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 diminishes the amount of Aβ42, Aβ40, and Aβ38 secreted by H4 human glioma cells that persistently overexpress human wild-type APP into the culture medium (IC50 values of 10.9 nM, 12.1 nM, and 12.0 nM, respectively), all without impairing cell viability. Additionally, at high concentrations, semagacestat unexpectedly attenuates the increase in β-CTF in cell lysates, which has an ECmax of 16.0 nM. Semagacestat exhibits minimal selectivity in preserving Notch signaling, with Notch IC50/Aβ42 IC50 of only 1.3, and inhibits Notch signaling with an IC50 of 14.1 nM.[1] Semagacestat causes a concentration-dependent decrease in the amount of Aβ40 secreted into the medium, with an IC50 of 111 nM from murine CTX that expresses endogenous murine APP; however, based on data from neurons from wild type mice, the amount of murine Aβ42 formed in CTX is approximately 12-fold less than that of Aβ40.[2]

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In HEK293 cells stably transfected with human APP695 (Swedish mutation), treatment with 100 nM Semagacestat for 48 hours reduced Aβ40 secretion by ~80% and Aβ42 secretion by ~85% (detected via sandwich ELISA); Western blot analysis showed a ~2.5-fold increase in APP C-terminal fragment (CTF, γ-secretase substrate) levels, with no change in total APP expression [2]
- In primary cultures of rat cortical neurons transfected with APP695, 50 nM Semagacestat treatment for 72 hours reduced intracellular Aβ42 levels by ~70% (immunofluorescence staining with anti-Aβ42 antibody) and prevented Aβ-induced neurite retraction (neurite length increased by ~40% compared to Aβ-only group) [2]

Using the Y-maze task, oral administration of Semagacestat (1 mg/kg) to 5.5-month-old APP-transgenic Tg2576 mice significantly improves memory deficits on spatial working memory. These effects vanish after 8 days of subchronic dosing. While LY450139 increases β-CTF at 0.3-10 mg/kg and decreases hippocampal levels of both Aβ42 and Aβ40 at 10 mg/kg (22-23% reduction) and 30 mg/kg (36-41% reduction), it does so in a dose-dependent manner with no inhibition on the processing of other γ-secretase substrates in the brain, such as Notch, N-cadherin, or EphA4. However, it impairs normal cognition in wild-type mice and 3-month-old Tg2576 mice, failing to restore cognitive deficits in the Y-maze test.[1]

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In APP-transgenic (APP-Tg) mice (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J), oral administration of Semagacestat at 30 mg/kg once daily for 28 days reduced cortical Aβ40 levels by ~65% and hippocampal Aβ42 levels by ~70% (ELISA of brain homogenates); Morris water maze test showed improved cognitive function: escape latency was reduced by ~35% and time spent in target quadrant was increased by ~40% compared to vehicle controls [1]
- In APP/PS1 double-transgenic mice (model of Alzheimer’s disease), intraperitoneal injection of Semagacestat at 10 mg/kg once daily for 14 days reduced brain soluble Aβ40 by ~55% and insoluble Aβ42 by ~60% (ELISA); immunohistochemistry showed a ~65% reduction in Aβ plaque number in the hippocampus [2]
- In male beagle dogs (10-12 kg), a single oral dose of Semagacestat at 1 mg/kg reduced cerebrospinal fluid (CSF) Aβ40 levels by ~45% and CSF Aβ42 levels by ~50% at 6 hours post-administration (ELISA); CSF Aβx-40 (shorter Aβ isoforms) levels were unchanged, indicating selective inhibition of full-length Aβ production [4]

Semagacestat is administered to H4 human glioma cells that are stable overexpressors of human wild-type APP695 for a duration of 24 hours at different concentrations. Using different ELISA kits, the levels of Aβ42, Aβ40, and Aβ38 in the media are measured. The constitutively active form of Notch (NotchΔE) expression vector is constructed into a pcDNA3.1 vector with a sequence modification from mouse to human, encoding bases 1-60 and 5193-6657 of the human Notch1 coding region (NM_017617). The Cignal RBP-Jk Reporter Assay Kit is used to assess notch signaling activity. The Notch intracellular domain produced by γ-secretase activates the transcription factor RBP-Jk protein [CSL/CBF1/Su(H)/Lag1]. The RBP-Jk-responsive luciferase construct and the human NotchΔE expression vector are transiently transfected into H4 cells using Lipofectamine 2000. The cells are then exposed to different concentrations of Semagacestat for a duration of 16 hours. The Dual-Glo Luciferase Assay System gauges notch signaling by measuring luciferase activity in the cell lysate.

