Glucosylceramide synthase

用 Ibiglustat (SAR402671) 琥珀酸盐（1 μM，15 天）处理的法布里病 (FD) 细胞中的 GL-3 水平与未处理的 WT 细胞接近生理水平，表明 Ibiglustat 琥珀酸盐可以阻止进一步的 GL-3 积累，并可能有助于改善FD 心肌细胞中这种鞘脂含量很高[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. 2017.

[4]. Effective clearance of GL-3 in a human iPSC-derived cardiomyocyte model of Fabry disease. J Inherit Metab Dis. 2014;37(6):1013-1022.

C24H30FN3O6S

507.57

507.18

C, 56.79; H, 5.96; F, 3.74; N, 8.28; O, 18.91; S, 6.32

1629063-80-4

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

91618117

White to off-white

157Ų

3

10

8

35

626

1

CC(C)(C1=CSC(=N1)C2=CC=C(C=C2)F)NC(=O)O[C@@H]3CN4CCC3CC4.C(CC(=O)O)C(=O)O

TWRYSLPYKQOKAO-PKLMIRHRSA-N

InChI=1S/C20H24FN3O2S.C4H6O4/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;5-3(6)1-2-4(7)8/h3-6,12-13,16H,7-11H2,1-2H3,(H,23,25);1-2H2,(H,5,6)(H,7,8)/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;butanedioic acid

Venglustat succinate; SAR-402671 succinate; SAR402671 succinate; GZ-402671 succinateGZ402671 succinate

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: ≥ 250 mg/mL (492.54 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。