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.

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.

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.

[1]. Mito-tempo protects against acute liver injury but induces limited secondary apoptosis during the late phase of acetaminophen hepatotoxicity. Arch Toxicol. 2019 Jan;93(1):163-178.

[2]. Targeting antioxidants to mitochondria by conjugation to lipophilic cations. Annu Rev Pharmacol Toxicol. 2007;47:629-56.

Previous studies have reported that delayed treatment with the mitochondrial-targeted superoxide dismutase mimic Mito-tempo (MT) can protect mice from early APAP hepatotoxicity by inhibiting the production of peroxynitrite. However, it remains unclear whether this protective effect persists into the later stages of toxicity. To investigate this late-stage protection, we treated C57Bl/6J mice with 300 mg/kg APAP and administered 20 mg/kg MT at 1.5 hours and 3 hours, respectively. The results showed that both MT treatments protected mice from late APAP hepatotoxicity (12 hours and 24 hours). Surprisingly, hepatocyte apoptosis was significantly increased in the MT-treated mice, while necrosis was almost exclusively observed in mice treated with APAP alone. Furthermore, caspase-3 activity and lysis were significantly increased in the livers of the MT-treated mice. Immunohistochemical staining revealed that active caspase-3-positive hepatocytes were located only in the centrilobular region. Two hours after administration of acetaminophen (APAP), treatment with the pan-caspase inhibitor ZVD-fmk (10 mg/kg) neutralized caspase activation and further protected the liver from APAP hepatotoxicity. The current standard treatment for APAP poisoning, N-acetylcysteine (NAC), is protective but does not induce an apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and similar caspase activation and apoptotic morphology were observed in hepatocytes of RIP3-deficient mice. These data suggest that although necrosis is the main cause of cell death after APAP hepatotoxicity, antioxidant MT treatment may shift the cell death pattern to secondary apoptosis in some cells. Mitochondrial oxidative stress and regulation of RIP3 kinase expression play a key role in this shift. [1] Mitochondrial oxidative damage leads to a variety of degenerative diseases. Therefore, selective inhibition of mitochondrial oxidative damage is a promising therapeutic strategy. One approach is to develop antioxidants that can selectively accumulate in the mitochondria of patients. These mitochondrial-targeting antioxidants are developed by coupling lipophilic triphenylphosphine cations with antioxidant moieties such as panthenol or α-tocopherol. These compounds can easily cross all biological membranes, including the blood-brain barrier, and enter muscle cells, thereby reaching tissues most affected by mitochondrial oxidative damage. Furthermore, due to their positive charge, they accumulate hundreds of times within mitochondria under the drive of membrane potential, thereby enhancing the protective effect of mitochondria against oxidative damage. These compounds can protect mitochondria from damage after oral administration, and therefore may form the basis of mitochondrial protective therapy. This article reviews the background and research progress to date of these mitochondrial-targeting antioxidants. [2]

C29H37CLN2O3P

528.04

528.23

1569257-94-8

Mito-TEMPO;1334850-99-5

73504624

Orange to red solid powder

2

4

6

36

612

0

C([P+](C1C=CC=CC=1)(C1C=CC=CC=1)C1C=CC=CC=1)C(NC1CC(C)(C)N([O])C(C)(C)C1)=O.[Cl-].O |^1:28|

AIAJCDXYZBOVGT-UHFFFAOYSA-N

InChI=1S/C29H34N2O2P.ClH.H2O/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;;/h5-19,23H,20-22H2,1-4H3;1H;1H2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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