ROS; mitochondria-targeted antioxidant

Mitoquinone (MitoQ) is an antioxidant that targets mitochondria. During four hours of cold storage (CS), dose-response tests were used to determine the ideal dosages of mitoquinone (MitoQ) and DecylTPP therapy. In order to investigate the potential protective impact of mitoquinone treatment on CS damage, MitoSOX Red—a fluorescent dye that targets and detects mitochondrial superoxide production—was first used. When CS was applied to normal rat kidney (NRK) cells, mitochondrial superoxide caused the cells' fluorescence to increase by about two times when compared to the control group. When it came to CS-induced mitochondrial superoxide production, quinone from mitochondria significantly defended against it, while DecylTPP, the control chemical, had no such effect. Treatment for mitochondrial superoxide greatly decreased the generation of mitochondrial superoxide, and kidneys treated with DecylTPP had levels of mitochondrial superoxide that were on par with kidneys receiving CS alone [1].

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Protective Effect of MitoQ on Mitochondrial Superoxide Generation during CS. [1]
The optimal doses for MitoQ and DecylTPP treatment were selected from dose-response experiments during 4-h CS. For the cellular-based studies, 1 μM was chosen from a range of 0.5, 0.75, 1.0, 1.5, and 2 μM, whereas the most efficacious dose for the ex vivo studies was 100 μM from a dose-response study of 50, 100, and 500 μM (data not shown). The potential protective benefits of MitoQ treatment against CS injury were tested initially using MitoSOX Red, a mitochondrial-targeted fluorescent dye that measures mitochondrial superoxide generation. As shown in Fig. 2A, NRK cells exposed to CS resulted in a ∼2-fold increase in fluorescence due to mitochondrial superoxide compared with untreated cells. MitoQ offered significant protection against CS-induced mitochondrial superoxide generation; whereas the control compound DecylTPP did not offer any protection. This was further confirmed using a spectrofluorometric-based assay detecting MitoSOX Red fluorescence excitation at 396 and 510 nm, where 396 nm is a specific indicator of mitochondrial superoxide, and 510 nm detects nonspecific oxidant generation (Robinson et al., 2006) (Fig. 2B). In addition, kidneys exposed to CS alone displayed increased mitochondrial superoxide generation compared with control kidneys (Fig. 2C). MitoQ treatment markedly decreased mitochondrial superoxide generation, whereas kidneys treated with DecylTPP had comparable levels of mitochondrial superoxide to kidneys exposed to CS alone (Fig. 2C).

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MitoQ Attenuates Nitrotyrosine Adduct Formation during CS. [1]
Immunocytochemistry and immunohistochemistry were used to evaluate nitrotyrosine protein adducts during CS. As shown in Fig. 3A, NRK cells exposed to CS alone had a significant increase in nitrotyrosine staining (red fluorescence) compared with untreated cells. MitoQ attenuated nitrotyrosine formation ∼2-fold, whereas the control compound DecylTPP did not decrease CS-mediated nitrotyrosine formation (Fig. 3A). Peroxynitrite-treated cells had intense nitrotyrosine formation (positive control) and was blocked when the nitrotyrosine antibody was preabsorbed with excess 3-nitrotyrosine (negative control; ONOO− + block). Rat kidney immunohistochemistry data were consistent with the in vitro study findings regarding the effect of MitoQ and nitrotyrosine. Figure 3B shows an increase in nitrotyrosine (brown staining) in the distal and proximal tubules and to a lesser extent in the glomeruli of CS kidneys compared with control kidneys. Kidneys treated with MitoQ had less nitrotyrosine formation. In contrast, DecylTPP-treated kidneys had similar amounts of nitrotyrosine compared with CS kidneys. The specificity of nitrotyrosine staining was also confirmed using antibody preabsorbed with excess 3-nitrotyrosine (CS + block).

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MitoQ Prevents Mitochondrial Respiratory Complex Inactivation during CS. [1]
Mitochondrial respiratory complex activity was evaluated in both the in vitro and ex vivo renal models to investigate whether 4-h CS alters mitochondrial respiratory function. Complexes I and II were significantly inactivated after CS of NRK cells compared with untreated cells (Fig. 4). MitoQ completely prevented complex I and II inactivation, whereas DecylTPP had no significant effect on complex activity. Complexes III and IV were not assessed in this study because we have previously shown both complex activities to be unchanged with 24-h CS (Mitchell et al., 2010); therefore evaluation of these complex activities was not warranted. Consistent with the in vitro findings, CS of rat kidneys led to partial inactivation of complexes I and II activity and had no effect on complexes III and IV (Fig. 4). MitoQ protected against complex I and II inactivation of CS kidneys, whereas DecylTPP did not have any effect.

