mitochondria-targeted superoxide dismutase mimetic

Mito-tempo (MT) is a mitochondria-targeted superoxide dismutase mimetic that protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation.

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].

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.

Animals
Male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) 8-12 weeks of age were kept in an environmentally controlled room with a 12h light/dark cycle. RIP3-deficient mice (C57BL/6N background) were kindly provided by Dr. Vishva Dixit (Genentech, Inc). C57BL/6N wild type animals were from Charles River, Frederick, Maryland, USA). The mice were acclimated before experiments with free access to diet and water. All experimental protocols followed the criteria of the National Research Council for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center.
Experimental design
Overnight fasted mice (16-18h) were treated i.p. with 300 mg/kg APAP (Sigma-Aldrich, St. Louis, MO) 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; a generous gift from Dr. S.X. Cai, Epicept Corp., San Diego, CA) 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
All vivo-morpholinos were from Gene Tools, LLC (Philomath, OR, USA). 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.

[1]. Du K, et al. Mito-tempo protects against acute liver injury but induces limited secondary apoptosis during the late phase of acetaminophen hepatotoxicity. Arch Toxicol. 2018 Oct 15

C29H36CLN2O2P

511.03510761261

510.22029

C, 68.16; H, 7.10; Cl, 6.94; N, 5.48; O, 6.26; P, 6.06

1334850-99-5

MitoTEMPO hydrate;1569257-94-8

Light yellow to pink solid powder

52.6Ų

[Cl-].[P+](C1C=CC=CC=1)(C1C=CC=CC=1)(C1C=CC=CC=1)CC(NC1CC(C)(C)N(C(C)(C)C1)O)=O

QJEOOHMMSUBNGG-UHFFFAOYSA-N

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

[2-[(1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)amino]-2-oxoethyl]-triphenylphosphanium Chloride

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~245.08 mM)
H2O : ~60 mg/mL (~117.64 mM)

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.

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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.

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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.
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 corn oil and mix evenly.

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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

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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.

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