Changes in Glial Support of the Hippocampus during the Development of an Alzheimer's Disease-like Pathology and Their Correction by Mitochondria-Targeted Antioxidant SkQ1.

Astrocytes and microglia are the first cells to react to neurodegeneration, e.g., in Alzheimer's disease (AD); however, the data on changes in glial support during the most common (sporadic) type of the disease are sparse. Using senescence-accelerated OXYS rats, which simulate key characteristics of sporadic AD, and Wistar rats (parental normal strain, control), we investigated hippocampal neurogenesis and glial changes during AD-like pathology. Using immunohistochemistry, we showed that the early stage of the pathology is accompanied by a lower intensity of neurogenesis and decreased astrocyte density in the dentate gyrus. The progressive stage is concurrent with reactive astrogliosis and microglia activation, as confirmed by increased cell densities and by the acquisition of cell-specific gene expression profiles, according to transcriptome sequencing data. Besides, here, we continued to analyze the anti-AD effects of prolonged supplementation with mitochondria-targeted antioxidant SkQ1. The antioxidant did not affect neurogenesis, partly normalized the gene expression profile of astrocytes and microglia, and shifted the resting/activated microglia ratio toward a decrease in the activated-cell density. In summary, both astrocytes and microglia are more vulnerable to AD-associated neurodegeneration in the CA3 area than in other hippocampal areas; SkQ1 had an anti-inflammatory effect and is a promising modality for AD prevention and treatment.

Mitochondrial targeted antioxidants, mitoquinone and SKQ1, not vitamin C, mitigate doxorubicin-induced damage in H9c2 myoblast: pretreatment vs. co-treatment.

BACKGROUND: Preconditioning of the heart ameliorates doxorubicin (Dox)-induced cardiotoxicity. We tested whether pretreating cardiomyocytes by mitochondrial-targeted antioxidants, mitoquinone (MitoQ) or SKQ1, would provide better protection against Dox than co-treatment. METHODS: We investigated the dose-response relationship of MitoQ, SKQ1, and vitamin C on Dox-induced damage on H9c2 cardiomyoblasts when drugs were given concurrently with Dox (e.g., co-treatment) or 24 h prior to Dox (e.g., pretreatment). Moreover, their effects on intracellular and mitochondrial oxidative stress were evaluated by 2,7-dichlorofluorescin diacetate and MitoSOX, respectively. RESULTS: Dox (0.5-50 µM, n = 6) dose-dependently reduced cell viability. By contrast, co-treatment of MitoQ (0.05-10 µM, n = 6) and SKQ1 (0.05-10 µM, n = 6), but not vitamin C (1-2000 µM, n = 3), significantly improved cell viability only at intermediate doses (0.5-1 µM). MitoQ (1 µM) and SKQ1 (1 µM) significantly increased cell viability to 1.79 ± 0.12 and 1.59 ± 0.08 relative to Dox alone, respectively (both p < 0.05). Interestingly, when given as pretreatment, only higher doses of MitoQ (2.5 µM, n = 9) and SKQ1 (5 µM, n = 7) showed maximal protection and improved cell viability to 2.19 ± 0.13 and 1.65 ± 0.07 relative to Dox alone, respectively (both p < 0.01), which was better than that of co-treatment. Moreover, the protective effects were attributed to the significant reduction in Dox-induced intracellular and mitochondrial oxidative stress. CONCLUSION: The data suggest that MitoQ and SKQ1, but not vitamin C, mitigated DOX-induced damage. Moreover, MitoQ pretreatment showed significantly higher cardioprotection than its co-treatment and SKQ1, which may be due to its better antioxidant effects.

Mitochondria-Targeted Drugs.