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MitoQ Decreases Nitrotyrosine Formation and Cell Death during CS Plus Rewarming of NRK Cells. [1]
To test whether MitoQ could potentially offer protection against oxidant production and cell death after reperfusion/transplantation, cells were exposed to CS plus RW. As shown in Fig. 6A, 4-h CS plus overnight (18 h) RW of NRK cells resulted in significant nitrotyrosine formation. Adding MitoQ during CS attenuated nitrotyrosine staining significantly during CS.RW, whereas the control compound DecylTPP had no effect. LDH cytotoxicity revealed a significant increase in cell death of NRK cells exposed to CS.RW compared with untreated cells (Fig. 6B). MitoQ decreased cell death approximately 2-fold during CS alone and CS.RW. DecylTPP did not reduce cell death in either treatment group.

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MitoQ Scavenged ROS Production in Isolated Pancreatic Acinar Cells [2]
Application of 1 mM H2O2 caused a steady rise of ROS in the cells (control group) as reflected by increased intensity of CM-H2DCFDA fluorescence; Cells pretreated with 1 μM MitoQ showed a significantly reduced ROS production compared with cells in the control group, whereas pretreatment of 1 μM dTPP, the lipophilic cation of MitoQ without antioxidant activity was without effect (Figure 1(a)). Neither MitoQ nor dTPP induced ROS production per se (Figure 1(b)).

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MitoQ Did Not Protect against Mitochondrial Depolarisation Caused by AP Precipitants in Isolated Pancreatic Acinar Cells [2]
Neither pretreatment with 1 μM MitoQ nor dTPP caused depolarisation of ΔΨm in isolated pancreatic acinar cells, in contrast to the protonophore CCCP applied at the end of the experiment to elicit complete depolarisation. (Figure 2(a)). However, at 10 μM both MitoQ and dTPP induced a steady decrease of ΔΨm per se that was more profound for MitoQ (Figures 2(b) and 2(c)). CCK (10 nM) induced depolarisation of ΔΨm compared with control cells treated with HEPES alone (Figure 2(d)), an effect that was not significantly affected by pretreatment with either 1 μM MitoQ or dTPP (Figure 2(e)). Similarly, perfusion of TLCS (500 μM) depolarised ΔΨm (Figure 2(f)) that was unaffected by 1 μM MitoQ or dTPP (Figure 2(g)).

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MitoQ Caused Pancreatic Acinar Cell Death and Aggravated CCK-Induced Necrosis [2]
Both 1 μM MitoQ and dTPP caused an increased PI uptake indicative of necrosis; significant differences were observed at 2, 6, and 10 h between cells pretreated with either MitoQ or dTPP and cells in the control group treated with HEPES alone (Figure 3(a)). Addition of 10 nM CCK induced a time-dependent increase of necrosis. However, 1 μM MitoQ did not exert any protection against CCK-induced cell death. Rather both MitoQ and dTPP significantly worsened necrosis compared with CCK alone at 2 h, whereas dTPP, but not MitoQ, aggravated CCK-induced cell death at later time-points (Figure 3(b)).

Treatment with mitoquinone (MitoQ) dramatically decreased neutrophil infiltration and pancreatic edema. Serum amylase is dose-dependently increased by MitoQ; at higher doses, it roughly doubles. When administered at 10 mg/kg (dose 1), MitoQ treatment dramatically raised serum IL-6 levels and nearly doubled Caerulein-induced lung MPO activity [2].

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MitoQ Ameliorated Overall Pancreatic Histopathology in CER-AP but Aggravated Systemic Injury [2]
Figure 4(a) shows representative histopathology slides for control and different treatment groups, with the overall histopathology score and breakdown scores for individual components summarised in Figure 4(b). Intraperitoneal saline injections did not cause any significant histopathological changes of the pancreas, whereas hyperstimulation with caerulein induced typical features of AP; marked oedema, vacuolisation, neutrophil infiltration in the ductal margins, and parenchyma of the pancreas, with focal acinar cell necrosis evident 12 h after the first caerulein injection. The CER-AP was also characterised by significantly increased serum amylase, pancreatic trypsin and MPO activity, and lung MPO activity compared to saline controls.

