Semagacestat (LY-450139; LY-4501)   中文名称: 司马西特

Aβ42 (IC50 = 10.9 nM); Aβ38 (IC50 = 12 nM); Aβ40 (IC50 = 12.1 nM); Notch (IC50 = 14.1 nM)
Semagacestat (LY-450139; LY-4501) is a selective, reversible inhibitor of γ-secretase (a multi-subunit protease complex), with an IC50 of 18 nM for human γ-secretase-mediated Aβ40 production and 22 nM for Aβ42 production in cell-free assays [2]
- Semagacestat does not significantly inhibit other serine proteases (e.g., Notch1 cleavage, cathepsin G) at concentrations up to 1 μM, showing high selectivity for γ-secretase [2]

Semagacestat 减少稳定过表达人野生型 APP 的 H4 人胶质瘤细胞向培养基中分泌 Aβ42、Aβ40 和 Aβ38，IC50 分别为 10.9 nM、12.1 nM 和 12.0 nM，且不影响细胞活力。 Semagacestat 还能增加细胞裂解物中的 β-CTF，ECmax 为 16.0 nM，并且在高浓度下这种增加会意外减弱。 Semagacestat 抑制 Notch 信号传导，IC50 为 14.1 nM，并显示出最小的 Notch 保留选择性，Notch IC50/Aβ42 IC50 仅 1.3。 Semagacestat 导致分泌到培养基中的 Aβ40 浓度依赖性减少，表达内源性鼠 APP 的鼠 CTX 的 IC50 为 111 nM，但根据野生型小鼠神经元的数据，CTX 中鼠 Aβ42 的形成大约比 Aβ40 少 12 倍。激酶测定：用不同浓度的Semagacestat处理稳定过表达人野生型APP695的H4人神经胶质瘤细胞24小时。使用单独的 ELISA 试剂盒测量培养基中 Aβ42、Aβ40 和 Aβ38 的水平。将编码人Notch1编码区(NM_017617)的碱基1-60和5193-6657的Notch组成型活性形式(NotchΔE)的表达载体构建成具有从小鼠到人类的序列修饰的pcDNA3.1载体。使用 Cignal RBP-Jk Reporter Assay 试剂盒评估 Notch 信号传导活性。 RBP-Jk 蛋白 [CSL/CBF1/Su(H)/Lag1] 是一种由 γ-分泌酶产生的 Notch 胞内结构域激活的转录因子。使用 Lipofectamine 2000 用人 NotchΔE 表达载体和 RBP-Jk 响应荧光素酶构建体瞬时转染 H4 细胞，然后暴露于不同浓度的 Semagacestat 16 小时。 Notch 信号传导是使用 Dual-Glo 荧光素酶测定系统根据细胞裂解液中的荧光素酶活性进行测量的。细胞测定：将细胞（小鼠皮质神经元和小脑颗粒细胞）与 Semagacestat 一起孵育 24 小时。为了检测细胞活力，用 0.5 mg/mL MTT 孵育后，通过其还原 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物 (MTT) 的能力来量化活细胞的百分比60分钟。为了检测 sAPP 种类，需要裂解细胞并通过蛋白质印迹进行分析。

---

在稳定转染人APP695（瑞典突变）的HEK293细胞中，100 nM Semagacestat 处理48小时可使Aβ40分泌减少约80%，Aβ42分泌减少约85%（通过夹心ELISA检测）；Western blot分析显示APP C端片段（CTF，γ-分泌酶底物）水平增加约2.5倍，总APP表达无变化[2]
- 在转染APP695的原代培养大鼠皮质神经元中，50 nM Semagacestat 处理72小时可使细胞内Aβ42水平减少约70%（抗Aβ42抗体免疫荧光染色），并阻止Aβ诱导的神经突回缩（神经突长度较仅Aβ处理组增加约40%）[2]

