Three-Year Follow-Up of High-Dose Ubiquinol Supplementation in a Case of Familial Multiple System Atrophy with Compound Heterozygous COQ2 Mutations

We report a 3-year follow-up of high-dose ubiquinol supplementation in a case of familial multiple system atrophy (MSA) with compound heterozygous nonsense (R387X) and missense (V393A) mutations in COQ2. A high-dose ubiquinol supplementation substantially increased total coenzyme Q10 levels in cerebrospinal fluid as well as in plasma. The patient was at the advanced stage of MSA, and the various scores of clinical rating scales remained stable without changes during the 3 years. The cerebral metabolic ratio of oxygen measured by 15O2 PET, however, increased by approximately 30% after administration of ubiquinol, suggesting that ubiquinol can improve mitochondrial oxidative metabolism in the brain. It also suggests the therapeutic potential of ubiquinol for patients with MSA with COQ2 mutations. Further clinical trials of administration of high-dose ubiquinol to MSA patients are warranted.


Introduction
Multiple system atrophy (MSA) is a progressive neurodegenerative disease clinically characterized by autonomic failure in addition to various combinations of parkinsonism, cerebellar ataxia, and pyramidal dysfunction [1]. Whole-genome sequence analysis in combination with linkage analysis has revealed homozygous or compound heterozygous mutations in COQ2 in two of the six multiplex Japanese families with MSA [2]. COQ2 encodes an enzyme in the biosynthetic pathway of coenzyme Q 10 (CoQ 10 ) [3]. Indeed, the total CoQ 10 levels in frozen brain tissues and lymphoblastoid cell lines from patients with MSA carrying homozygous (M128V-V393A/M128V-V393A) and compound heterozygous mutations (R387X/V393A) were substantially lower than those from control subjects [2]. These observations suggest the efficacy of CoQ 10 supplementation for patients with MSA, in particular, for those with COQ2 mutations.
CoQ 10 is a lipophilic molecule that functions as an essential carrier for electron transport in the mitochondrial respiratory chain and as an endogenous antioxidant [4]. Patients with primary CoQ 10 deficiency, a severer phenotype than MSA, caused by genetic defects in genes involved in the CoQ 10 biosynthetic pathway, have been reported to respond well to CoQ 10 supplementation [5][6][7][8][9][10][11]. Herein, we present the detailed clinical features of a patient with familial MSA carrying compound heterozygous COQ2 mutations (R387X/V393A) [2] and report the outcome of a high-dose ubiquinol (reduced form of CoQ 10 ) supplementation to this patient.
The patient gradually noticed slurring of speech, unsteadiness of gait, increased urinary frequency, and erectile dysfunction at the age of 44. Examination at the age of 45 revealed jerky pursuit of eye movements, gazeevoked nystagmus, and scanning speech. Lower limb movements were uncoordinated when performing the heel-knee-shin test. His gait was ataxic, and he was unable to perform tandem gait. He had urinary urgency, frequent urination, and erectile dysfunction. He also had frequent orthostatic symptoms attributable to hypotension. His cognition and visual acuity were normal. Brain magnetic resonance imaging (MRI) showed the hot cross bun sign in the pontine base and mild atrophy of the pons and cerebellum. He was then diagnosed as having Bfamilial MSA^because his elder sister had a similar presentation with dysarthria and unsteady gait with the onset at the age of 50. She died at the age of 61. He started intermittent catheterization at the age of 47 and had frequent orthostatic symptoms once a week at the age of 48. He became wheelchair-bound at the age of 50 and subsequently became bed-ridden owing to his severe orthostatic symptoms. At the age of 56, his speech was unintelligible most of the time due to the severe ataxic dysarthria. At the age of 58, endoscopic gastrostomy and laryngotracheal separation were performed owing to his recurrent aspiration pneumonia. At the age of 59, his renal function gradually worsened (serum creatinine level, 1.0-1.5 mg/dl).

Study Design
An open-label dose escalation trial for this patient with familial MSA with compound heterozygous COQ2 mutations (R387X/V393A) was designed to evaluate the safety and tolerability of high-dose ubiquinol, to assess pharmacokinetics, and to obtain the clinical data including the scores of clinical rating scales [Barthel Index [13], Scale for the Assessment and Rating of Ataxia (SARA) [14], International Cooperative Ataxia Rating Scale (ICARS) [15], and Unified Multiple System Atrophy Rating Scale (UMSARS) [16]]. Positron emission tomography (PET) was carried out to measure the cerebral blood flow (CBF) and the cerebral metabolic rate of oxy g e n ( C M R O 2 ) . T h e p r o t o c o l o f t h i s s t u d y (UMIN000010712) was reviewed and approved by the institutional review board of the participating institutions. Informed consent was obtained from the patient and his legal representative prior to the initiation of this study, in accordance with the Declaration of Helsinki. Ubiquinol in the powder form (Kaneka QH ubiquinol) was provided by Kaneka Corporation (Tokyo, Japan) and was administered via a gastrostomy tube.
After baseline assessment, supplementation was started at 600 mg of ubiquinol/day (given once a day), with the dosage increased to 840 mg/day at week 2 and to 1200 mg/day at week 6. The 1200-mg/day dosage was maintained until week 8. When no adverse events were observed during this period, the patient resumed taking 1200 mg of ubiquinol/day after an interval of 8 weeks and remained taking ubiquinol at this same dose for over 3 years to date.

