Glucosylceramide synthase

编码溶酶体酶葡糖脑苷酶（GCase）的基因GBA的突变是发展包括帕金森病（PD）在内的突触核蛋白疾病的最大遗传风险因素。此外，携带突变GBA等位基因的帕金森病患者发病较早，运动和非运动症状的疾病进展加快。对突触核蛋白病小鼠模型的临床前研究表明，使用中枢神经系统渗透剂小分子通过抑制葡糖神经酰胺合酶（GCS）来调节鞘脂代谢途径可能是突触核蛋白疾病的潜在治疗方法。在这里，我们的目的是通过抑制GCase的主要糖脂底物葡糖基神经酰胺（GlcCr）的从头合成来减轻脂质储存负担。我们之前已经表明，系统性GCS抑制减少了GlcCr和葡糖基鞘氨醇（GlcSph）的积累，减缓了海马中α-突触核蛋白的积累，并改善了认知缺陷。[1]
用 Ibiglustat (SAR402671)（1 μM，15 天）L-苹果酸处理的法布里病 (FD) 细胞中的 GL-3 水平与未处理的 WT 细胞几乎相同，表明 Ibiglustat L-苹果酸可以防止进一步的 GL-3 积累并增加 FD 心肌细胞中这种鞘脂的含量 [4]。

Venglustat降低了Gaucher相关突触核蛋白病小鼠的糖脂含量。[1]
Venglustat，一种脑渗透GCS抑制剂，在GBA和Gaucher相关突触核蛋白病的小鼠模型中减少了葡萄糖神经酰胺。[1]
venglustat对GCS的抑制减少了Gaucher相关突触核蛋白病小鼠海马中异常蛋白质聚集体的积累。[1]
Venglustat给药防止了Gaucher相关突触核蛋白病小鼠海马相关记忆缺陷的发展。[1]

Venglustat是一种小分子葡萄糖神经酰胺合酶（GCS）抑制剂，旨在减少葡萄糖神经酰胺（GL-1）的产生，因此有望显著减少基于葡萄糖神经酰胺的鞘糖脂的形成。由于其对糖脂形成的影响，GCS抑制在许多影响糖脂代谢的疾病中具有治疗潜力。因此，venglustat正在开发用于多种疾病的底物还原治疗，包括3型戈谢病、与GBA突变相关的帕金森病、Fabry病、GM2神经节苷脂病和常染色体显性遗传的多囊肾病[2]。

通过液相色谱法和串联质谱法（LC–MS/MS）对鞘脂进行定量分析28。简言之，将脑组织在10体积的水（w/v）中匀浆。用1ml提取溶液（50∶50乙腈/甲醇）通过蛋白质沉淀提取10微升匀浆或血浆。如前所述，通过液-液提取法提取小鼠CSF鞘脂37。使用Waters Acquity UPLC和Cortecs HILIC柱（2.1mm × 100毫米、2.7微米颗粒），并通过API 5000三重四极质谱仪以MRM模式进行分析。通过Waters Acquity UPLC和BEH HILIC柱（2.1mm × 100毫米，1.7微米颗粒），并通过API 6500三重四极质谱仪以MRM模式进行分析。GlcCr和GlcSph标准品分别购自Matreya，LLC和Avanti Polar Lipids。所有程序都是在不了解基因型或治疗的情况下进行的[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.[1]

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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 (Thermo Fisher Scientific; Waltham, MA) 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 (World Precision Instruments; Sarasota County, FL), 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 (Eppendorf; Hamburg, Germany). CSF samples with visible blood contamination were excluded from analyses.[1]

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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 (BD Biosciences; Billerica, MA) 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 use27. 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]. Preclinical pharmacology of glucosylceramide synthase inhibitor venglustat in a GBA-related synucleinopathy model. Sci Rep. 2021;11(1):20945. Published 2021 Oct 22.

[2]. Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of Oral Venglustat in Healthy Volunteers. Clin Pharmacol Drug Dev. 2021;10(1):86-98.

[3]. Molecular mechanisms of α-synuclein and GBA1 in Parkinson’s disease. Cell Tissue Res. 2017.

[4]. 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.

C24H30FN3O7S

523.574308872223

523.178

1629063-78-0

Ibiglustat;1401090-53-6;Ibiglustat succinate;1629063-80-4

60199241

White to off-white solid

178Ų

4

11

8

36

662

2

S1C(C2C=CC(=CC=2)F)=NC(=C1)C(C)(C)NC(=O)O[C@@H]1CN2CCC1CC2.OC(C(=O)O)CC(=O)O

SQXUKOJKIWCALK-AAXLQGCPSA-N

InChI=1S/C20H24FN3O2S.C4H6O5/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;5-2(4(8)9)1-3(6)7/h3-6,12-13,16H,7-11H2,1-2H3,(H,23,25);2,5H,1H2,(H,6,7)(H,8,9)/t16-;2-/m10/s1

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

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 : ≥ 100 mg/mL (~191.00 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.77 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.77 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (4.77 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。