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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 | |
Additional Infomation |
Aloxistatin is an L-leucine derivative that is the amide obtained by formal condensation of the carboxy group of (2S,3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid with the amino group of N-(3-methylbutyl)-L-leucinamide. It has a role as a cathepsin B inhibitor and an anticoronaviral agent. It is a L-leucine derivative, a monocarboxylic acid amide, an epoxide and an ethyl ester.
Aloxistatin is an inhibitor of cysteine protease with blood platelet aggregation inhibiting activity. Aloxistatin is an irreversible, membrane-permeable inhibitor of lysosomal and cytosolic cysteine proteases with the ability to inhibit calpain activity in intact platelets. E-64 isolated from a culture of Aspergillus japonicus is a specific inhibitor of cysteine proteinases. E-64-c, a synthetic analog of E-64, was effective in model animals of muscular dystrophy only when it was given intraperitoneally and by means of osmotic minipump. It showed no effects due to its low absorbability from intestine when it was administered orally. EST, the ethyl ester of E-64-c, was expected to be readily absorbed through intestinal membrane, since it is more lipophilic than E-64-c. Both EST and E-64-c have a high specificity to cysteine proteinase similar to E-64 but E-64-c was 100 to 1000 times stronger than EST in in vitro cathepsin inhibition. However, EST was stronger than E-64-c in cathepsin inhibition when given orally. The cathepsin B&L activities (whole activities of cathepsins B and L) in the skeletal muscle, heart and liver of hamsters were strongly inhibited soon after oral administration of 100 mg/kg body weight of EST. The inhibition continued for at least 3 h and then disappeared gradually. E-64-c was found in plasma of hamster treated with EST, but unchanged EST was not found. These results suggested that EST was converted to E-64-c, a more active form, during the permeation through intestinal membrane. The conversion of EST to E-64-c was also indicated by the absorption experiment using in situ loop method. EST was thus shown to be useful as an oral drug and expected to be effective in therapeutic trials using model animals.[1] E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor (Tamai et al. (1986) J. Pharmacobio-Dyn. 9, 672-677), was tested for ability to inhibit calpain activity in intact platelets. Calpain activity was measured by proteolysis of actin-binding protein and talin, two known substrates of calpain. Incubation of platelets with E-64c (not permeant) or E-64d before lysis prevented proteolysis after lysis. When the platelets were incubated with E-64c or E-64d and then washed to remove the drugs before lysis, only E-64d inhibited proteolysis. When platelets were incubated with E-64c or E-64d and then activated with A23187 plus calcium, a treatment that activates intraplatelet calpain, only E-64d inhibited proteolysis. These results indicate that E-64d can enter the intact cell and inhibit calpain.[2] Parathyroid hormone (PTH) activates calpains I and II (calcium-activated papain-like proteases) and stimulates the synthesis and secretion of cathepsin B (a lysosomal cysteine protease) in osteoblastic cells. Anabolic doses of PTH also stimulate osteoprogenitor cell proliferation and differentiation into mature, fully functional osteoblasts capable of elaborating bone matrix, whereas catabolic doses of PTH stimulate calcium mobilization and matrix turnover. Previous investigations in other cell types have demonstrated that calcium-activated calpains play a major role in regulating proliferation and differentiation by catalyzing limited regulatory proteolysis of nuclear proteins, transcription factors, and enzymes. We tested the hypothesis that inhibition of intracellular cysteine proteases such as the calpains will ablate PTH-mediated osteoblast proliferation and differentiation, two fundamental indices of bone anabolism. A brief preincubation with the membrane-permeable, irreversible cysteine protease inhibitor E64d (10 micrograms/mL) before short-term PTH treatment blunted PTH-induced cell proliferation in subconfluent cultures and also attenuated proliferation and inhibited differentiation in longer-term confluent cultures. This confirms the hypothesis that cysteine proteases such as the calpains are important in mediating the proliferative and prodifferentiating or anabolic effects of PTH on MC3T3-E1 cells in culture. Immunofluorescent localization demonstrated that calpain I, calpain II, and calpastatin (the endogenous calpain inhibitor) are abundant and widely distributed within actively proliferating MC3T3-E1 preosteoblasts. Since the calpains are active and stable at neutral intracellular pH levels in osteoblasts, whereas cathepsins are not, our results support a role for these calcium-activated regulatory proteases in mediating the anabolic effects of PTH in bone.[3] |
Molecular Formula |
C17H30N2O5
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Molecular Weight |
342.43
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Exact Mass |
342.215
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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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Related CAS # |
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PubChem CID |
65663
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Appearance |
White to off-white solid powder
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Density |
1.2±0.1 g/cm3
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Boiling Point |
470.5±55.0 °C at 760 mmHg
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Melting Point |
126.2°C
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Flash Point |
238.4±31.5 °C
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Vapour Pressure |
0.0±2.6 mmHg at 25°C
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Index of Refraction |
1.530
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LogP |
3.64
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Hydrogen Bond Donor Count |
2
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Hydrogen Bond Acceptor Count |
5
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Rotatable Bond Count |
11
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Heavy Atom Count |
24
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Complexity |
450
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Defined Atom Stereocenter Count |
3
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SMILES |
C([C@H]1O[C@@H]1C(=O)OCC)(=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) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.30 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.30 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (7.30 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 2.08 mg/mL (6.07 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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. Solubility in Formulation 5: ≥ 2.08 mg/mL (6.07 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 6: 2% DMSO+corn oil: 5mg/mL |
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.