Glucosylceramide synthase

在未经处理的 WT 细胞中，Ibiglustat（1 μM，15 天；法布里病 (FD) 细胞）产生的 GL-3 水平几乎达到生理水平，表明 Ibiglustat 进一步抑制 GL-3 积累，并可以增强 FD 细胞中 GL-3 的存在。这种鞘脂的很大一部分[4]

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。在这里，我们研究了脑渗透临床候选GCS抑制剂venglustat在GBA相关突触核蛋白病小鼠模型中的疗效，包括更紧密地复制典型GBA-PD患者基因型的杂合GBA小鼠模型。总之，这些数据支持了调节GCase相关鞘脂代谢作为治疗GBA相关突触核蛋白病的治疗策略的基本原理[1]。

糖脂水平的测量[1]
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。

为了暴露不溶性α-突触核蛋白聚集体，一些组织在室温下用蛋白酶K（1:4稀释）预处理7分钟以消化可溶性α-突触蛋白23。在室温下用10%（vol/vol）正常驴血清封闭脑切片1小时，并与以下抗体孵育：小鼠抗泛素（1:300；猫#MAB1510）、兔抗α-突触核蛋白（1:300）和小鼠抗τ（1:500，tau-5）。然后将脑切片与驴抗小鼠Alexa Fluor-488（1:250稀释液）或驴抗兔生物素化第二抗体（1:200稀释液）孵育1小时。对于α-突触核蛋白聚集物的定量，使用花青3-酪酰胺信号扩增试剂盒。细胞核用4’，6-二氨基-2-苯基吲哚（DAPI）复染。切片用aqua-poly/mount覆盖，CA1海马细胞体层外部的放射层用SPOT相机（SPOT Imaging）与配备有20 × 物镜，如前所述22。对每只动物的两到三个切片进行成像，并通过MetaMorph软件上的阈值荧光区域定量测量免疫荧光。所有程序都是在不了解治疗或基因型的情况下进行的，阈值面积百分比表示为平均值 ± 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]. 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. 2018;373(1):51-60. doi:10.1007/s00441-017-2704-y.

[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.

Venglustat is under investigation in clinical trial NCT01674036 (Safety, Tolerability and Pharmacokinetics of Genz-682452 in Healthy Men).

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Drug Indication
Treatment of autosomal dominant polycystic kidney disease
Treatment of galactosialidosis, Treatment of GM1 gangliosidosis, Treatment of GM2 gangliosidosis, Treatment of sialidosis
Treatment of polycystic kidney disease
Treatment of Parkinson's disease

C20H24FN3O2S

389.4869

389.157

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)

60199242

White to off-white solid

1.3±0.1 g/cm3

1.613

4.13

82.7Ų

1

6

5

27

533

1

S1C(C2C([H])=C([H])C(=C([H])C=2[H])F)=NC(=C1[H])C(C([H])([H])[H])(C([H])([H])[H])N([H])C(=O)O[C@]1([H])C([H])([H])N2C([H])([H])C([H])([H])C1([H])C([H])([H])C2([H])[H]

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.42 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 (6.42 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 (6.42 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。