BACKGROUND: Targeting of drugs to the subcellular compartments represents one of the modern trends in molecular pharmacology. The approach for targeting mitochondria was developed nearly 50 years ago, but only in the last decade has it started to become widely used for delivering drugs. A number of pathologies are associated with mitochondrial dysfunction, including cardiovascular, neurological, inflammatory and metabolic conditions. OBJECTIVE: This mini-review aims to highlight the role of mitochondria in pathophysiological conditions and diseases, to classify and summarize our knowledge about targeting mitochondria and to review the most important preclinical and clinical data relating to the antioxidant lipophilic cations MitoQ and SkQ1. METHODS: This is a review of available information in the PubMed and Clinical Trials databases (US National Library of Medicine) with no limiting period. RESULTS AND CONCLUSION: Mitochondria play an important role in the pathogenesis of many diseases and possibly in aging. Both MitoQ and SkQ1 have shown many beneficial features in animal models and in a few completed clinical trials. More clinical trials and research efforts are needed to understand the signaling pathways influenced by these compounds. The antioxidant lipophilic cations have great potential for the treatment of a wide range of pathologies.

Neuroprotective Effects of Mitochondria-Targeted Plastoquinone in a Rat Model of Neonatal Hypoxic⁻Ischemic Brain Injury.

Neonatal hypoxia⁻ischemia is one of the main causes of mortality and disability of newborns. To study the mechanisms of neonatal brain cell damage, we used a model of neonatal hypoxia⁻ischemia in seven-day-old rats, by annealing of the common carotid artery with subsequent hypoxia of 8% oxygen. We demonstrate that neonatal hypoxia⁻ischemia causes mitochondrial dysfunction associated with high production of reactive oxygen species, which leads to oxidative stress. Targeted delivery of antioxidants to the mitochondria can be an effective therapeutic approach to treat the deleterious effects of brain hypoxia⁻ischemia. We explored the neuroprotective properties of the mitochondria-targeted antioxidant SkQR1, which is the conjugate of a plant plastoquinone and a penetrating cation, rhodamine 19. Being introduced before or immediately after hypoxia⁻ischemia, SkQR1 affords neuroprotection as judged by the diminished brain damage and recovery of long-term neurological functions. Using vital sections of the brain, SkQR1 has been shown to reduce the development of oxidative stress. Thus, the mitochondrial-targeted antioxidant derived from plant plastoquinone can effectively protect the brain of newborns both in pre-ischemic and post-stroke conditions, making it a promising candidate for further clinical studies.

[MITOCHONDRIA-TARGETED ANTIOXIDANTS IN THE PREVENTION OF THE CORNEA EROSION WHEN PERFORMING SURGERY UNDER GENERAL ANESTHESIA.]

Despite the use of modern methods of prevention, at least 10% of patients operated on for ophthalmic indications not develop corneal erosion as the indirect complication of general anesthesia. OBJECTIVE: To reduce the number of ophthalmic complications of general anesthesia by prophylactic use of new mito- chondria-targeted antioxidants - Vizomitin (eye drops). MATERIALS AND METHODS: 70 patients, which was supposed to perform the average duration of operations under general anesthesia were randomized into 3 groups depending on the method specific (pharmacological) prevention of corneal erosions: (1) control (specic (pharmacological) prevention was not carried out), (2), using preparation "natural tear, and (3) "Vizomitin" preparation. Postoperative biomicroscopy was performed to assess the condition of the cornea, tear film stability was measured and the height of the tear meniscus. RESULTS: When using eye drops "Vizomitin" value is an indicator of stability of the tear film on the 3rd day after the operation more than in the control group of patients by 51% (p = 0.012) and patients groups, natural tear by 57% (p = 0.013). Surgical interventions performed under general anesthesia, leading to an increase in the number ofpatients with decreased tear meniscus height index of the control group with 4 to 7 patients (p = 0.30) in the group of natural tear from 3 to 11 patients (p = 0.008) . In the group with drug "Vizomitin" the number of such patients is reduced from 7 to 1 (p = 0.018). CONCLUSION: In the surgical procedures under general anesthesia eye drops "Vizomitin" effectively prevents the devel- opment of corneal erosion.

The novel mitochondria-targeted antioxidant SkQ1 modulates angiogenesis and inflammatory micromilieu in a murine orthotopic model of pancreatic cancer.

