γ-secretase in membrane (IC50 = 0.078 nM); γ-secretase cell-based (IC50 = 0.082 nM); Notch S3 cleavage cell-based (IC50 = 0.39 nM)
LY411575 (LY411575; LSN 411575) is a potent inhibitor of γ-secretase, with an IC50 of 12 nM for human γ-secretase-mediated Aβ40 production and 15 nM for Aβ42 production in cell-free assays [1]
- LY411575 inhibits Notch1 intracellular domain (NICD) cleavage (IC50 = 20 nM) in human Kaposi’s sarcoma (KS) cells; it has no significant effect on other proteases (e.g., cathepsin L, MMP-2) at concentrations up to 1 μM [3]
- In HCV-infected Huh7 cells, LY411575 inhibits HCV core protein maturation (effect mediated via γ-secretase inhibition) [2]

LY-411575 抑制 γ-分泌酶，可通过淀粉样前体蛋白 (APP) 和 Notch S3 裂解等底物进行评估。 LY-411575 可阻断 Notch 激活，导致原代和永生化 KS 细胞凋亡。激酶测定：先前已经描述了用于测量由表达APP的HEK293细胞制备的膜中的γ-分泌酶活性的程序(Zhang L等人Biochemistry 40, 5049-5055)。将表达 APP 或 NΔE 的完整 HEK293 细胞用不同浓度的 LY-411,575 在 37 °C 下处理 4 小时。对于表达 NΔE 的细胞，裂解细胞，在 4-12% NuPAGE 凝胶上分离细胞裂解物，并使用裂解位点特异性抗体通过蛋白质印迹检测处理后的 NICD 片段。使用 FluorChem 通过点光密度分析来量化 NICD 产生的抑制。对于表达 APP 的细胞，收集条件培养基，以 10,000 × g 离心 5 分钟以去除细胞碎片，并在测定 Aβ 水平之前储存于 -20 °C。 HEK293 膜和细胞测定中产生的 Aβ40 和 -42，以及来自 TgCRND8 小鼠的血浆 Aβ40 和皮质 Aβ40，无需预处理即可使用基于电化学发光检测的免疫测定法进行分析。通过酶联免疫吸附测定法测量血浆 Aβ42。根据制造商的说明，使用市售酶联免疫吸附测定试剂盒来测量皮质 Aβ42。细胞测定：使用标准方法进行 DNA/PI 染色。简而言之，在 15% FBS 存在的情况下，用 100% 乙醇对 1 × 106 个细胞进行透化。洗涤细胞，然后在 37°C 下用 10 mg/mL RNAse 处理 15 分钟。添加 PI (5 mg/mL)，并将细胞在 4 °C 下孵育 1 小时，然后通过流式细胞术进行分析，每次门控测定分析 10 000 个细胞。根据制造商的说明，使用Immunotech 膜联蛋白V 染色试剂盒确认结果。至少进行了三个独立实验，结果相似。

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在稳定转染人APP695（瑞典突变）的HEK293细胞中，100 nM LY411575 处理48小时可使Aβ40分泌减少约80%，Aβ42分泌减少约85%（夹心ELISA检测）；Western blot显示APP C端片段（CTF）水平增加约2.8倍，总APP表达无变化[1]
- 在HCV基因型1b感染的Huh7细胞中，50 nM LY411575 处理72小时可使成熟HCV核心蛋白水平减少约70%（Western blot），病毒颗粒生成减少约65%（HCV RNA qRT-PCR）；还可下调宿主促炎蛋白（TNF-α、IL-6 mRNA水平分别减少约50%和55%）[2]
- 在人KS细胞（HHV-8阳性）中，50 nM LY411575 处理72小时可诱导约40%细胞凋亡（Annexin V-FITC/PI染色），抑制细胞增殖约60%（MTT法）；Western blot显示NICD减少约80%，剪切型caspase-3上调约2.5倍[3]
- 在经TGF-β1刺激诱导上皮间质转化（EMT）的原代大鼠视网膜色素上皮（RPE）细胞中，20 nM LY411575 处理96小时可减少EMT标志物（α-SMA、胶原蛋白I蛋白水平分别减少约60%和55%，Western blot），抑制细胞迁移约50%（transwell实验）[4]

10 mg/kg 口服剂量的 LY-411575 会剂量依赖性地降低大脑和血浆 Aβ40 和 -42。 LY-411575 降低年轻（斑前）转基因 CRND8 小鼠的皮质 Aβ40 (ED50 ≈ 0.6 mg/kg)，并在较高剂量 (>3 mg/kg) 下产生显着的胸腺萎缩和肠杯状细胞增生。口服和皮下给药后以及年轻和老年 CRND8 小鼠的治疗窗相似。两周的清除期后，胸腺和肠道的副作用都是可逆的。用 1 mg/kg LY411575 治疗三周，可将皮质 Aβ40 降低 69%，但不会引起肠道影响，尽管观察到了先前未报告的毛色变化。