对 5.5 个月大的 APP 转基因 Tg2576 小鼠口服 Semagacestat (1 mg/kg)，可显着改善 Y 迷宫任务中空间工作记忆的记忆缺陷，亚慢性给药 8 天后消失。 LY450139 在 10 mg/kg（减少 22-23%）和 30 mg/kg（减少 36-41%）时降低海马 Aβ42 和 Aβ40 水平，并以剂量依赖性方式在 0.3-10 mg/kg 时增加 β-CTF对大脑中其他 γ 分泌酶底物（例如 Notch、N-钙粘蛋白或 EphA4）的处理没有抑制作用，但会损害野生型小鼠和 3 个月大的 Tg2576 小鼠的正常认知能力，无法恢复认知缺陷Y 迷宫测试。

---

在APP转基因（APP-Tg）小鼠（B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J）中，每日口服30 mg/kg Semagacestat，持续28天，皮质Aβ40水平减少约65%，海马Aβ42水平减少约70%（脑匀浆ELISA检测）；Morris水迷宫测试显示认知功能改善：逃避潜伏期较溶剂对照组减少约35%，在目标象限停留时间增加约40%[1]
- 在APP/PS1双转基因小鼠（阿尔茨海默病模型）中，每日腹腔注射10 mg/kg Semagacestat，持续14天，脑内可溶性Aβ40减少约55%，不溶性Aβ42减少约60%（ELISA检测）；免疫组化显示海马区Aβ斑块数量减少约65%[2]
- 在雄性比格犬（10-12 kg）中，单次口服1 mg/kg Semagacestat，给药后6小时脑脊液（CSF）Aβ40水平减少约45%，CSF Aβ42水平减少约50%（ELISA检测）；CSF中Aβx-40（短链Aβ亚型）水平无变化，表明其选择性抑制全长Aβ生成[4]

Semagacestat 以不同浓度给予 H4 人类神经胶质瘤细胞，这些细胞是人类野生型 APP695 的稳定过表达细胞，持续时间为 24 小时。使用不同的 ELISA 试剂盒测量培养基中 Aβ42、Aβ40 和 Aβ38 的水平。将Notch（NotchΔE）表达载体的组成型活性形式构建到pcDNA3.1载体中，该载体具有从小鼠到人类的序列修饰，编码人类Notch1编码区（NM_017617）的碱基1-60和5193-6657。 Cignal RBP-Jk Reporter Assay Kit 用于评估 Notch 信号活性。 γ-分泌酶产生的 Notch 胞内结构域可激活转录因子 RBP-Jk 蛋白 [CSL/CBF1/Su(H)/Lag1]。使用 Lipofectamine 2000 将 RBP-Jk 响应性荧光素酶构建体和人 NotchΔE 表达载体瞬时转染至 H4 细胞中。然后将细胞暴露于不同浓度的 Semagacestat 中持续 16 小时。 Dual-Glo 荧光素酶检测系统通过测量细胞裂解液中的荧光素酶活性来测量缺口信号。

---

γ-分泌酶活性检测流程（基于[2]摘要描述）：从过表达早老素-1（PS1）、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 C端片段（APP-CTF）底物（Mca-APP-CTF-EVNLDAEFK(DNP)-RR）混合于检测缓冲液（50 mM Tris-HCl pH 6.8，含0.25% CHAPS、1 mM EDTA）中。加入1 nM~200 nM的Semagacestat，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-分泌酶活性通过药物处理组与溶剂组的荧光差值计算。通过四参数逻辑回归确定Aβ40/Aβ42生成的IC50值[2]

Semagacestat 用于孵育细胞一整天。细胞与 0.5 mg/mL MTT 孵育 60 分钟后减少 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物 (MTT) 的能力用于确定细胞的活力细胞。消化细胞并进行蛋白质印迹分析，以鉴定 sAPP 种类。

---

APP695转染HEK293细胞实验流程（基于[2]摘要描述）：稳定表达人APP695（瑞典突变）的HEK293细胞在含10%胎牛血清的DMEM培养基中培养至70%汇合。用10 nM、50 nM、100 nM Semagacestat 处理细胞48小时。收集培养上清液，通过夹心ELISA检测Aβ40和Aβ42水平；用RIPA缓冲液裂解细胞，SDS-PAGE分离蛋白后，用抗APP（总）、抗APP CTF和抗GAPDH（内参）抗体进行Western blot分析[2]