Our understanding in the last few years about reactive oxygen species (ROS) has changed from being harmful substances to crucial intra- and extracellular messengers as well as important regulators controlling a wide spectrum of signaling pathways, including those in cancer immunology. Therefore, these multiple essential roles of ROS and especially of mitochondria-derived ROS in malignant transformation and cancer progression make them a promising target for anticancer therapy. Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in the world. A link between ROS, antioxidants and the PDAC development and progression has been recently established. Therefore, usage of specific highly efficient antioxidants could bring an option for treatment and/or prevention of PDAC. 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) is a new antioxidant with the highest mitochondrion membrane penetrating ability and potent antioxidant capability. In this work, we investigated an impact of SkQ1 on tumor angiogenesis, immune micromilieu, and oncological parameters in the orthotopic Panc02 murine model of PDAC. We showed that in this model SkQ1 treatment leads to the elevation of pro-angiogenic factors and to building of mainly an anti-inflammatory cytokine milieu. On the cellular level we showed an increase in a percentage of memory T cells and a decrease in frequency on natural killer T (NKT) cells. At the same time, SkQ1 was ineffective in the improvement of oncological parameters of PDAC-bearing mice. New studies are needed to clarify the absence of therapeutic and/or prophylactic benefits of the antioxidant.

SkQ1 Ophthalmic Solution for Dry Eye Treatment: Results of a Phase 2 Safety and Efficacy Clinical Study in the Environment and During Challenge in the Controlled Adverse Environment Model.

INTRODUCTION: This Phase 2 clinical trial assessed the efficacy and safety of the novel antioxidative, renewable compound SkQ1 for topical treatment of dry eye signs and symptoms. METHODS: In a single-center, randomized, double-masked, placebo-controlled, 29-day study, 91 subjects with mild to moderate dry eye instilled the study drug twice daily and recorded dry eye symptoms daily. Subjects were randomized 1:1:1 into one of three ophthalmic solution treatment groups: SkQ1 1.55 µg/mL, SkQ1 0.155 µg/mL, or 0.0 µg/mL (placebo). Subjects were exposed to a controlled adverse environment chamber at 3 of the 4 study visits (Day -7, Day 1, and Day 29). Investigator assessments occurred at all study visits. RESULTS: SkQ1 was safe and efficacious in treating dry eye signs and symptoms. Statistically significant improvements with SkQ1 compared to placebo occurred for the dry eye signs of corneal fluorescein staining and lissamine green staining in the central region and lid margin redness, and for the dry eye symptoms of ocular discomfort, dryness, and grittiness. In addition, SkQ1 demonstrated greater efficacy compared to placebo, although the differences were not statistically significant, for corneal fluorescein staining in other regions and/or time points (total staining score, central region, corneal sum score, and temporal region), lissamine green staining for the central and nasal regions, and blink rate scores. CONCLUSIONS: This Phase 2 study indicated that SkQ1 is safe and efficacious for the treatment of dry eye signs and symptoms and supported previous study results. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT02121301. FUNDING: Miotech S.A.

Results of a Multicenter, Randomized, Double-Masked, Placebo-Controlled Clinical Study of the Efficacy and Safety of Visomitin Eye Drops in Patients with Dry Eye Syndrome.

INTRODUCTION: This article presents the results of an international, multicenter, randomized, double-masked, placebo-controlled clinical study of Visomitin (Mitotech LLC, Moscow, Russian Federation) eye drops in patients with dry eye syndrome (DES). Visomitin is the first registered (in Russia) drug with a mitochondria-targeted antioxidant (SkQ1) as the active ingredient. METHODS: In this multicenter (10 sites) study of 240 subjects with DES, study drug (Visomitin or placebo) was self-administered three times daily (TID) for 6 weeks, followed by a 6-week follow-up period. Seven in-office study visits occurred every 2 weeks during both the treatment and follow-up periods. Efficacy measures included Schirmer's test, tear break-up time, fluorescein staining, meniscus height, and visual acuity. Safety measures included adverse events, slit lamp biomicroscopy, tonometry, blood pressure, and heart rate. Tolerability was also evaluated. RESULTS: This clinical study showed the effectiveness of Visomitin eye drops in the treatment of signs and symptoms of DES compared with placebo. The study showed that a 6-week course of TID topical instillation of Visomitin significantly improved the functional state of the cornea; Visomitin increased tear film stability and reduced corneal damage. Significant reduction of dry eye symptoms (such as dryness, burning, grittiness, and blurred vision) was also observed. CONCLUSION: Based on the results of this study, Visomitin is effective and safe for use in eye patients with DES for protection from corneal damage. FUNDING: Mitotech LLC.