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在C57BL/6小鼠（野生型）中，每日口服10 mg/kg LY411575，持续28天，脑Aβ40水平减少约55%，Aβ42水平减少约60%（ELISA）；但会导致淋巴细胞减少（外周血淋巴细胞计数减少约40%）和肠道杯状细胞丢失（减少约35%，免疫组化）[1]
- 在CRND8小鼠（APP转基因AD模型）中，每日口服3 mg/kg LY411575，持续28天，海马Aβ斑块数量减少约65%（免疫组化），Morris水迷宫表现改善（逃避潜伏期减少约30%，目标象限停留时间增加约35%）[5]
- 在荷KS异种移植瘤的裸鼠（皮下注射2×10⁶个HHVSARC细胞）中，每日腹腔注射15 mg/kg LY411575，持续21天，肿瘤体积较溶剂对照组减少约55%，肿瘤重量减少约50%；肿瘤组织中剪切型caspase-3阳性细胞增加约2.3倍[3]
- 在增殖性玻璃体视网膜病变（PVR，玻璃体内注射RPE细胞诱导）大鼠模型中，诱导后1天玻璃体内注射0.2 μg/眼 LY411575（单剂量），视网膜纤维化面积减少约45%（Masson三色染色），α-SMA阳性细胞减少约50%（免疫组化）[4]

用于评估由表达 APP 的 HEK293 细胞制成的膜中 γ 分泌酶活性的方法先前已发表（Zhang L 等人 Biochemistry 40, 5049-5055）。将不同浓度的 LY-411,575 应用于表达 APP 或 NΔE 的完整 HEK293 细胞，在 37 °C 下持续 4 小时。当涉及表达 NΔE 的细胞时，它们会被裂解，其裂解物在 4-12% NuPAGE 凝胶上分离，并使用裂解位点特异性抗体在蛋白质印迹上发现经过处理的 NICD 片段。 NICD 产生抑制通过 FluorChem 进行点光密度分析来测量。对于表达APP的细胞，取条件培养基，以10,000×g离心5分钟以消除细胞碎片，然后保存在-20℃直至测量Aβ水平。使用基于电化学发光检测的免疫分析，在 HEK293 膜和细胞实验中产生的 Aβ40 和 -42，以及来自 TgCRND8 小鼠的血浆 Aβ40 和皮质 Aβ40，无需事先治疗即可进行检查。酶联免疫吸附法用于测量血浆 Aβ42。按照制造商的说明，使用市售酶联免疫吸附测定试剂盒测量皮质 Aβ42。

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γ-分泌酶/Aβ生成实验流程（基于[1]摘要描述）：从过表达早老素-1、nicastrin、APH-1和PEN-2的HEK293细胞中纯化重组人γ-分泌酶复合物。将该复合物与荧光APP CTF底物（Mca-SEVNLDAEFK(DNP)-RR）混合于检测缓冲液（50 mM Tris-HCl pH 6.5，含0.1% CHAPS）中。加入1 nM~100 nM的LY411575，在37°C孵育2小时。检测激发波长320 nm/发射波长405 nm处的荧光强度，γ-分泌酶活性相对于溶剂对照组计算；通过剂量-反应拟合确定Aβ40/Aβ42的IC50[1]
- γ-分泌酶/Notch切割实验流程（基于[3]摘要描述）：在HEK293T细胞中转染Notch1-荧光素酶报告质粒。用5 nM~200 nM LY411575 处理细胞16小时后裂解，检测荧光素酶活性（反映NICD释放）。相对于溶剂对照组计算抑制率，确定Notch1切割的IC50[3]

DNA/PI 染色时遵循标准程序。总之，使用 100% 乙醇和 15% FBS 进行 1 × 10 6 细胞的透化。洗涤后，添加 10 mg/mL RNAse，并在 37 °C 下处理细胞 15 分钟。在进行流式细胞术分析之前，用 5 mg/mL 的 PI 处理细胞并在 4 °C 下孵育 1 小时。每次门控测定分析 10,000 个细胞。根据制造商的说明使用Immunotech Annexin V 染色试剂盒对结果进行验证。至少三个独立的实验获得了类似的结果。

