NQO1-Dependent Redox Cycling of Idebenone: Effects on Cellular Redox Potential and Energy Levels

Short-chain quinones are described as potent antioxidants and in the case of idebenone have already been under clinical investigation for the treatment of neuromuscular disorders. Due to their analogy to coenzyme Q10 (CoQ10), a long-chain quinone, they are widely regarded as a substitute for CoQ10. However, apart from their antioxidant function, this provides no clear rationale for their use in disorders with normal CoQ10 levels. Using recombinant NAD(P)H:quinone oxidoreductase (NQO) enzymes, we observed that contrary to CoQ10 short-chain quinones such as idebenone are good substrates for both NQO1 and NQO2. Furthermore, the reduction of short-chain quinones by NQOs enabled an antimycin A-sensitive transfer of electrons from cytosolic NAD(P)H to the mitochondrial respiratory chain in both human hepatoma cells (HepG2) and freshly isolated mouse hepatocytes. Consistent with the substrate selectivity of NQOs, both idebenone and CoQ1, but not CoQ10, partially restored cellular ATP levels under conditions of impaired complex I function. The observed cytosolic-mitochondrial shuttling of idebenone and CoQ1 was also associated with reduced lactate production by cybrid cells from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) patients. Thus, the observed activities separate the effectiveness of short-chain quinones from the related long-chain CoQ10 and provide the rationale for the use of short-chain quinones such as idebenone for the treatment of mitochondrial disorders

Pharmacokinetic analysis of idebenone in eye fluids.

<p>Concentrations of idebenone in aqueous humor (open circle) and vitreous humor (filled triangle) following once daily administration of idebenone in the diet for 21 days (A) and following single oral administration of idebenone at 60 mg/kg (B) were determined. Concentrations of idebenone in the eye fluids are expressed as ng/ml (left y axis) and nM (right y axis). For (A), sampling time was more than 8h after last dose of idebenone, the values therefore represent trough levels.</p

Idebenone Protects against Retinal Damage and Loss of Vision in a Mouse Model of Leber’s Hereditary Optic Neuropathy

<div><p>Leber’s hereditary optic neuropathy (LHON) is an inherited disease caused by mutations in complex I of the mitochondrial respiratory chain. The disease is characterized by loss of central vision due to retinal ganglion cell (RGC) dysfunction and optic nerve atrophy. Despite progress towards a better understanding of the disease, no therapeutic treatment is currently approved for this devastating disease. Idebenone, a short-chain benzoquinone, has shown promising evidence of efficacy in protecting vision loss and in accelerating recovery of visual acuity in patients with LHON. It was therefore of interest to study suitable LHON models <i>in vitro</i> and <i>in vivo</i> to identify anatomical correlates for this protective activity. At nanomolar concentrations, idebenone protected the rodent RGC cell line RGC-5 against complex I dysfunction <i>in vitro.</i> Consistent with the reported dosing and observed effects in LHON patients, we describe that in mice, idebenone penetrated into the eye at concentrations equivalent to those which protected RGC-5 cells from complex I dysfunction <i>in vitro</i>. Consequently, we next investigated the protective effect of idebenone in a mouse model of LHON, whereby mitochondrial complex I dysfunction was caused by exposure to rotenone. In this model, idebenone protected against the loss of retinal ganglion cells, reduction in retinal thickness and gliosis. Furthermore, consistent with this protection of retinal integrity, idebenone restored the functional loss of vision in this disease model. These results support the pharmacological activity of idebenone and indicate that idebenone holds potential as an effective treatment for vision loss in LHON patients.</p></div

Idebenone prevents rotenone-induced RGC death. Analysis of RGCs in the mouse retina 7 days after intravitreal injection of rotenone or DMSO (Sham).

<p>Mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-Brn3a primary antibody to visualize RGCs. Scale bar = 25 µm. (B) Quantification of RGCs (RGC number/mm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data expressed as mean ± SEM.</p

Idebenone time-dependently restores rotenone-induced loss of vision.

<p>Visual acuity was evaluated by counting the number of head movements at a velocity of 3 rpm 7 days prior injection (day -7) and 1 to 70 days after injection (day 1, 7, 21, 35, 70) of rotenone (5 mM). Mice were treated with dietary idebenone 2000 mg/kg or vehicle for the time of the experiment. (A) Schematic representation of the experimental setup. After vehicle (DMSO) injection, head movements in both directions of drum rotation (clockwise: CW; counterclockwise CCW) remain intact. Injection of rotenone into the left eye however, affects only the CW responses (dashed arrow) whereas CCW responses remain unaffected. (B) Quantification of clockwise (CW) head movement (number of head movements/2 min) following vehicle treatment and rotenone injection (vehicle + rotenone, n = 10 animals), and idebenone 2000 mg/kg treatment and rotenone injection (IDE 2000+ rotenone, n = 11 animals). Data are expressed as mean ± SEM. Statistical significance relative to vehicle + rotenone group: p≤0.05 (*); (C) Percentage of mice showing clockwise (CW) head movements for each treatment group 70 days after injection. Responder: group of mice showing head movements; Non-responder: group of mice without head movements.</p

Dose-effect of idebenone treatment on multiple endpoints in an <i>in vivo</i> mouse model for LHON.

<p>Dose-effect of idebenone treatment on multiple endpoints in an <i>in vivo</i> mouse model for LHON.</p

Idebenone protects against rotenone-induced gliosis.

<p>For analysis of gliosis 7 days after intravitreal injection of rotenone or DMSO (Sham), mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-GFAP primary antibody to visualize Müller glial cell projections (white arrows). White arrowheads indicate outer plexiform layer and also illustrate rotenone induced reduction in retinal thickness (i.e. distance between white arrow heads and ganglion cell layer at the top of the images). Scale bar = 15 µm. (B) Quantification of gliosis (GFAP signal based on relative fluorescence units, RFU) following idebenone treatment and rotenone injection (n = 3 to 7 per group). Data are expressed as mean ± SEM.</p

Protective effect of idebenone against complex I inhibition in RGC-5 cells <i>in vitro</i>.

<p>(A) Scheme of the experimental design. RGC-5 cells were pre-incubated with idebenone or vehicle (DMSO) for 1 or 2 days before the complex I inhibitor rotenone was applied to the cells for 6 hours. Cell viability was then measured after 1 day post-incubation with idebenone or vehicle. (B) Treatment with rotenone caused a 59% reduction in cell viability in vehicle-treated RGC-5 cells. (C) Idebenone pre-treatment for 1 (white bars) and 2 days (black bars) significantly rescued cell viability (expressed as % rescue). Data are expressed as mean ± SD (n = 6 wells per group). Statistical significance relative to vehicle group: p≤0.05 (*), p≤0.001 (***).</p