Glucosylceramide synthase

In untreated WT cells, Ibiglustat (1 μM, 15 days; Fabry disease (FD) cells) produced GL-3 levels that were almost physiological, suggesting that Ibiglustat inhibits further GL-3 accumulation and could enhance GL-3 presence in FD cells. A significant portion of this sphingolipid[4]

Mutations in GBA, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), represent the greatest genetic risk factor for developing synucleinopathies including Parkinson's disease (PD). Additionally, PD patients harboring a mutant GBA allele present with an earlier disease onset and an accelerated disease progression of both motor and non-motor symptoms. Preclinical studies in mouse models of synucleinopathy suggest that modulation of the sphingolipid metabolism pathway via inhibition of glucosylceramide synthase (GCS) using a CNS-penetrant small molecule may be a potential treatment for synucleinopathies. Here, we aim to alleviate the lipid storage burden by inhibiting the de novo synthesis of the primary glycosphingolipid substrate of GCase, glucosylceramide (GlcCer). We have previously shown that systemic GCS inhibition reduced GlcCer and glucosylsphingosine (GlcSph) accumulation, slowed α-synuclein buildup in the hippocampus, and improved cognitive deficits. Here, we studied the efficacy of a brain-penetrant clinical candidate GCS inhibitor, venglustat, in mouse models of GBA-related synucleinopathy, including a heterozygous Gba mouse model which more closely replicates the typical GBA-PD patient genotype. Collectively, these data support the rationale for modulation of GCase-related sphingolipid metabolism as a therapeutic strategy for treating GBA-related synucleinopathies [1].

Measurement of glycosphingolipid levels[1]
Quantitative analysis of sphingolipids was performed by liquid chromatography and tandem mass spectrometry (LC–MS/MS)28. Briefly, brain tissue was homogenized in 10 volumes of water (w/v). Ten microliters of homogenate or plasma was extracted with 1 ml of extraction solution (50:50 acetonitrile/methanol) by protein precipitation. Mouse CSF sphingolipids were extracted by liquid–liquid extraction, as previously described37. GlcCer and galactosylceramide were separated using a Waters Acquity UPLC and Cortecs HILIC column (2.1 mm × 100 mm, 2.7 µm particles) and analyzed by an API 5000 triple quadrupole mass spectrometer in MRM mode. GlcSph and psychosine were separated by a Waters Acquity UPLC and BEH HILIC column (2.1 mm × 100 mm, 1.7 µm particles) and analyzed by an API 6500 triple quadrupole mass spectrometer in MRM mode. GlcCer and GlcSph standards were purchased from Matreya, LLC and Avanti Polar Lipids, respectively. All procedures were performed blinded to the genotype or treatment.

To expose insoluble α-synuclein aggregates, some tissues were pretreated with proteinase K (1:4 dilution) for 7 min at room temperature to digest soluble α-synuclein23. Brain sections were blocked with 10% (vol/vol) normal donkey serum for 1 h at room temperature and incubated with the following antibodies: mouse anti-ubiquitin (1:300; cat# MAB1510), rabbit anti-α-synuclein (1:300), and mouse anti-tau (1:500, Tau-5). Brain sections were then incubated for 1 h with either a donkey anti-mouse Alexa Fluor-488 (1:250 dilution,) or donkey anti-rabbit biotinylated secondary antibody (1:200 dilution). For α-synuclein aggregate quantification, a cyanine 3-tyramide signal amplification kit was used. Cell nuclei were counterstained with 4’, 6-diamino-2-phenylindole (DAPI). Sections were coverslipped with aqua-poly/mount and the stratum radiatum external to the CA1 hippocampal cell body layer was imaged with a SPOT camera (SPOT Imaging) paired with a Nikon Eclipse E800 fluorescence microscope equipped with a 20 × objective lens, as previously described22. Two to three sections were imaged per animal and immunofluorescence was quantitatively measured via threshold fluorescent area on MetaMorph Software. All procedures were performed blinded to the treatment or genotype and the percent threshold area is expressed as the mean ± SE. [1]

Administration of the glucosylceramide synthase inhibitors: venglustat and tool compound GZ667161[1]
A subset of animals received glucosylceramide synthase inhibitors, venglustat (aka GZ402671) or GZ667161, via pelleted diet at 0.03%- or 0.033%-wt/wt, respectively. For each experiment, sex and siblings were randomly matched for group assignment. Target engagement and exposure confirmation studies included GbaD409V/D409V or GbaD409V/WT mice administered venglustat for two consecutive weeks beginning at approximately 4 months of age. Mice included in sustained GCS inhibition studies were administered either GZ667161 or venglustat upon weaning at ~ 4 weeks of age. Wild-type, baseline, and control groups were fed vehicle rodent chow. GCS inhibitor and vehicle diets were continuously provided to mice until necropsy and tissue collection.