Biochemical Analysis
Peripheral blood was collected into heparinized tubes to obtain plasma samples. BD Vacutainer CPT Cell Preparation Tubes (BD, Franklin Lakes, NJ) with sodium heparin were used for separation of peripheral blood mononuclear cells (PBMCs). The total CoQ 10 levels (sum of ubiquinol and ubiquinone levels) in plasma, PBMCs, and cerebrospinal fluid (CSF) were measured by high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) [17] or with tandemmass spectrometry (LC-MS/MS). For measurement of the total CoQ 10 level in CSF by HPLC with ECD, 400 μl of CSF was mixed with 1600 μl of isopropanol. An aliquot of 1500 μl was evaporated to dryness and resolved in 120 μl of isopropanol and 30 μl of distilled water. An aliquot of 80 μl of the supernatant was directly injected onto the HPLC equipped with the separation column (SHISEIDO CQ-S: 150 mm × 2.0 mm I.D.) with the mobile phase of 50 mM sodium perchlorate in methanol/ distilled water (95/5, v/v). Measurement of the total CoQ 10 level in additional CSF samples obtained after 12, 24, and 36 months of ubiquinol supplementation was conducted as follows. Twenty microliters of CSF was diluted to 5 ml with phosphate-buffered saline and mixed with 8 ml of diethyl ether and 10 μl of internal standard solution (200 ng/ml of CoQ 8 in isopropanol). After the solution was vigorously mixed, the supernatant separated by centrifugation was evaporated to dryness and resolved in 20 μl of isopropanol. Quantitation of CoQ 10 was accomplished by LC-MS/MS using Nexera X2 and LCMS-8060 (Shimadzu, Japan).
For quantitative analysis, brain PET images before and after ubiquinol supplementation were intrasubjectively coregistered and morphologically normalized to the brain PET template using statistical parametric mapping 8 and MATLAB version R2014a (MathWorks Inc., Natick, MA, USA). The regions of interest (ROIs) were manually placed on the cortical ribbon of the upper frontal, lower frontal, Rolandic, lateral and medial parietal, temporal, and occipital areas and on the striatum, thalamus, cerebellar hemisphere, and cerebellar vermis on the morphologically normalized CBF PET images obtained before supplementation. These ROIs were automatically applied to other PET images, and each ROI value was then computationally calculated.

Ubiquinol Supplementation and Clinical Outcome
The analysis of the total CoQ 10 levels in plasma and PBMCs revealed a significant increase after 2 weeks of ubiquinol supplementation at 600 mg/day ( Table 1). The total CoQ 10 levels in plasma and PBMCs remained similar for another 4 weeks at 840 mg/day, and a subsequent 2-week administration of ubiquinol at 1200 mg/day led to substantial increases in the total CoQ 10 levels in the plasma and the PBMCs. The CoQ 10 level in CSF increased from 0.22 to 3.79 ng/ml after 2 weeks of 840 mg/day, and a similar level of 3.64 ng/ml was observed after 2 weeks of 1200 mg/day. Eight weeks after the last supplementation of ubiquinol, the total CoQ 10 levels in plasma, PBMCs, and CSF returned to baseline levels.
Since we did not observe any adverse events at 1200 mg/ day dosage and ubiquinol supplementation at 1200 mg/day led to higher total CoQ 10 levels in the plasma and the PBMCs compared with those observed with 840 mg/day, we decided to maintain the 1200-mg/day dosage. After supplementation of 1200 mg/day was resumed, the CoQ 10 levels in plasma and PBMCs returned to levels similar to those observed after the initial 2 weeks of 1200 mg/day and maintained at similar levels throughout the following period. The CoQ 10 levels in CSF were 7.36, 9.14, and 14.06 ng/ml after 12, 24, and 36 months of 1200 mg/day, respectively. Thus, the total CoQ 10 levels in CSF gradually increased after resuming ubiquinol supplementation at 1200 mg/day for the following period ( Table 1). The patient continued to take 1200 mg of ubiquinol/day for over 3 years (Fig. 1). During the entire course, we did not observe any adverse events that were considered to be associated with the ubiquinol supplementation throughout the entire study period. After 36 months of supplementation, evaluation of scores of clinical rating scales (Barthel index, SARA, ICARS, and UMSARS) showed no remarkable changes ( Table 2). The brain MRI findings also remained unchanged for the 3 years (Fig. 2). It was notable that his serum creatinine level gradually declined over 36 months (from 1.45 to 0.95 mg/dl) (Fig. 1). His body weight decreased in the first 16 weeks (48.5 to 41.0 kg), but gradually increased over 36 months (41.0 to 46.0 kg) after increasing his daily calorie intake (from 900 to 1500 kcal/day).  Measurement of the CBF and the CMRO 2 by PET Figures 3 and 4 show CBF and CMRO 2 images, respectively, and Table 3 summarizes the quantitative measurements before and after 2 weeks of ubiquinol supplementation at 1200 mg/day. At the baseline, CBF decreased and CMRO 2 markedly decreased in the entire brain. For example, the CBF and CMRO 2 in the Rolandic area were 30.6 ml/100 ml/min [reference level [19], 44.6, 5.6 (mean, standard deviation)] and 1.81 ml/100 ml/min [reference level [19], 3.3, 0.5 (mean, standard deviation)], respectively. After 2 weeks of ubiquinol supplementation at 1200 mg/day, CBF remained unchanged as compared with the baseline level ( Fig. 3 and Table 3). In contrast, the CMRO 2 markedly increased by approximately 30% in the entire brain ( Fig. 4 and Table 3), although it did not reach normal levels. For example, the CMRO 2 in the Rolandic area increased from 1.81 to 2.15 ml/100 ml/min [reference level, 3.3, 0.5 (mean, standard deviation) [19]].