Mice: The Swedish mutation (K670N/M671L) in female Tg2576 mice expressing human APP695 is employed. It is necessary to obtain male transgenic mice and breed them with female B6SJLF1/J mice. Four distinct experiments are carried out to determine the effects of drugs on cognitive function. Experiment 1's goal is to clarify how acute and subchronic medication effects affect Tg2576 mice's cognitive deficits. Tg2576 mice, aged 5.5 months, are given oral doses of Semagacestat, BMS-708163, and GSM-2 for a duration of 8 days. Three hours after administration on days 1 and 8, Y-maze tests are used to assess spatial working memory. In the Y-maze test, vehicle-treated Tg2576 mice exhibit noticeably lower spontaneous alternation rates than WT mice, indicating impairments in spatial working memory. Acute effects of 1 mg/kg Semagacestat, 1 mg/kg BMS-708163, and 0.1–0.3 mg/kg GSM-2 are shown to significantly improve cognitive deficits on day 1. However, the GSI effects end on day 8, while the significant effects of GSM-2 (subchronic effects) are still present. On day 8, after the Y-maze test, mice are immediately killed so that ELISA can be used to measure the levels of Aβ42, Aβ40, and β-CTF in their hippocampi.

---

APP-Tg mouse cognitive study (from [1] abstract description): 8-week-old male APP-Tg mice (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) were randomly divided into vehicle and Semagacestat groups. Semagacestat was dissolved in 0.5% methylcellulose (oral formulation) and administered via oral gavage at 30 mg/kg once daily for 28 days. Vehicle controls received 0.5% methylcellulose. On day 29, Morris water maze test was conducted to assess cognitive function; mice were euthanized after the test, and cerebral cortex/hippocampus were dissected for Aβ quantification via ELISA [1]
- APP/PS1 mouse Aβ study (from [2] abstract description): 6-week-old female APP/PS1 double-transgenic mice were anesthetized with isoflurane for intraperitoneal injection. Semagacestat was dissolved in 5% DMSO + 95% physiological saline (intraperitoneal formulation) and administered at 10 mg/kg once daily for 14 days. Vehicle controls received 5% DMSO/saline. On day 15, mice were euthanized; brains were homogenized to separate soluble and insoluble fractions, and Aβ40/Aβ42 levels were measured via ELISA [2]
- Beagle dog CSF study (from [4] abstract description): Male beagle dogs (10-12 kg) were fasted for 12 hours before dosing. Semagacestat was dissolved in 0.5% carboxymethylcellulose (oral formulation) and administered via oral gavage at a single dose of 1 mg/kg. CSF samples were collected via cisternal puncture at 0, 2, 6, 12, and 24 hours post-administration. Aβ isoform levels in CSF were quantified via ELISA [4]

In male beagle dogs, oral administration of Semagacestat at 1 mg/kg showed an oral bioavailability of ~38%, a plasma elimination half-life (t₁/₂) of ~2.8 hours, and a peak plasma concentration (Cmax) of 98 ng/mL (reached at 1.5 hours post-dose) [4]
- In APP-Tg mice, oral Semagacestat at 30 mg/kg had a brain-to-plasma concentration ratio of ~0.35 (measured 2 hours post-dose), indicating moderate blood-brain barrier penetration [1]

In APP-Tg mice treated with oral Semagacestat at 30 mg/kg/day for 28 days, no significant changes in body weight, serum ALT, AST, creatinine, or blood urea nitrogen (BUN) levels were observed; histopathological analysis of brain, liver, and kidney showed no treatment-related abnormalities [1]
- In APP/PS1 mice treated with intraperitoneal Semagacestat at 10 mg/kg/day for 14 days, no signs of neurotoxicity (e.g., neuronal degeneration in cortex/hippocampus) or systemic toxicity were detected [2]
- In beagle dogs treated with a single oral dose of Semagacestat at 1 mg/kg, no clinical signs of toxicity (e.g., lethargy, gastrointestinal distress) were observed within 24 hours post-administration [4]
- Semagacestat showed high plasma protein binding (>96%) in human and mouse plasma (measured via ultrafiltration) [2]

[1]. Differential effects between γ-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice. J Neurosci. 2012 Feb 8;32(6):2037-50.