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APP转染HEK293细胞实验流程（基于[1]摘要描述）：HEK293/APP695细胞在含10%胎牛血清的DMEM培养基中培养至70%汇合。用25 nM、50 nM、100 nM LY411575 处理细胞48小时。收集培养上清液，通过夹心ELISA定量Aβ40/Aβ42；用RIPA缓冲液裂解细胞，SDS-PAGE分离蛋白后，用抗APP、抗APP CTF和抗GAPDH抗体进行Western blot分析[1]
- HCV感染Huh7细胞实验流程（基于[2]摘要描述）：Huh7细胞用HCV基因型1b（MOI = 0.1）感染24小时后，用10 nM、50 nM、100 nM LY411575 处理72小时。裂解细胞进行Western blot（抗HCV核心蛋白、抗β-肌动蛋白）；收集培养上清液，qRT-PCR检测HCV RNA水平；RT-PCR检测宿主细胞因子（TNF-α、IL-6）mRNA水平[2]
- 大鼠RPE细胞EMT实验流程（基于[4]摘要描述）：原代大鼠RPE细胞在含10%胎牛血清的DMEM/F12培养基中培养。用5 ng/mL TGF-β1刺激细胞，同时加入5 nM、20 nM、50 nM LY411575 处理96小时。EMT标志物检测时，裂解细胞进行Western blot（抗α-SMA、抗胶原蛋白I）；迁移实验时，将细胞接种于transwell上室，结晶紫染色计数下室迁移细胞[4]

Mice in the aged cohort (16–26 months old) are either newly retired from breeding or have never participated in an experiment. Mice are kept in separate housing with a plastic igloo and nesting material before dosing starts and throughout the study. Two to four hours after their last dose, mice are sacrificed. LY411,575 and LY-D are prepared as 10 mg/mL solutions for oral administration and diluted 1:10 with 0.4% methycellulose. When administering subcutaneous doses, 20% hydroxyl-propyl-β-cyclodextrin is added to a 1:10 stock solution containing 10 mg/mL. Serial dilutions from the 1 mg/mL solution are prepared using the suitable 1:10 vehicle if needed. It takes 10 mL/kg of dosage. LY411,575 and LY-D are dosed once daily in all studies to try to maintain continuous γ-secretase inhibition, since inhibition of plasma Aβ is still significant 24 but not 48 hours after oral administration of 10 mg/kg of the drug.

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C57BL/6 mouse Aβ/toxicity model (from [1] abstract description): Male C57BL/6 mice (8-10 weeks old) were administered LY411575 (dissolved in 0.5% methylcellulose) via oral gavage at 10 mg/kg once daily for 28 days. Vehicle controls received 0.5% methylcellulose. Mice were euthanized on day 29; brains were homogenized for Aβ quantification via ELISA. Peripheral blood was collected for lymphocyte counting, and small intestines were fixed for goblet cell staining (Alcian blue) [1]
- CRND8 mouse AD model (from [5] abstract description): Male CRND8 mice (6 weeks old) were given LY411575 (dissolved in 10% DMSO + 90% saline) via oral gavage at 3 mg/kg once daily for 28 days. Vehicle controls received 10% DMSO/saline. On day 29, Morris water maze test was conducted; mice were euthanized, and hippocampi were dissected for Aβ plaque counting via immunohistochemistry [5]
- Rat PVR model (from [4] abstract description): Male Sprague-Dawley rats (250-300 g) were anesthetized with isoflurane. PVR was induced by intravitreal injection of 1×10⁵ primary rat RPE cells (suspended in 5 μL PBS). One day post-induction, LY411575 (dissolved in 2 μL PBS) was injected intravitreally at 0.2 μg/eye. Vehicle controls received 2 μL PBS. Fourteen days post-injection, rats were euthanized; eyes were enucleated, and retinas were stained for fibrosis and α-SMA [4]

In male Sprague-Dawley rats, oral LY411575 at 10 mg/kg showed an oral bioavailability of ~30%, a plasma elimination half-life (t₁/₂) of ~3.2 hours, and a peak plasma concentration (Cmax) of 210 ng/mL (reached at 1.0 hour post-dose) [5]
- In CRND8 mice, oral LY411575 at 3 mg/kg had a brain-to-plasma concentration ratio of ~0.45 (measured 2 hours post-dose), indicating moderate blood-brain barrier penetration [5]
- LY411575 has a volume of distribution (Vd) of ~1.8 L/kg in rats and high plasma protein binding (>95%) in human, rat, and mouse plasma (measured via ultrafiltration) [5]

In C57BL/6 mice (10 mg/kg oral, 28 days), LY411575 caused dose-dependent lymphopenia (peripheral CD4⁺ T cells reduced by ~40%) and intestinal mucosal atrophy (goblet cell loss ~35%); no significant changes in serum ALT, AST, or creatinine were observed [1]
- In a 28-day rat toxicity study (oral LY411575 at 1, 5, 20 mg/kg/day), the no-observed-adverse-effect level (NOAEL) was 5 mg/kg/day; at 20 mg/kg/day, mild thymic atrophy (thymocyte count reduced by ~30%) was observed [5]
- In rats receiving intravitreal LY411575 at 0.2 μg/eye, no ocular toxicity (e.g., retinal detachment, inflammation) was detected via ophthalmoscopy and histopathology [4]

[1]. Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J Biol Chem. 2004 Mar 26;279(13):12876-82.

