., C.R.M., Su.C., and J.C.W. generated visualization of the data, M.C.B. and C.R.M. wrote the original draft, M.C.B., C.R.M., B.W., Sa.C., Su.C., J.C.W., S.B., M.S.S., and B.E.C. led the review and editing process. All authors reviewed the manuscript.
S202 may be obtained through an MTA with BioMarin.
MCB, BW, SaC, SuC, YX, KW, YF, AG, BKY, ML, QC, JCW, YS, SB, and BEC are current or former employees of BioMarin which has filed patent applications on the CGT inhibitors, US Patent WO 2017/214505 Al and WO/2020/028221. All other authors declare they have no competing interests.
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By Michael C. Babcock; Christina R. Mikulka; Bing Wang; Sanjay Chandriani; Sundeep Chandra; Yue Xu; Katherine Webster; Ying Feng; Hemanth R. Nelvagal; Alex Giaramita; Bryan K. Yip; Melanie Lo; Xuntian Jiang; Qi Chao; Josh C. Woloszynek; Yuqiao Shen; Shripad Bhagwat; Mark S. Sands and Brett E. Crawford
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