MitoQ treatment at both doses tested significantly reduced pancreatic oedema and neutrophil infiltration. However, pancreatic necrosis was not prevented, with a trend toward greater necrosis at the higher dose although this did not attain significance. MitoQ dose-dependently increased serum amylase with an approximate doubling at the higher dose (Figure 5(a)). Pancreatic trypsin activity and MPO activity were not significantly affected by MitoQ at either dose (Figures 5(b) and 5(c)). In addition, MitoQ treatment nearly doubled lung MPO activity induced by caerulein (Figure 5(d)) with a significant increase of serum IL-6 levels also evident at dose 1 (Figure 5(e)).

The nonantioxidant analogue dTPP significantly reduced oedema, neutrophil infiltration, and necrosis at both doses, resulting in an overall reduction of the histopathological score. Serum amylase was not significantly affected (Figure 5(a)), although dTPP reduced pancreatic trypsin and MPO activity (Figures 5(b) and 5(c)). Similar to the results obtained with MitoQ, dTPP also significantly increased caerulein-induced lung MPO activity and serum IL-6 levels (Figures 5(d) and 5(e)).

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MitoQ Did Not Protect against TLCS-AP [2]
Sham operation only induced mild oedema of the pancreatic acinar cells without discernible signs of inflammation and necrosis. Infusion of 3 mM TLCS into the pancreas via the pancreatic duct resulted in marked histopathological changes of the head of the pancreas at 24 h, characterised by significantly increased oedema, inflammation, necrosis and thus, overall histopathology score (Figure 6(a)(i–iv)). However, the body and tail of the pancreas were much less affected (data not shown). The TLCS-AP was associated with increased serum amylase, pancreatic MPO activity, and serum IL-6 levels compared to the sham group (Figures 6(b)–6(d)).

Neither MitoQ nor dTPP at the lower dose induced histopathological changes of the pancreas, with oedema, inflammation, necrosis, and overall histopathological score unaltered (Figure 6(a)(i–iv)). Similarly, there were no significant changes of serum amylase and pancreatic MPO activity when TLCS-AP mice were treated with either MitoQ or dTPP (Figures 6(b) and 6(c)). Serum IL-6 levels were marginally increased by MitoQ or dTPP treatment but this did not attain statistical significance (Figure 6(d)). Application of MitoQ or dTPP at both doses alone to mice in the absence of caerulein injections or TLCS infusion showed that both MitoQ and dTPP significantly increased lung MPO activity per se (data not shown).

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Biphasic Effects of MitoQ on PMA-Induced ROS Production in PMNs [2]
Figure 7(a) illustrates the effect of 1 μM MitoQ or dTPP on PMA-induced ROS production in isolated PMNs. The NAD(P)H oxidase stimulator PMA (50 ng/mL) induced a dramatic increase of ROS in the extracellular solution around PMNs within the first few minutes that peaked at 8 mins and declined to a plateau after approximately 20 mins. Application of the NAD(P)H oxidase inhibitor DPI reduced the peak phase and completely inhibited the ROS plateau (Figure 7(c)). MitoQ treatment caused a biphasic effect on ROS production in PMNs. Thus, a concentration-dependent inhibition of the initial ROS peak induced by PMA was observed, with the peak time delayed to 10 minutes (Figures 7(a)–7(c)). Application of 1 μM dTPP had no significant effect on the peak of PMA-induced ROS production in PMNs compared to cells treated by PMA alone. Interestingly, MitoQ caused a concentration-dependent potentiation of PMA-induced ROS production at 40 mins (Figure 7(c)), an action shared by dTPP only at the higher concentration.

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CS Induced Renal Injury and Cell Death Is Decreased with MitoQ Treatment. [1]
Periodic acid-Schiff staining was performed to examine histopathological changes during CS of rat kidneys. Significant widespread tubular damage such as dilation, brush border loss, and cellular debris/cast formation occurred with CS exposure (Fig. 5A). MitoQ improved renal histology significantly, whereas DecylTPP treatment did not reverse renal injury. TUNEL staining, indicated by brown staining of the nuclei, was used as a marker of cell death. As shown in Fig. 5B, CS led to a significant increase in cell death (red arrows) compared with control kidneys. MitoQ treatment decreased cell death by ∼2-fold compared with CS kidneys, whereas DecylTPP offered no protective benefits against cell death.