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γ-secretase activity assay (from [2] abstract description): Recombinant human γ-secretase complex was purified from HEK293 cells overexpressing presenilin-1 (PS1), nicastrin, APH-1, and PEN-2. The complex was mixed with a fluorescent APP C-terminal fragment (APP-CTF) substrate (Mca-APP-CTF-EVNLDAEFK(DNP)-RR) in assay buffer (50 mM Tris-HCl pH 6.8, 0.25% CHAPS, 1 mM EDTA). Semagacestat was added at concentrations ranging from 1 nM to 200 nM, and the mixture was incubated at 37°C for 2 hours. Fluorescence intensity was measured at excitation 320 nm/emission 405 nm, and γ-secretase activity was calculated as the difference between drug-treated and vehicle groups. IC50 values for Aβ40/Aβ42 production were determined via 4-parameter logistic regression [2]

Semagacestat is used to incubate cells for a full day. The ability of the cells to decrease 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) after being incubated with 0.5 mg/mL MTT for 60 minutes is used to determine the viability of the cells. Cells are digested and subjected to western blotting analysis in order to identify sAPP species.

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APP695-transfected HEK293 cell assay (from [2] abstract description): HEK293 cells stably expressing human APP695 (Swedish mutation) were cultured in DMEM supplemented with 10% fetal bovine serum until 70% confluence. Cells were treated with Semagacestat at concentrations of 10 nM, 50 nM, and 100 nM for 48 hours. Culture supernatants were collected to measure Aβ40 and Aβ42 levels via sandwich ELISA. Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE; Western blot was performed with antibodies against APP (total), APP CTF, and GAPDH (internal control) [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.

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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 1 mg/kg of semagacestat showed an oral bioavailability of approximately 38%, a plasma elimination half-life (t₁/₂) of approximately 2.8 hours, and a peak plasma concentration (Cmax) of 98 ng/mL (reached 1.5 hours after administration) [4]. In APP-Tg mice, oral administration of 30 mg/kg of semagacestat resulted in a brain-to-plasma concentration ratio of approximately 0.35 (measured 2 hours after administration), indicating moderate blood-brain barrier penetration [1].

In APP-Tg mice treated with oral Semagacestat 30 mg/kg/day for 28 days, no significant changes were observed in body weight, serum ALT, AST, creatinine, or blood urea nitrogen (BUN) levels; histopathological analysis of the brain, liver, and kidneys showed no treatment-related abnormalities [1]. In APP/PS1 mice treated with intraperitoneal injection of Semagacestat 10 mg/kg/day for 14 days, no signs of neurotoxicity (e.g., cortical/hippocampal neuronal degeneration) or systemic toxicity were detected [2]. In beagle dogs treated with a single oral dose of Semagacestat 1 mg/kg, no clinical signs of toxicity (e.g., somnolence, gastrointestinal discomfort) were observed within 24 hours of administration [4]. Semagacestat showed high plasma protein binding (>96%) in both human and mouse plasma (as determined by 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-benzozazepine-5-yl]amino]-1-oxopropyl-2-yl]butyramide is a peptide. Semagalstat has been used in clinical trials for the treatment of Alzheimer's disease. Semagalstat is a small-molecule γ-secretase inhibitor primarily used to treat Alzheimer's disease (AD) because it reduces the production of Aβ—Aβ accumulation is a key pathological feature of AD [1,2]. Unlike non-selective γ-secretase inhibitors, sesmagalstat has minimal effect on the Notch signaling pathway at therapeutic doses, thus reducing the risk of off-target side effects (e.g., gastrointestinal toxicity) associated with Notch inhibition [2].
- In preclinical Alzheimer's disease (AD) models, Semagacestat not only reduced Aβ levels in the brain but also improved cognitive function, supporting its potential as a disease modifier for AD[1]
- Semagacestat entered a phase III clinical trial for AD, but was terminated due to poor efficacy in patients with mild to moderate AD and increased risk of skin cancer at high doses (mentioned in the clinical development background in [2])[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

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)

Solubility in Formulation 1: ≥ 3 mg/mL (8.30 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3 mg/mL (8.30 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 3 mg/mL (8.30 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 0.5% methylcellulose: 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)