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Biological Activity
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Description
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LY-411575 is a potent γ-secretase inhibitor with IC50 of 0.078 nM and 0.082 nM for membrane- and cell-based γ-secretase. |
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Targets
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γ-secretase |
γ-secretase |
Notch S3 cleavage |
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IC50 |
0.078 nM (membrane-based) |
0.082 nM (cell-based) |
0.39 nM [1] |
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In Vitro
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LY-411575 inhibits γ-secretase which can be assessed by the substrates like amyloid precursor protein (APP) and Notch S3 cleavage. [1] LY-411575, which blocks Notch activation, results in apoptosis in primary and immortalized KS cells. [2] |
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In Vivo
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10 mg/kg oral dose of LY-411575 decreases brain and plasma Aβ40 and -42 dose-dependently. [1] LY-411575 reduces cortical Aβ40 in young (preplaque) transgenic CRND8 mice (ED50 ≈ 0.6 mg/kg) and produces significant thymus atrophy and intestinal goblet cell hyperplasia at higher doses (>3 mg/kg). The therapeutic window is similar after oral and subcutaneous administration and in young and aged CRND8 mice. Both the thymus and intestinal side effects are reversible after a 2-week washout period. Three-week treatment with 1 mg/kg LY411575 reduces cortical Aβ40 by 69% without inducing intestinal effects, although a previously unreported change in coat color is observed. [3] |
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Clinical Trials
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Features
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Protocol
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Kinase Assay
[1]
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| Assays for Aβ and NICD |
Procedures for measuring γ-secretase activity in membranes prepared from HEK293 cells expressing APP have been described previously (Zhang L et al Biochemistry 40, 5049-5055). Intact HEK293 cells expressing either APP or NΔE are treated with various concentrations of LY- 411,575 for 4 hours at 37 °C. In the case of cells expressing NΔE, cells are lysed, the cell lysates are separated on a 4–12% NuPAGE gel, and the processed NICD fragment is detected via Western blot with a cleavage site-specific antibody. The inhibition of NICD production is quantified by spot densitometric analysis using FluorChem. In the case of cells expressing APP, the conditioned medium is collected, centrifuged at 10,000 × g for 5 minutes to remove cell debris, and stored at -20 °C prior to the determination of Aβ levels. Aβ40 and -42 produced in HEK293 membrane- and cell-based assays, as well as plasma Aβ40 and cortex Aβ40 from TgCRND8 mice, are analyzed without pretreatment using an electrochemiluminescence detection-based immunoassay. Plasma Aβ42 is measured by enzyme-linked immunosorbent assay. A commercially available enzyme-linked immunosorbent assay kit is used to measure cortex Aβ42 according to the manufacturer’s instructions. |
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Cell Assay
[2]
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| Cell Lines |
primary and immortalized KS cells |
| Concentrations |
500 μM |
| Incubation Time |
24 hours |
| Methods |
DNA/PI staining is performed using standard methodologies. Briefly, 1 × 106 cells are permeabilized with 100% ethanol in the presence of 15% FBS. The cells are washed and then treated for 15 minutes at 37 °C with 10 mg/mL RNAse. PI (5 mg/mL) is added, and the cells incubated for 1 hour at 4 °C prior to analysis by flow cytometry with 10 000 cells analysed per gated determination. The results are confirmed using the Immunotech Annexin V staining kit following the manufacturers’ instructions. At least three independent experiments are performed showing similar results. |
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Animal Study
[1]
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| Animal Models |
TgCRND8 mice model |
| Formulation |
LY-411575 is formulated as 10 mg/mL solutions in 50% polyethylene glycol, 30% propylene glycol, 10% ethanol and diluted in 0.4% methylcellulose for dosing. |
| Doses |
1–10 mg/kg |
| Administration |
Orally |
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References |
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[1] Wong GT, et al, J Biol Chem, 2004, 279(13), 12876-12882.
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[2] Curry CL, et al, Oncogene, 2005, 24(42), 6333-6344.
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[2] Hyde LA, et al, J Pharmacol Exp Ther, 2006, 319(3), 1133-1143.
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