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Mitochondrial dysfunctions induced by oxidative stress could play a pivotal role in the development of testicular damage and degeneration, leading to impaired fertility in adulthood. MitoQ as mitochondria-targeted antioxidant has been used in many diseases for a long time, but its therapeutic effects on testicular injury 'have not been reported yet. Here, we examined the protective action mechanism of MitoQ on testicular injury from oxidative stress induced by triptolide (TP). Mice were orally administrated with MitoQ (1.3, 2.6 and 5 .2mg/kg, respectively) in a TP-induced model of testicular damage for 14 days. And then testis injuries were comprehensively evaluated in terms of morphological changes, spermatogenesis assessment, blood-testis barrier (BTB) integrity, and apoptosis. The results demonstrated MitoQ effectively increased testicular weight, maintained the integrity of BTB, protected microstructure of testicular tissue and sperm morphology by inhibition of oxidative stress. Further mechanism studies revealed that MitoQ markedly activates the Keap1-Nrf2 antioxidative defense system characterized by increasing the expression of Nrf2 and its target genes HO-1 and NQO1. Meanwhile, MitoQ upregulated the expression of mitochondrial dynamics proteins Mfn2 and Drp-1and exerted a protective effect on mitochondria. On this basis, the results from pharmacokinetic study indicated that the MitoQ could enter into testis tissues after oral administration in despite of the low absolute bioavailability, which provided the material basis for MitoQ in the treatment of testicular damage. More importantly, MitoQ reached mitochondria quickly and had an outstanding feature of mitochondria targeting in Sertoli cells. Therefore, these results provide information for the application of MitoQ against testicular injury diseases [3].

Cold Storage In Vitro Model. [1]
Normal rat kidney proximal tubular cells were maintained in six-well 100 or 150-mm, or 150-mm plates in a humidified incubator gassed with 5% CO2 and 95% air at 37°C in DMEM containing 5% fetal calf serum (FCS). Cells were grown to 60% confluence and divided into four treatment groups: 1) untreated (Untx), 2) CS, 3) CS + MitoQ, and 4) CS + DecylTPP. Untreated cells remained at 37°C in DMEM containing 5% FCS (group 1). CS was initiated by washing cells with cold PBS twice and storing them in UW/Viaspan solution alone (4 h at 4°C) (group 2), CS + MitoQ (1 μM) (group 3), or CS + DecylTPP (1 μM) (group 4). In separate experiments, cells were exposed to CS plus RW by replacing UW solution alone or UW solution containing MitoQ or DecylTPP with DMEM containing 5% FCS overnight (18 h at 37°C).

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Measurement of ROS Production [2]
Real-time ROS production and redox changes in pancreatic acinar cells were measured with the probe 5-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl (CM-H2DCFDA) using a Zeiss LSM510 confocal microscope as previously described. Freshly isolated murine pancreatic acinar cells were incubated with either 1 μM MitoQ or dTPP and simultaneously loaded with 10 μM CM-H2DCFDA for 30 minutes. The cells were then perfused with H2O2 to induce ROS. The fluorescence of CM-H2DCFDA was excited at 488 nm and the emission was collected at 505–550 nm.

For ROS measurement in PMNs, a peroxidase-enhanced luminol chemiluminescent assay was employed using a POLARstar Omega Plate Reader. Cells were plated at a density of 500,000 per well and pretreated with 1 μM MitoQ or dTPP for 10 minutes, before adding 50 μM luminol and 75 units/mL horseradish peroxidase. Activation of NAD(P)H oxidase was induced by 50 ng/mL phorbol myristate acetate (PMA) while inhibition was achieved by using 1 μM diphenylene iodonium (DPI). The luminescence emission at 440 nm for the ROS dye was recorded for 40 min. The chemiluminescence intensity was normalised to negative controls for each mouse/run.

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Measurement of ΔΨm in Pancreatic Acinar Cells [2]
In separate experiments, ΔΨm of pancreatic acinar cells was determined by tetramethyl rhodamine methyl ester assay as described previously. Briefly, the cells were loaded with 40 nM TMRM for 30 minutes prior incubation with either 1 μM or 10 μM of MitoQ or dTPP. Cholecystokinin-8 (CCK-8, 10 nM) or bile acid taurolithocholic acid 3-sulphate (TLCS, 500 μM) was used to induce ΔΨm depolarisation. At the end of the perfusion, the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 10 μM) was added to induce complete depolarisation of ΔΨm. The fluorescence of TMRM was excited at 543 nm and the emission was collected at 560–650 nm.