Discussion
In the present single case study of a patient with familial MSA carrying compound heterozygous mutations in COQ2, administration of high-dose ubiquinol led to a substantial increase in the total CoQ 10 levels not only in the plasma and PBMC but also in the CSF. Although previous reports have failed to show the increase in total CSF CoQ 10 level by ubiquinone or ubiquinol supplementation, which was caused possibly due to the insufficient dose (300 mg/day) [25], this is the first study showing that ubiquinol supplementation at 840 and 1200 mg/ day clearly elevated the total CSF CoQ 10 level. CoQ 10 has been reported to be poorly absorbed, and its bioavailability varies among formulations [26]. Previous dose escalation studies (up to 3000 mg/day) using chewable tablets of ubiquinone in patients with Parkinson disease, amyotrophic lateral sclerosis, and Huntington disease concordantly showed that the total plasma CoQ 10 levels reached the plateau levels of approximately 7.0-7.5 μg/ml after multiple doses of After 36 months of ubiquinol 1,200 mg/day Baseline assessment Fig. 2 Brain MR images of patient. Upper images were obtained at 60 years of age (before supplementation of ubiquinol), and lower images were obtained at 63 years of age (after 36 months of ubiquinol 1200 mg/day). T1-weighted sagittal images (left) and T2-weightd axial images at the middle regions of the pontine base (right). They show gross atrophy of the infratentorial structures especially in the pons, middle cerebellar peduncle, and cerebellum 2400 mg/day [22][23][24]. When assessing the bioavailability of ubiquinol in this study, the trough concentrations of total CoQ 10 in plasma were 5.04 μg/ml for 600 mg/day, 4.02 μg/ ml for 840 mg/day, and 7.86 μg/ml for 1200 mg/day 2 weeks after the daily intake of ubiquinol. Furthermore, another previous study using ubiquinol showed that mean total plasma CoQ 10 levels were 2.61 μg/ml for 90 mg/day, 3.66 μg/ml for 150 mg/day, and 6.53 μg/ml for 300 mg/day 2 weeks after a daily intake of ubiquinol [27]. These observations indicate that ubiquinol is better absorbed in the gastrointestinal tract than ubiquinone, and we conclude that the ubiquinol dose of 1200 mg/day is sufficient for achieving a plateau of total CoQ 10 level in plasma.
Remarkably, CMRO 2 increased without an increase in CBF after administration of 1200 mg of ubiquinol, which suggests that ubiquinol improved cerebral mitochondrial oxidative metabolism. Despite the increase in the CMRO 2 , however, we did not detect any obvious neurological improvements as determined by the rating scales, presumably owing to the advanced stage of neurodegeneration. Notably, his serum creatinine level gradually declined during the ubiquinol supplementation over 36 months (from 1.45 to 0.95 mg/dl). Because renal involvement has been frequently observed in patients with primary CoQ 10 deficiency caused by genetic defects in CoQ 10 biosynthesis [28][29][30], the renal dysfunction in the patient was likely caused by CoQ 10 deficiency and was ameliorated by ubiquinol supplementation. He also showed weight loss in the first 16 weeks of supplementation (48.5 to 41.0 kg). We extensively investigated the cause of his weight loss. However, we did not find chronic infectious diseases, malignancies, extremity edema, pleural effusion, or ascites in this patient during the entire study period. He gradually regained his body weight over 36 months (41.0 to 46.0 kg) after increasing his daily calorie intake. Despite the body weigh changes, his general health condition remained stable.

Conclusions
The current study suggests that high-dose ubiquinol supplementation (up to 1200 mg/day) is tolerable and improves cerebral mitochondrial oxidative metabolism, which may alter mg/day 1,200 ubiquinol of 2 weeks After Baseline assessment Fig. 3 CBF images before and after ubiquinol supplementation at 1200 mg/day the natural history of MSA progression especially when applied in the early phase of MSA in patients with genetic defects in the CoQ 10 biosynthetic pathway. Further clinical trials including administration of ubiquinol to MSA patients carrying heterozygous COQ2 mutations as well as to patients without mutations in COQ2 are warranted. Prospective randomized controlled trials will be undertaken to further extend these initial promising observations.