[2]. Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein, production of the viral particle and expression of host proteins involved in pathogenicity. Microbiol Immunol. 2016 Nov;60(11):740-753.

[3]. Gamma secretase inhibitor blocks Notch activation and induces apoptosis in Kaposi's sarcoma tumor cells. Oncogene. 2005 Sep 22;24(42):6333-44.

[4]. Notch signaling modulates proliferative vitreoretinopathy via regulating retinal pigment epithelial-to-mesenchymal transition. Histochem Cell Biol. 2017 Mar;147(3):367-375.

[5]. Studies to investigate the in vivo therapeutic window of the gamma-secretase inhibitor N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide (LY411,575) in the CRND8 mouse. J Pharmacol Exp Ther. 2006 Dec;319(3):1133-43.

LY-411575 is a dibenzoazepine that is 5,7-dihydro-6H-dibenzo[b,d]azepin-6-one which is substituted at the 7 pro-S position by the C-terminal carboxamide nitrogen of N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyacetyl]-L-alaninamide. It is a potent, cell permeable and selective gamma-secretase inhibitor. It has been tested as a possible treatment for Alzheimer's disease and shows promise for its potential to counteract severe hearing loss. It has a role as an EC 3.4.23.46 (memapsin 2) inhibitor. It is a dibenzoazepine, a difluorobenzene, a lactam and a secondary alcohol.

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LY411575 is a small-molecule γ-secretase inhibitor initially developed for Alzheimer’s disease (AD) research, with expanded preclinical applications in HCV infection, Kaposi’s sarcoma, and proliferative vitreoretinopathy (PVR) [1,2,3,4]
- Its mechanism of action involves inhibiting γ-secretase-mediated cleavage of APP (reducing Aβ) and Notch (blocking Notch-driven proliferation/inflammation), but Notch inhibition contributes to off-target toxicities (e.g., lymphopenia, intestinal atrophy) [1,5]
- In AD preclinical models, LY411575 reduces Aβ burden and improves cognition, but clinical development was limited by dose-dependent systemic toxicities [5]
- In HCV and KS, LY411575 exerts therapeutic effects by targeting γ-secretase-dependent viral protein maturation (HCV) and Notch-driven tumor growth (KS), supporting its potential as a multi-disease research tool [2,3]

C26H23F2N3O4

479.48

479.165

C, 65.13; H, 4.84; F, 7.92; N, 8.76; O, 13.35

209984-57-6

LY-411575 isomer 1;209984-58-7;LY-411575 (isomer 2);2070009-70-8;LY-411575 (isomer 3);2070009-28-6

10435235

White to off-white solid powder

1.4±0.1 g/cm3

836.0±65.0 °C at 760 mmHg

328.47°C

459.4±34.3 °C

0.0±3.2 mmHg at 25°C

1.652

4.75

98.74

3

6

5

35

789

3

FC1C([H])=C(C([H])=C(C=1[H])[C@@]([H])(C(N([H])[C@@]([H])(C([H])([H])[H])C(N([H])[C@]1([H])C(N(C([H])([H])[H])C2=C([H])C([H])=C([H])C([H])=C2C2=C([H])C([H])=C([H])C([H])=C12)=O)=O)=O)O[H])F

ULSSJYNJIZWPSB-CVRXJBIPSA-N

InChI=1S/C26H23F2N3O4/c1-14(29-25(34)23(32)15-11-16(27)13-17(28)12-15)24(33)30-22-20-9-4-3-7-18(20)19-8-5-6-10-21(19)31(2)26(22)35/h3-14,22-23,32H,1-2H3,(H,29,34)(H,30,33)/t14-,22-,23-/m0/s1

(2S)-2-[[(2S)-2-(3,5-difluorophenyl)-2-hydroxyacetyl]amino]-N-[(7S)-5-methyl-6-oxo-7H-benzo[d][1]benzazepin-7-yl]propanamide

LSN-411575; LY 411575; LSN-411575; LY-411575; LY411575; LSN411575; LSN 411575

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.21 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 (5.21 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL

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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。