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In Vitro Cell Death Assay for Pancreatic Acinar Cells [2]
Pancreatic acinar cell death was detected as the intensity of fluorescent dye propidium iodide (PI) taken up by the nuclei of necrotic cells. For cell death induced by CCK-8, a time-course fluorescent plate reader method was used. Briefly, cells were isolated from one murine pancreas, centrifuged, and resuspended into 1 mL solution. Cells were carefully pipetted into individual wells to ensure homogeneity. Cells were treated with CCK-8 (10 nM) alone, or in the presence of 1 μM of either MitoQ or dTPP. For normal control groups, cells treated with equal volume of extracellular solution. After 5 min, PI (50 μM) was then added to all wells mixed by automated agitation. The microplate was then placed in the POLARstar Omega Plate Reader (preheated to 37°C), and fluorescence determined by excitation 543 nm and emission 620 nm with bottom reading. The assay was set to run with a cycle time of 600 seconds. All fluorescence measurements are expressed as changes from basal fluorescence (F/F 0 ratio), where F 0 represents the initial fluorescence recorded at the start of the experiment, and F the fluorescence recorded at specific time points.

Cold Storage Ex Vivo Model. [1]
Male Fischer 344 inbred rats weighing between 250 and 300 g were anesthetized with ethrane, followed by shaving and prepping with betadine. A 2-ml bolus of 0.9% (w/v) NaCl was administered intravenously, and an incision was made 1 cm superior to the symphysis pubis to the tip of the xiphoid process. Bulldog clamps were placed on the aorta and vena cava (proximal and distal to the renal vessels) to prevent blood flow to the kidneys. A 22-gauge surgical needle was used to puncture the rat's aorta to flush the renal grafts with saline (10 ml per kidney) using a small catheter. Once the kidneys started to turn light brown (perfusion), another vent was formed in the vena cava to allow blood flow from the kidneys. Once both kidneys were completely flushed, the right kidney was recovered and served as a control (group 1), and the left kidney was exposed to CS alone for 4 h at 4°C (group 2). In additional experiments, kidneys were flushed with saline through the aorta using a small catheter followed by flushing the right kidney with saline containing MitoQ (100 μM) and the left kidney with saline containing DecylTPP (100 μM) (10 ml per kidney). Tissues were recovered and stored in CS + MitoQ (100 μM; right kidney) or CS + DecylTPP (100 μM; left kidney) for 4 h at 4°C (groups 3 and 4, respectively). A thin middle section from all of the kidneys were cut and immediately fixed in 10% formalin before being embedded in paraffin for sectioning (4 μm) and histological evaluation. The remaining portion of the kidneys were quickly frozen in liquid nitrogen and stored in −80°C until needed for biochemistry analyses.

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Experimental AP Models [2]
Seven intraperitoneal injections of a supramaximal dose (50 μg/kg) of caerulein, a CCK-8 analogue, were given on an hourly basis to induce hyperstimulation acute pancreatitis (CER-AP). Control mice received equal volumes of PBS injection. In the MitoQ treatment groups, MitoQ at 10 mg/kg (dose 1) or 25 mg/kg (dose 2) was given at the first and third injections of caerulein. Similarly, dTPP at 9.6 mg/kg (dose 1) or 24 mg/kg (dose 2) was given for the dTPP treatment group. MitoQ and dTPP were at the same molar concentration at doses 1 and 2. Mice were sacrificed at 12 h after the first caerulein injection to collect samples.

Bile acid-induced AP was achieved by retrograde infusion of TLCS into the pancreatic duct (TLCS-AP). After induction of anesthesia, TLCS applied using a mini infusion pump at a speed of 5 μL/min for 10 minutes. Successful infusion of TLCS into pancreas was demonstrated by a diffuse light blue colour (methylene blue) appearing in the pancreatic head. Control mice received sham surgery without TLCS infusion. In the treatment groups, MitoQ (10 mg/kg) or dTPP (9.6 mg/kg) was given at 1 h and 3 h after TLCS infusion. Mice were sacrificed at 24 h after the TLCS infusion or sham surgery. In both animal models, analgesia was achieved by administration of 0.1 mg/kg buprenorphine hydrochloride.

