| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 50mg |
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| 100mg |
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| Other Sizes |
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| Targets |
mitochondria-targeted superoxide dismutase mimetic
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| ln Vitro |
Mito-tempo (MT) is a mitochondria-targeted superoxide dismutase mimetic that protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation.
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| ln Vivo |
At both time points, Mito-TEMPO (MT) significantly inhibited the rise in ALT activity and decreased the necrotic region, suggesting that Mito-TEMPO's protection persisted for at least 24 hours following APAP. In the latter phases of APAP hepatotoxicity, Mito-Tempo can cause secondary apoptosis. By blocking RIP3, Mito-Tempo causes secondary apoptosis in response to excessive APAP[1].
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| Enzyme Assay |
Caspase activity measurements and western blotting
Liver caspase activity was measured as described (Lawson et al. 1999). In brief, frozen liver tissue was homogenized in 25 mM HEPES buffer containing 5 mM EDTA, 2 mM DTT and 0.1% CHAPS, and then centrifuged to get the homogenate. A fluorogenic substrate (Ac-DEVD-AFC) was added to the homogenate and fluorescence was measured with or without the presence of pan-caspase inhibitor (z-VAD-fmk). Results are expressed as RFU per unit time per mg protein concentration. Western blotting was performed as described (Bajt et al. 2000) using a rabbit anti-caspase 3 antibody and a rabbit anti-beta-actin antibody, and an anti-RIP3 antibody. The proteins were visualized using a goat anti-rabbit HRP conjugated antibody. |
| Cell Assay |
Histology[1]
Liver tissue samples embedded in paraffin were cut in 5 μm sections and stained with hematoxylin and eosin (H&E) for assessment of apoptosis versus necrosis (Gujral et al. 2002). Nitrotyrosine staining was performed as previously described (Knight et al. 2002), using a rabbit polyclonal anti-nitrotyrosine antibody and the Dako LSAB peroxidase kit. Active caspase-3 staining was performed using a cleaved caspase-3 (Asp175) antibody. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed for cell death using the In Situ Cell Death Detection Kit, AP following manufacturer’s instructions. |
| Animal Protocol |
Animals
Male C57BL/6J mice 8-12 weeks of age were kept in an environmentally controlled room with a 12h light/dark cycle. RIP3-deficient mice (C57BL/6N background) and C57BL/6N wild type animals were were acclimated before experiments with free access to diet and water. Experimental design Overnight fasted mice (16-18h) were treated i.p. with 300 mg/kg APAP dissolved in warm saline. Some mice were treated with 200mg/kg APAP in experiments evaluating effect of RIP3 deficiency. A dose of 20 mg/kg Mito-Tempo dissolved in saline was administered i.p. 1.5 or 3 h after APAP. Some mice were subsequently treated (i.p.) with 10 mg/kg Z-VD fmk (EP1013) dissolved in Tris-buffered saline or vehicle 2 h after APAP. To mimic the clinical care of APAP-overdose patients, some mice received the antidote NAC (i.p., 500 mg/kg) at 1.5 or 3 h after APAP overdose. Groups of mice were euthanized at 0-24 h post-APAP by exsanguination under isoflurane anesthesia. Additional mice were treated i.p. with 100 μg/kg Salmonella abortus equi endotoxin (ET) and 700 mg/kg galactosamine (Gal) for 6 h. Blood was drawn into a heparinized syringe and centrifuged to obtain plasma. Plasma ALT activities were measured using the ALT assay kit from Pointe Scientific, MI. The liver tissue was cut into pieces and fixed in 10% phosphate-buffered formalin for histology or flash frozen in liquid nitrogen and subsequently stored at −80°C. In vivo morpholino treatment The antisense sequence used for RIP3 was 5’-TAGGCCATAACTTGACAGAAGACAT-3’. The standard control in vivo oligo sequence from Gene Tools was used for all control morpholino treatments. Morpholinos were used as provided by the manufacturer and administered ip to mice (12.5 mg/kg body weight) every 24h for 2 days. Treatment with APAP was then done on day 3. |
| References | |
| Additional Infomation |
Researchers previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300 mg/kg APAP followed by 20 mg/kg MT 1.5 h or 3 h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24 h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10 mg/kg) 2 h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.[1]
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| Molecular Formula |
C29H36CLN2O2P
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|---|---|
| Molecular Weight |
511.03510761261
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| Exact Mass |
510.22
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| Elemental Analysis |
C, 68.16; H, 7.10; Cl, 6.94; N, 5.48; O, 6.26; P, 6.06
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| CAS # |
1334850-99-5
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| Related CAS # |
MitoTEMPO hydrate;1569257-94-8
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| PubChem CID |
134828258
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| Appearance |
Light yellow to pink solid powder
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
35
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| Complexity |
612
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
QJEOOHMMSUBNGG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H35N2O2P.ClH/c1-28(2)20-23(21-29(3,4)31(28)33)30-27(32)22-34(24-14-8-5-9-15-24,25-16-10-6-11-17-25)26-18-12-7-13-19-26;/h5-19,23,33H,20-22H2,1-4H3;1H
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| Chemical Name |
[2-[(1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)amino]-2-oxoethyl]-triphenylphosphanium Chloride
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| Synonyms |
Mito-TEMPO; 1334850-99-5; [2-[(1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)amino]-2-oxoethyl]-triphenylphosphanium;chloride; (2-(2,2,6,6-TETRAMETHYLPIPERIDIN-1-OXYL-4-YLAMINO)-2-OXOETHYL)TRIPHENYLPHOSPHONIUM CHLORIDE; Mito-TEMPO?; DTXSID601044299; MFCD26406410;
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
DMSO : ~125 mg/mL (~245.08 mM)
H2O : ~60 mg/mL (~117.64 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.25 mg/mL (4.41 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 22.5 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. Solubility in Formulation 2: ≥ 2.25 mg/mL (4.41 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 22.5 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. View More
Solubility in Formulation 3: ≥ 2.25 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 2.2 mg/mL (4.4 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline ≥ 2.2 mg/mL (4.4 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline) ≥ 2.2 mg/mL (4.4 mM) in 10% DMSO + 90% Corn oil Solubility in Formulation 5: 50 mg/mL (98.03 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9568 mL | 9.7840 mL | 19.5679 mL | |
| 5 mM | 0.3914 mL | 1.9568 mL | 3.9136 mL | |
| 10 mM | 0.1957 mL | 0.9784 mL | 1.9568 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.
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