Effect of Long-Term Treatment with Antioxidant SkQ1 Added to Drinking Water on Cytochromes P450 Level in Rat Liver.

Mitochondria-targeted cationic antioxidant plastoquinonyl decyltriphenylphosphonium (SkQ1) added to drinking water in therapeutic doses (250 nmol/kg per day) for a long time (up to 24 months) does not induce cytochromes P450 in rat liver.

Effect of SkQ1 eye drops on the rat lens metabolomic composition and the chaperone activity of α-crystallin.

The ability of SkQ1 eye drops to slow down the cataract development is demonstrated on the senescence-accelerated OXYS rats: the SkQ1 treatment leads to the considerable improvement of the lens condition as compared to the control group. The comparison of the chaperone activities of α-crystallins isolated from the rat lenses did not reveal significant difference between SkQ1-treated and control rats. The contents of major metabolites (23 compounds) in lenses of SkQ1-treated and untreated rats are also very similar, though the concentration of reduced glutathione (GSH) in lenses of SkQ1-treated rats is 12% lower. This difference may be attributed to the reduction of the oxidative stress under action of SkQ1 eye drops, and to the decreased requirement to produce high amounts of this antioxidant.

Ameliorative effects of SkQ1 eye drops on cataractogenesis in senescence-accelerated OXYS rats.

BACKGROUND: Antioxidant supplements have been suggested as a strategy to decrease the risk of age-related cataract, but there is no evidence that antioxidants can reduce the signs of the disease. Recently, we showed that the mitochondrial antioxidant SkQ1 can partially reverse cataract signs in senescence-accelerated OXYS rats. The aim of the present study was the histomorphological examination of the influence of SkQ1 eye drops on the cataract development in OXYS rats. METHODS: OXYS rats received SkQ1 eye drops (250 nM) from 9 to 12 months of age. Ophthalmoscopic examination was carried out before and after treatment. Light and electron microscopy were used for histomorphological examination. Expression of the Cryaa and Cryab genes was determined using real-time PCR. αB-crystallin expression was detected using Western blotting. RESULTS: SkQ1 completely prevented the cataract development in OXYS rats, and in some of the animals diminished the signs of the disease. Light and electron microscopy showed that SkQ1 attenuated the (typical for cataract) alterations in the lens capsule and epithelial cells, ameliorated disturbances of the hexagonal packing geometry of lens fibers, and improved ultrastructure of the epithelial cells. The levels of mRNA of α-crystallins genes which encode small heat shock proteins αA- and αB-crystallin that play a central role in maintaining lens transparency were significantly lower in the OXYS rats' lenses than in Wistar rats (control). SkQ1 normalized the level of mRNA of Cryaa, and significantly increased the level of Cryab mRNA as well as αB-crystallin protein in the lens of OXYS rats to the level of the control Wistar rats. CONCLUSION: SkQ1 eye drops hold promise as a treatment of cataract.

Therapeutic doses of SkQ1 do not induce cytochromes P450 in rat liver.

The effect of SkQ1 (a mitochondria-targeted antioxidant) on the level of cytochromes P450 in rat liver was studied. It was found that administration of therapeutic dose of SkQ1 with drinking water for 5 days (250 nmol/kg of body weight per day) did not alter the level of cytochromes P450. Under the same conditions, the standard dose of phenobarbital used for the induction of cytochromes P450 caused the 2.7-fold increase in the content of these cytochromes. We conclude that therapeutic doses of SkQ1 do not induce cytochromes P450 in rats.

Effect of the mitochondria-targeted antioxidant SkQ1 on development of spontaneous tumors in BALB/c mice.

The mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyldecyl)triphenylphosphonium) is a new pharmaceutical substance with a wide spectrum of effects including increase in lifespan of laboratory animals (for example, of BALB/c mice males) and inhibition of development of some experimental tumors and also of tumor cell growth. In this work, the effects of SkQ1 on development of spontaneous tumors in female and male BALB/c mice housed in an SPF-class vivarium were studied. We found that the addition of SkQ1 to drinking water at the dose of 1 and 30 nmol/kg body weight per day throughout the lifespan modified the spectrum of spontaneous tumors in the female mice, decreasing the incidence of follicular lymphomas. SkQ1 at the dose of 1 nmol/kg per day also suppressed the dissemination of these neoplasms, but it did not significantly influence the overall incidence of benign and malignant tumors (including primary multiple tumors) or the lifespan of the tumor-bearing mice (both males and females). Hence, the previously described ability of SkQ1 to increase the lifespan of laboratory BALB/c mice is not related to its anticarcinogenic activity.

The mitochondria-targeted antioxidant SkQ1 restores αB-crystallin expression and protects against AMD-like retinopathy in OXYS rats.

Age-related macular degeneration (AMD), a neurodegenerative and vascular retinal disease, is the leading cause of blindness in the developed world. Accumulating evidence suggests that alterations in the expression of a small heat shock protein (αB-crystallin) are involved in the pathogeneses of AMD. Here we demonstrate that senescence-accelerated OXYS rats-an animal model of the dry form of AMD-develop spontaneous retinopathy against the background of reduced expression of αB-crystallin in the retina at the early preclinical stages of retinopathy (age 20 days) as well as at 4 and 24 months of age, during the progressive stage of the disease. The level of αA-crystallin expression in the retina of OXYS rats at all the ages examined was no different from that in disease-free Wistar rats. Treatment with the mitochondria-targeted antioxidant SkQ1 (plastoquinonyl-decyltriphenylphosphonium) from 1.5 to 4 months of age, 250 nmol/kg, increased the level of αB-crystallin expression in the retina of OXYS rats. SkQ1 slowed the development of retinopathy and reduced histological aberrations in retinal pigment epithelium cells. SkQ1 also attenuated neurodegenerative changes in the photoreceptors and facilitated circulation in choroid blood vessels in the retina of OXYS rats; this improvement was probably linked with the restoration of αB-crystallin expression.

[Effect of plastoquinone derivative SkQ1 on benzo(a)pyrene-induced soft tissue carcinogenesis].

Ninety female SHR mice were subcutaneously injected with a single dose of 2 mg benzo(a)pyrene (BaP) dissolved in 0.2 ml of vegetable oil. Since the next day after BaP injection mice were started to treat with mitochondria-targeted antioxidant SkQ1 at the doses of 5 and 50 nmol/kg/day in drinking water. Control animals received tap water. Study was finished by 358th day. Number of tumor-bearing mice increased in all groups exposed to BaP but retarded since 20th week in SkQ1-treated groups in comparison with control. Maximal tumor volume gain was observed in control mice resulting in premature death. By the 30th week of study only 20% of control animals survived, whereas SkQ1 treatment increased survival up to 30% at the dose of 5 nmol and 40% at the dose of 50 nmol. By the 40th week mean tumor volume in 5 and 50 nmol SkQ1-treated mice was 13 and 21 cm3 respectively, whereas in control--40 cm3. In SkQ1-treated mice pneumonia was observed rarely as compared with controls. It could be supposed, SkQ1 at the doses of 5 and 50 nmol/kg/day retarted BaP-induced soft tissue carcinogenesis in SHR mice.

SkQ1 slows development of age-dependent destructive processes in retina and vascular layer of eyes of wistar and OXYS rats.