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Pharmacokinetic and tissue distribution study in mice [3]
Diet was prohibited for 12 h before the experiment while water was taken freely. All mice were randomly assigned to two groups for intravenous or oral administration of 2.6 mg/kg MitoQ, respectively. Blood sample was collected into heparinized tubes at 0 (pro-drug), 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h after tail injection (i.v.) administration. Meanwhile, blood sample was collected into heparinized tubes at 0 (pro-drug), 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h after orally administration. Blood samples of each time point were taken from 6 mice. Blood sample was immediately centrifuged at 3000 ×g for 10 min at 4 °C, and plasma was transferred into a new 1.5 mL Eppendorf tube and then stored at −80 °C until analysis. The pharmacokinetic parameters of MitoQ were calculated by DAS Software (version 3.0, China State Drug Administration) using non-compartmental methods. Absolute bioavailability was calculated by comparing the area under the curve (AUC) of oral administration with AUC of the same drug following intravenous administration at the same dose, as a previous report (Ma et al., 2014). The unit of the concentration (nmol/L) of MitoQ derived from the calibration curve was converted to ng/mL by multiplying relative molecular mass. Eighteen mice were randomly assigned to three groups (6 mice/group) to carry out tissue distribution study. Mice in three groups were sacrificed at 10 min, 1 h and 2 h respectively after intravenous injection 2.6 mg/kg pure MitoQ. Subsequently, testes were immediately removed, washed in normal saline and blotted dry with filter paper. An accurately weighed amount of the soft tissue samples (0.2 g) was individually homogenized with normal saline (0.5 ml) and stored at −80 °C until analysis. The unit of the concentration (nmol/L) of MitoQ derived from the calibration curve was converted to ng/mL by multiplying relative molecular mass.

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Dosage information/dosage regimen [3]
Animals were randomly assigned to five groups (n = 10 per group). Control group: mice were given vehicle by gavage and intraperitoneal (i.p.) injection orderly once a day (ethanol/0.9% saline =1:9, vol); TP model group: mice were given an intraperitoneal injection with TP at a dose of 120 μg/kg after pre-treatment with above-mentioned vehicle; MitoQ group: mice were orally administrated with MitoQ at the dose of 1.3, 2.6, 5.2 mg/kg, respectively,for 1 h and then an i.p. injection with TP (120 μg/kg)every day. After the continuous treatment of MitoQ for 14 days, mice were euthanized, testicular tissues were removed and weighted to calculate the testis index (testis weight/body weight) and stored in liquid nitrogen for future studies.

Pharmacokinetic study of MitoQ in mice [3]
The validated LC–MS/MS method was successfully applied to the pharmacokinetic study of MitoQ after the single oral, tail injection administration of MitoQ at 2.6 mg/kg to mice. The main pharmacokinetic parameters calculated by the non-compartmental model are listed in Fig. 6 F. The mean plasma concentration-time curves of MitoQ are shown in Fig. 6 D and E.
The time to peak concentration (Tmax) were observed at about 0.74 h aft
er oral administration that indicated that MitoQ could be quickly absorbed into blood circulatory system. But peak plasma concentrations (Cmax) were found at a low level for only 9.77 ± 2.76 ng/ml. The half-lives (T1/2) of MitoQ were 1.066 ± 0.319 h and 0.74 ± 0.49 h after intravenous or oral administration, respectively. Low absolute bioavailability (17.95% ± 5.98%) of MitoQ was calculated after intravenous and oral administration. And the apparent volumes of distribution were 76.268 ± 13.534 L/kg and 324.52 ± 241.44 L/kg after intravenous or oral administration, respectively.
The distribution of MitoQ in the testis tissues is listed in Fig. 6 G. The highest concentrations in testis tissues were 35.82 ± 8.233 ng/g and 194.55 ± 8.59 ng/g at 10 min following the orally and intravenous administration, respectively. However, MitoQ could not be detected in any tissue samples beyond 2 h. The data suggested that no accumulation was observed in tissues. The result also showed MitoQ could cross the blood-testis barrier and provided the material basis for testis injury.

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Mitoquinone (MitoQ10 mesylate) is a mitochondria-targeted antioxidant undergoing development for the treatment of neurodegenerative diseases. The aim of this study was to develop and validate an assay based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) to determine mitoquinone and to detect and identify the metabolites of MitoQ10 in rat plasma after an oral dose. After a simple protein precipitation step, plasma samples were analyzed by reversed-phase liquid chromatography using gradient elution with acetonitrile/water/formic acid. Electrospray ionization in the positive ion mode with multiple reaction monitoring (MRM) was used to analyze mitoquinone employing the deuterated compound (d3-MitoQ10 mesylate) as internal standard. The calibration curve for mitoquinone was linear over the concentration range 0.5-250 ng/mL with a correlation coefficient>0.995. The method was sensitive (limit of quantitation 0.5 ng/mL) and had acceptable accuracy (relative error<8.7%) and precision (intra- and inter-day coefficient of variation<12.4%). Recoveries of mitoquinone at concentrations of 1.5, 20 and 200 ng/mL were in the range 87-114%. The method was successfully applied to a pharmacokinetic study in rat after a single oral dose in which four metabolites of MitoQ10 were tentatively identified as hydroxylated MitoQ10, desmethyl MitoQ10 and the glucuronide and sulfate conjugates of the quinol form of MitoQ10. [4]

[1]. The mitochondria-targeted antioxidant mitoquinone protects against cold storage injury of renaltubular cells and rat kidneys. J Pharmacol Exp Ther. 2011 Mar;336(3):682-92.