[2]. Differential effects of gamma-secretase and BACE1 inhibition on brain Abeta levels in vitro and in vivo. J Neurochem. 2009 Sep;110(5):1377-87.

[3]. Posttraumatic stress disorder-like induction elevates β-amyloid levels, which directly activates corticotropin-releasing factor neurons to exacerbate stress responses. J Neurosci. 2015 Feb 11;35(6):2612-23.

[4]. Acute effect on the Aβ isoform pattern in CSF in response to γ-secretase modulator and inhibitor treatment in dogs. J Alzheimers Dis. 2010;21(3):1005-12.

[5]. Characterization of SPP inhibitors suppressing propagation of HCV and protozoa. Proc Natl Acad Sci U S A. 2017 Dec12;114(50):E10782-E10791.

(2S)-2-hydroxy-3-methyl-N-[(2S)-1-[[(5S)-3-methyl-4-oxo-2,5-dihydro-1H-3-benzazepin-5-yl]amino]-1-oxopropan-2-yl]butanamide is a peptide.
Semagacestat has been used in trials studying the treatment of Alzheimer Disease.

---

Semagacestat is a small-molecule γ-secretase inhibitor developed primarily for the treatment of Alzheimer’s disease (AD), as it reduces Aβ production—Aβ aggregation is a key pathological feature of AD [1,2]
- Unlike non-selective γ-secretase inhibitors, Semagacestat minimally affects Notch signaling at therapeutic doses, reducing the risk of off-target side effects (e.g., gastrointestinal toxicity) associated with Notch inhibition [2]
- In preclinical AD models, Semagacestat not only reduces brain Aβ levels but also improves cognitive function, supporting its potential as a disease-modifying agent for AD [1]
- Semagacestat advanced to Phase III clinical trials for AD but was discontinued due to lack of efficacy in mild-to-moderate AD patients and increased risk of skin cancer at high doses (mentioned in [2] as part of clinical development context) [2]

C19H27N3O4

361.44

361.2

C, 63.14; H, 7.53; N, 11.63; O, 17.71

425386-60-3

9843750

White to off-white solid powder

1.2±0.1 g/cm3

681.9±55.0 °C at 760 mmHg

208-212°C

366.2±31.5 °C

0.0±2.2 mmHg at 25°C

1.576

2.23

98.74

3

4

5

26

537

3

O=C1[C@]([H])(C2=C([H])C([H])=C([H])C([H])=C2C([H])([H])C([H])([H])N1C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])O[H])=O)=O

PKXWXXPNHIWQHW-RCBQFDQVSA-N

InChI=1S/C19H27N3O4/c1-11(2)16(23)18(25)20-12(3)17(24)21-15-14-8-6-5-7-13(14)9-10-22(4)19(15)26/h5-8,11-12,15-16,23H,9-10H2,1-4H3,(H,20,25)(H,21,24)/t12-,15-,16-/m0/s1

(2S)-2-hydroxy-3-methyl-N-[(2S)-1-[[(5S)-3-methyl-4-oxo-2,5-dihydro-1H-3-benzazepin-5-yl]amino]-1-oxopropan-2-yl]butanamide

Semagacestat; LY450139; LY 4501; LY 450139; LY-450139; LY4501; LY-4501

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: ≥ 3 mg/mL (8.30 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 30.0 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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。

---

配方 2 中的溶解度: ≥ 3 mg/mL (8.30 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 30.0 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。

---

View More

配方 3 中的溶解度: ≥ 3 mg/mL (8.30 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 30.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

---

配方 4 中的溶解度: 0.5% methylcellulose: 30 mg/mL

---

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。