We show the development of clearly pronounced age-related pathological changes in eye tissues of Wistar and OXYS rats. Photoreceptor cells were virtually absent in all OXYS rats in the age of 24 months. Massive accumulations of lipofuscin granules were detected in the pigmented epithelium cells. Flattening, overgrowing, and degradation of endothelial cells of choriocapillaries were also observed. Along with these changes, vessels without signs of degradation were detected in the pigmented epithelium. In 24-month-old Wistar rats these changes were local and were seen in only some of the animals. The mitochondria-targeted antioxidant SkQ1 (the rats were given SkQ1 daily with food at the dose of 250 nmol/kg for 5 months, starting from the age of 19 months) prevented the development of these pathological changes in both Wistar and OXYS rats. The data were subjected to mathematical processing and statistical analysis.

Mitochondria-targeted antioxidant SkQR1 ameliorates gentamycin-induced renal failure and hearing loss.

The influence of the mitochondria-targeted antioxidant SkQR1 on gentamycin-induced nephrotoxicity and ototoxicity has been analyzed. SkQR1 reduces the death of kidney epithelium cells and decreases the severity of renal failure caused by gentamycin application and also lowers the animals' mortality. Treatment with SkQR1 also decreases gentamycin-induced hearing loss. Mitochondria-targeted antioxidants, such as SkQR1, are new promising agents for preventing negative consequences of therapy with antibiotics.

The therapeutic effect of mitochondria-targeted antioxidant SkQ1 and Cistanche deserticola is associated with increased levels of tryptophan and kynurenine in the rat lens.

Supplementation of senescence-accelerated OXYS rats with the mitochondria-targeted antioxidant SkQ1 and with the powder from Cistanche deserticola results in the deceleration of the cataract development and even in the improvement of lens transparency. The therapeutic effect of these preparations correlates with a significant elevation of tryptophan and kynurenine levels in the lens. This finding is attributed to a deceleration of the tryptophan and kynurenine oxidation due to antioxidant-assisted reduction of oxidative stress in the lens.

In vivo injected mitochondria-targeted plastoquinone antioxidant SkQR1 prevents ß-amyloid-induced decay of long-term potentiation in rat hippocampal slices.

Addition of 200 nM ß-amyloid 1-42 (Abeta) to a rat hippocampal slice impairs the induction of a long-term post-tetanic potentiation (LTP) of population spike (PS) in pyramidal neurons of the CA1 field of hippocampus. Intraperitoneal injection into the rat of the mitochondria-targeted plastoquinone derivative SkQR1 (1 µmol/kg of weight given 24 h before the slices were made) abolishes the deleterious effect of Abeta on LTP. These data demonstrate that SkQR1 therapy is able to compensate the Abeta-induced impairments of long-term synaptic plasticity in the hippocampus, which are the main cause of loss of memory and other cognitive functions associated with Alzheimer's disease.

[The effect of mithochondria targeted antioxidant SkQ1 on aging, life span and spontaneous carcinogenesis in three mice strains].

Female outbred SHR mice, inbred 129/Sv mice and transgenic HER-2/neu mice were given mitochondria targeted antioxidant SkQ1 with drinking water in the various doses (0,5-2500 nmol/kg day) since the age of 2 months, whereas control animals received tap water. Age-related dynamics of the body weight and temperature, the amount of drinking water and consumed food, estrous function, as well as parameters of the life span and spontaneous carcinogenesis were estimated. As compared with controls, no difference in the parameters of body weight and temperature or amount of consumed food and water in the treated mice of all studied mice strains was revealed. In SkQ1-treated SHR mice, the tendencies of inhibition of the age-dependent disturbances of estrous function and aging appearance were observed. No effect of SkQ1 on estrous function and external view in inbred and transgenic mice was shown. SkQ1 treatment significantly decreased locomotor activity (in 12-15 months old SHR and 129/Sv mice) and exercise tolerance in old (20 months) SHR mice. The treatment with SkQ1 (0,5-50 nmol/kg day) increased parameters of the life span in SHR mice (mean life span, mean life span of the last 10% of survival, median and maximum life span) without significant effect on the life span in 129/Sv and HER-2/neu mice. There was no reliable difference in tumor development in all SkQ1-treated mice strains as compared with the control. The drug considerably inhibited the incidence of age-associated non-tumor pathology in SHR mice. Our data suggest geroprotective activity of SkQ1, and a lack of toxic or carcinogenic activities during long term use.