[2]. Effects of the mitochondria-targeted antioxidant mitoquinone in murine acute pancreatitis. Mediators Inflamm. 2015;2015:901780.

[3]. MitoQ ameliorates testis injury from oxidative attack by repairing mitochondria and promoting the Keap1-Nrf2 pathway. Toxicol Appl Pharmacol. 2019 May 1:370:78-92.

[4]. Quantitation and metabolism of mitoquinone, a mitochondria-targeted antioxidant, in rat by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2007;21(13):1958-64.

Mitoquinone is based on a novel technology, targeted lipophilic cations, that transport and concentrate antioxidants into the mitochondria -- organelles inside cells that provide energy for life processes -- where they accumulate up to a thousand fold. In 2004, a genomic study of hereditary early-onset Parkinson's disease demonstrated a direct molecular link between mitochondrial dysfunction and the pathogenesis of Parkinson's disease. Mitochondrial dysfunction also has been shown to represent an early critical event in the pathogenesis of the sporadic form of Parkinson's disease. Clinical studies by the Parkinson's Study Group show that very high doses of an antioxidant called Coenzyme Q (which Mitoquinone effectively targets into mitochondria) appear to slow the progression of Parkinson's disease symptoms.

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Drug Indication
Investigated for use/treatment in hepatitis (viral, C) and parkinson's disease.

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Mechanism of Action
Mitoquinone is targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation. Because of the large mitochondria membrane potential, the cations accumulate within cellular mitochondria up to 1,000 fold, compared to non-targeted antioxidants such as Coenzyme Q or its analogues, enabling the antioxidant moiety to block lipid peroxidation and protect mitochondria from oxidative damage. By selectively blocking mitochondrial oxidative damage, it prevents cell death.

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Pharmacodynamics
Mitoquinone may help to prevent the nerve cell damage that leads to Parkinson's disease. It aims to slow or halt Parkinson's disease at its cause by tackling cell damage caused when mitochondria cease to function normally. It is anticipated that MitoQ will slow or arrest the progression of Parkinson's disease symptoms. Hepatits C virus can directly alter mitochondrial function, leading to increased reactive oxygen species (free-radical) production that can lead to scarring of the liver and cirrhosis. Mitoquinone could be used to halt or decrease liver inflammation and fibrosis progression, even in the absence of sustained virologic response. Mitoquinone directly affects the mitochondria in two steps: a targeting component directs the drug to the mitochondria; and an antioxidant component helps to prevent cell damage.

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The majority of kidneys used for transplantation are obtained from deceased donors. These kidneys must undergo cold preservation/storage before transplantation to preserve tissue quality and allow time for recipient selection and transport. However, cold storage (CS) can result in tissue injury, kidney discardment, or long-term renal dysfunction after transplantation. We have previously determined mitochondrial superoxide and other downstream oxidants to be important signaling molecules that contribute to CS plus rewarming (RW) injury of rat renal proximal tubular cells. Thus, this study's purpose was to determine whether adding mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to University of Wisconsin (UW) preservation solution could offer protection against CS injury. CS was initiated by placing renal cells or isolated rat kidneys in UW solution alone (4 h at 4°C) or UW solution containing MitoQ or its control compound, decyltriphenylphosphonium bromide (DecylTPP) (1 μM in vitro; 100 μM ex vivo). Oxidant production, mitochondrial function, cell viability, and alterations in renal morphology were assessed after CS exposure. CS induced a 2- to 3-fold increase in mitochondrial superoxide generation and tyrosine nitration, partial inactivation of mitochondrial complexes, and a significant increase in cell death and/or renal damage. MitoQ treatment decreased oxidant production ~2-fold, completely prevented mitochondrial dysfunction, and significantly improved cell viability and/or renal morphology, whereas DecylTPP treatment did not offer any protection. These findings implicate that MitoQ could potentially be of therapeutic use for reducing organ preservation damage and kidney discardment and/or possibly improving renal function after transplantation. [1]

