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1mg |
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5mg |
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10mg |
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25mg |
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50mg |
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100mg |
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250mg |
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Purity: ≥98%
Aloxistatin (also known as E-64d, E-64c ethyl ester; EST, Loxistatin; NSC 694281) is a novel, potent, selective, irreversible, broad-spectrum and membrane-permeable cysteine protease inhibitor with anticoagulant properties. Aloxistatin suppresses autophagy and lysosomal activity in addition to acting by alkylating the cysteine thiol group in the protease's catalytic site.
Targets |
Cysteine protease
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ln Vitro |
Aloxistatin inhibits calpain, which prevents proteolysis by penetrating the intact platelet.[2]
Aloxistatin distorts Osteoblast differentiation is inhibited in vitro by parathyroid hormone (PTH)-induced cell proliferation.[3] |
ln Vivo |
Aloxistatin (100 mg/kg, p.o.) significantly reduces the activity of cathepsin B&L in hamster skeletal muscle, heart, and liver.[1]
Aloxistatin provides neuroprotection in SCI lesion and penumbra in spinal cord injury (SCI) rats.[4] Aloxistatin inhibits cathepsin B activity, which lowers brain amyloid-β and improves memory deficits in animal models of Alzheimer's disease.[5] |
Enzyme Assay |
The inhibitors L1 (10–20 μM) or Aloxistatin (20–30 μM) are applied to CTLs and NK cells (0.8×106/mL) for a duration of 24 hours at 37°C in 24-well plates. After that, cells are utilized in 51Cr-release experiments or lysed to look at perforin in Western blot analyses. Some 51Cr-release assays also add the inhibitor at the same concentration during the 4-hour reactions, as shown. NP-40 lysis buffer (25 mM HEPES, 250 mM NaCl, 2.5 mM ethylenediaminetetraacetic acid, 0.1% volume/volume Nonidet P-40) is used to prepare cell lysates, and the Bradford assay is used to measure the total protein concentration. Protein is loaded and resolved in equal amounts onto 8% SDS-PAGE gels. The right antibodies are used as indicated to detect human or mouse perforin. As a loading control, anti-actin antibody is employed.
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Cell Assay |
Staining for the proliferation marker Ki67 or the apoptotic marker cleaved caspase 3 allows for the assessment of cell proliferation and apoptosis. The procedure for the polarity markers is the same as before. For four days, MCF10 variants are grown in 3D rBM overlay cultures and are treated with either 5 μM CA074Me, 5 μM Aloxistatin, or 0.1% DMSO. By counting a total of 100 structures on two different coverslips using a Zeiss Axiophot epifluorescent microscope, the percentage of structures that are positive for Ki67 or cleaved caspase 3 can be ascertained. If a structure has at least one Ki67-staining cell, it is deemed to be Ki67 positive. When a structure has one or more cells that are positive for cleaved caspase 3 and those cells are not located in the center of a developing lumen, the structure is said to be caspase 3 positive[3].
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Animal Protocol |
Mice and Pigs: Male Hartley strain guinea pigs, weighing an average of 400 g, or approximately six weeks old, are used. The London mutant β-secretase site sequences and the wt β-secretase site-containing human AβPP are expressed in male transgenic mice. Although accurate dosage can be achieved by gavage delivery, this method is traumatic and should only be used for brief dosage intervals (up to approximately one week). In the studies using guinea pigs, gavage delivery is utilized. The recommended dosages of aloxistatin (0.1, 1.0, 5, and 10 mg/kg) are suspended in Me2SO and given orally once a day through a feeding tube. Me2SO alone is administered by gavage to vehicle control animals.
Rats: The rats are inbred male DS rats. Up until the age of seven weeks, weaned rats are given laboratory chow containing 0.3% NaCl. DS rats given an 8% NaCl diet for seven weeks show signs of compensating for concentric left ventricular (LV) hypertrophy, which is related to hypertension at twelve weeks. At nineteen weeks, the rats show signs of a distinct stage of fatal LV failure, accompanied by lung congestion. To that end, DS rats are started on an 8% NaCl diet at 7 weeks of age. From 12 to 19 weeks of age, they are randomized into three groups: HF, Aloxistatin (10 mg per kg of body mass per day, administered intraperitoneally every other day), and RNH-6270 (3 mg/kg per day in chow) (n=10 for each group). Preliminary experiments and prior research determine the doses of aloxistatin and RNH-6270, an ARB. Age-matched controls (control group, n = 10) were DS rats fed a diet containing 0.3% NaCl. All of the rats are killed at 19 weeks of age by injecting an excess of 50 mg/kg of NSC 10816 intraperitoneally, and their hearts are taken out for histological and biological examinations. To measure renin activity, arterial blood is drawn from the abdominal aorta. Every week starting at 7 weeks of age, conscious rats have their heart rate and systolic blood pressure measured using a noninvasive tail-cuff technique. In independent studies, n = 5 per group of 12-week-old DS rats fed a low-salt diet starting at 7 weeks of age are given vehicle, RNH-6270, or Aloxistatin in the same way as in the previous studies. The LV tissues used to measure targeting mRNAs and protein levels are then promptly frozen in liquid nitrogen and kept at -80°C. |
References |
Molecular Formula |
C17H30N2O5
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Molecular Weight |
342.43
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Exact Mass |
342.22
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Elemental Analysis |
C, 59.63; H, 8.83; N, 8.18; O, 23.36
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CAS # |
88321-09-9
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Appearance |
Solid powder
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SMILES |
CCOC(=O)[C@@H]1[C@H](O1)C(=O)N[C@@H](CC(C)C)C(=O)NCCC(C)C
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InChi Key |
SRVFFFJZQVENJC-IHRRRGAJSA-N
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InChi Code |
InChI=1S/C17H30N2O5/c1-6-23-17(22)14-13(24-14)16(21)19-12(9-11(4)5)15(20)18-8-7-10(2)3/h10-14H,6-9H2,1-5H3,(H,18,20)(H,19,21)/t12-,13-,14-/m0/s1
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Chemical Name |
ethyl (2S,3S)-3-[[(2S)-4-methyl-1-(3-methylbutylamino)-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylate
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Synonyms |
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
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Solubility (In Vivo) |
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Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 2.9203 mL | 14.6015 mL | 29.2030 mL | |
5 mM | 0.5841 mL | 2.9203 mL | 5.8406 mL | |
10 mM | 0.2920 mL | 1.4602 mL | 2.9203 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.