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CSF collection[1]
Animals were anesthetized via an intraperitoneal injection of a 10:1 Ketamine/Xylazine cocktail prior to being placed into a surgical ear bar rig. After making a midline cut to remove a small patch of skin from the head, the fat and muscle layers were opened using a cautery pen to reveal the base of the skull and occipital crest. The remaining tissue was then removed to expose the cisterna magna membrane. Using a pulled glass pipette, the cisterna magna membrane was punctured to allow CSF to flow freely into the pipette via capillary action. After collecting approximately 10–20 uL, CSF was transferred to a clean protein lo-bind tube . CSF samples with visible blood contamination were excluded from analyses.

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Animal perfusion and tissue and blood collection[1]
Prior to whole blood collection, mice were anesthetized via a 200 uL intraperitoneal injection of sodium pentobarbital. Following the loss of response to a foot-pinch and corneal reflex, approximately 250 uL of whole blood was collected from the retro-orbital sinus using a glass capillary tube into a Microtainer® tube containing K2 EDTA anticoagulant. Whole blood samples were collected retro-orbitally and immediately placed on ice. Plasma was isolated after 5 min centrifugation at 8000 RPM at 4 °C. Immediately following blood collection, animals were transcardially perfused with cold phosphate-buffered saline (PBS) at a rate of 18 mL/minute, for two minutes. After cutting the brains sagittally along the midline, the left hemisphere was microdissected into various regions, snap-frozen in liquid nitrogen, and stored at − 80 °C until use. The right hemisphere was post-fixed in 10% neutral-buffered formalin for 48–72 h. Right hemispheres were then washed three times in 1X PBS and transferred to 30% sucrose for 24–48 h. Right hemispheres were embedded in O.C.T. and sectioned into 20 µm sections using a cryostat, as previously described.

[1]. Viel C, et al. Preclinical pharmacology of glucosylceramide synthase inhibitor venglustat in a GBA-related synucleinopathy model. Sci Rep. 2021;11(1):20945. Published 2021 Oct 22.
[2]. Peterschmitt MJ, et al. Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of Oral Venglustat in Healthy Volunteers. Clin Pharmacol Drug Dev. 2021;10(1):86-98.
[3]. Stojkovska I, et al. Molecular mechanisms of α-synuclein and GBA1 in Parkinson's disease. Cell Tissue Res. 2018;373(1):51-60. doi:10.1007/s00441-017-2704-y
[4]. Itier JM, et al. Effective clearance of GL-3 in a human iPSC-derived cardiomyocyte model of Fabry disease. J Inherit Metab Dis. 2014 Nov;37(6):1013-22.

C20H24FN3O2S

389.4869

389.15731

C, 61.68; H, 6.21; F, 4.88; N, 10.79; O, 8.22; S, 8.23

1401090-53-6

Ibiglustat (L-Malic acid);1629063-79-1 (HCl); 1629063-78-0 (malate; Ibiglustat succinate;1629063-80-4; 1401090-53-6; 1629063-80-4 (succinic acid)

White to off-white solid

4.13

82.7Ų

O=C(O[C@@H]1CN2CCC1CC2)NC(C)(C3=CSC(C4=CC=C(F)C=C4)=N3)C

YFHRCLAKZBDRHN-MRXNPFEDSA-N

InChI=1S/C20H24FN3O2S/c1-20(2,17-12-27-18(22-17)14-3-5-15(21)6-4-14)23-19(25)26-16-11-24-9-7-13(16)8-10-24/h3-6,12-13,16H,7-11H2,1-2H3,(H,23,25)/t16-/m1/s1

(3S)-1-azabicyclo[2.2.2]octan-3-yl N-{2-[2-(4-fluorophenyl)- 1,3-thiazol-4-yl]propan-2-yl}carbamate

Venglustat; SAR402671; GZ402671; Genz-682452-AA; SAR-402671; GZ402671; Ibiglustat; SAR 402671; GZ-402671; GZ-452; Genz-682452

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~128.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.42 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)