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In summary, this is the first report showing that mitochondrial superoxide increases significantly during early CS and contributes to mitochondrial and renal damage. We have identified the mitochondria-targeted antioxidant MitoQ to significantly protect against CS-mediated oxidative stress, mitochondrial dysfunction, cell death, and renal injury of renal proximal tubular cells and isolated rat kidneys. These findings suggest that infusion of MitoQ to kidneys before transplantation may be of therapeutic use to reduce CS damage, improve outcome for transplant recipients, and also increase the numbers of donated organs available for transplant, all of which could lead to a decline in health-care costs.[1]

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Although oxidative stress has been strongly implicated in the development of acute pancreatitis (AP), antioxidant therapy in patients has so far been discouraging. The aim of this study was to assess potential protective effects of a mitochondria-targeted antioxidant, MitoQ, in experimental AP using in vitro and in vivo approaches. MitoQ blocked H2O2-induced intracellular ROS responses in murine pancreatic acinar cells, an action not shared by the control analogue dTPP. MitoQ did not reduce mitochondrial depolarisation induced by either cholecystokinin (CCK) or bile acid TLCS, and at 10 µM caused depolarisation per se. Both MitoQ and dTPP increased basal and CCK-induced cell death in a plate-reader assay. In a TLCS-induced AP model MitoQ treatment was not protective. In AP induced by caerulein hyperstimulation (CER-AP), MitoQ exerted mixed effects. Thus, partial amelioration of histopathology scores was observed, actions shared by dTPP, but without reduction of the biochemical markers pancreatic trypsin or serum amylase. Interestingly, lung myeloperoxidase and interleukin-6 were concurrently increased by MitoQ in CER-AP. MitoQ caused biphasic effects on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase but elevating later levels. Our results suggest that MitoQ would be inappropriate for AP therapy, consistent with prior antioxidant evaluations in this disease.[2]

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In conclusion, the findings of this study further emphasize the unsuitability of antioxidant therapy in the treatment of AP, previously highlighted by a randomised, double-blind, and placebo-controlled clinical trial. There was no protection of experimental TLCS-AP by MitoQ and mixed effects observed in the milder CER-AP model, including elevations of inflammation markers. These results are in accordance with previous studies showing that suppression of ROS enhances pancreatic acinar cell necrosis by inhibiting a protective apoptotic mechanism, an action that would promote local pancreatic damage in AP.[2]

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In conclusion, this study demonstrates the novel beneficial effects of MitoQ on testicular injury induced by TP in vivo. MitoQ effectively restored microstructure of testicular tissue, recovered the integrity of BTB and maintained spermatogenesis in a mouse model of testicular damage induced by TP. The mechanisms underlying these effects may involve protecting testicular tissues from two aspects. On one hand, MitoQ played an antioxidant role by regulating the mitochondrial dynamics. On the other hand, MitoQ reduced oxidative stress through activating Nrf2/Keap1 signaling. The potency, efficacy and pharmacokinetic characteristics make it a prominent candidate for potentially clinical use in treating mitochondrial-related testicular injury and male infertility disease.[3]

C37H44O4P

583.716631889343

583.297

444890-41-9

845959-50-4 (mesylate);845959-52-6 (beta-CD complex);444890-41-9 (cation);336184-91-9 (bromide);

11388332

Typically exists as solid at room temperature

9.4

0

4

16

42

886

0

[P+](C1C=CC=CC=1)(C1C=CC=CC=1)(C1C=CC=CC=1)CCCCCCCCCCC1C(C(=C(C(C=1C)=O)OC)OC)=O

OIIMUKXVVLRCAF-UHFFFAOYSA-N

InChI=1S/C37H44O4P/c1-29-33(35(39)37(41-3)36(40-2)34(29)38)27-19-8-6-4-5-7-9-20-28-42(30-21-13-10-14-22-30,31-23-15-11-16-24-31)32-25-17-12-18-26-32/h10-18,21-26H,4-9,19-20,27-28H2,1-3H3/q+1

10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)decyl-triphenylphosphanium

MitoQ; MitoQ10; Mito Q; MitoQ 10; mitoquinone; Mitoquinone cation; UNII-47BYS17IY0; MITOQUINONE [WHO-DD]; 10-(6'-ubiquinonyl)decyltriphenylphosphonium bromide; 444890-41-9; Mitoquinone ion; Mito-Q; Mito-Q; MitoQ-10

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.)