Ubiquinol (reduced Coenzyme Q10) in patients with severe sepsis or septic shock: a randomized, double-blind, placebo-controlled, pilot trial

This was a single-center, randomized, double-blind, pilot phase II trial comparing ubiquinol to placebo in patients with severe sepsis or septic shock. The study was conducted at Beth Israel Deaconess Medical Center (BIDMC), which is an urban tertiary care hospital in Boston, Massachusetts with approximately 55,000 emergency department visits annually and a total of 77 intensive care unit beds. The study was approved by the Committee on Clinical Investigations at BIDMC (approval number: 2012P-000267) and patients or their legally authorized surrogate provided written informed consent prior to enrollment. The trial was registered at Clinicaltrials.gov (identifier: NCT01948063) and was sponsored by Kaneka Corporation, Japan. The trial was investigator-initiated and the sponsor was not involved in study design or conduct, and had no role in manuscript preparation.

The hospital’s emergency department and intensive care units were screened for eligible patients between November 2012 and January 2014. Inclusion criteria were that patients were aged ≥18 years and were diagnosed with severe sepsis or septic shock. Severe sepsis was defined as the presence of two or more systemic inflammatory response syndrome criteria, documented or suspected infection, and evidence of sepsis-induced organ dysfunction or tissue hypoperfusion as defined by the Surviving Sepsis campaign [1]. Septic shock was defined as sepsis-induced hypotension persisting despite adequate fluid resuscitation, in accordance with the Surviving Sepsis campaign definitions [1].

We excluded patients based on the following criteria: 1) currently on CoQ10 supplementation, 2) unable to receive enteral medication per the clinical team, 3) enrolled in another ongoing study, 4) non-English speaking, 5) patient unable to consent and legally authorized surrogate not present, 6) ‘do not resuscitate’, ‘do not intubate’ or ‘comfort measures only’ designation, or 7) member of a protected population.

A master randomization list was created using SAS (SAS Institute, Cary, NC, USA), randomizing patients to ubiquinol or placebo in a 1:1 ratio. The list was located in the central pharmacy for the duration of the study. The study drug (placebo or ubiquinol) was administered as a pill by mouth or in liquid form per nasogastric tube if present. Given the distinct taste of the liquid, this was not provided to all patients. The study drug was administered twice per day after enrollment, and was continued daily for seven days or until hospital discharge, whichever came first. Ubiquinol (Kaneka Corporation, Japan) was administered at a dosage of 200 mg per dose. Ubiquinol was chosen over ubiquinone (oxidized CoQ10) due to its higher bioavailability [11]. The dose was chosen based on safety data showing that doses of 300 mg are well-tolerated in healthy volunteers [11], and the dose was comparable to that used in a previous post-cardiac arrest trial [12]. The placebo was in identical pills or liquids as the study drug and patients, healthcare personnel, and the research team remained blinded throughout the study period.

Venous blood was collected at enrollment immediately before administration of study drug (0 h) and at 12, 24, 48, and 72 h thereafter. Samples were centrifuged and serum and plasma were aliquoted into light-protected cryotubes and immediately frozen at −80 °C.

Upon enrollment, we recorded demographic data, co-morbid conditions, vital signs, clinical laboratory values, whether or not the patient was receiving mechanical ventilation or vasopressors, and calculated the Acute Physiology and Chronic Health Evaluation II (APACHE II) score. Vital signs and laboratory values were also collected at all subsequent time points and outcome data, including length of intensive care unit stay, length of hospital stay, and mortality, was collected at patient discharge. All data was entered into a secure, online database by trained research assistants.

The primary outcome of the study was change in plasma CoQ10 parameters (total CoQ10 levels, CoQ10 levels relative to cholesterol levels, levels of oxidized (ubiquinone) and reduced (ubiquinol) CoQ10, and the fraction of reduced CoQ10 (reduced CoQ10/total CoQ10 levels)).

CoQ10 levels were measured in plasma samples by the Department of Pathology and Laboratory Medicine at Cincinnati Children’s Hospital Medical Center (Cincinnati, Ohio, USA) using high-performance liquid chromatography as previously described in detail [13]. We measured three different CoQ10 parameters (total CoQ10, oxidized CoQ10, and reduced CoQ10 (all measured in μg/mL)), as well as total cholesterol levels. From these, two additional parameters were calculated: total CoQ10/cholesterol and reduced CoQ10/total CoQ10. CoQ10 levels were measured at the 0, 12, and 24 h time points.

Plasma samples were analyzed for multiple vascular endothelial and inflammatory markers (VEGF, VCAM-1, IL-2, IL-6, IL-10, and TNF-α) using customized Meso Scale Discovery Human Multiplex Panel (Rockville, Maryland, USA). All samples were measured in duplicate with the inter-assay coefficients of variability ranging from 2.2 to 5.8 %. VCAM-1 is reported in log-transformed μg/mL and the rest of the markers in log-transformed pg/mL. Cytokine levels were measured at the 0, 12, 24, 48 and 72 h time points.

Cytochrome c levels were measured by electrochemiluminescence in a Quickplex SQ 120 instrument (Meso Scale Discovery) at the Resuscitation Institute/Rosalind Franklin University of Medicine and Science (North Chicago, IL, USA). Cytochrome c levels had previously been reported to correlate inversely with survival after cardiac arrest in rodents [14]. Standard curves were prepared using commercially available human heart cytochrome c (cat# C3483-10UG, Sigma-Aldrich, St. Louis, MO, USA) diluted in 10 % pooled human plasma from healthy volunteers in assay buffer at concentrations of 1,200.0 ng/mL, 400.0 ng/mL, 133.3 ng/mL, 44.4 ng/mL, 14.8 ng/mL, 4.9 ng/mL, 1.6 ng/mL, and 0 ng/mL. Each plasma sample with an unknown cytochrome c concentration was thawed in ice and diluted 1:1 in the assay buffer. Samples and standards (in a volume of 25 μL) were run in duplicate. Data analysis was performed using Meso Scale Discovery software, which developed the standard curve used to calculate the unknown concentrations. The upper limit of detection was 1,200 ng/mL and the lower limit of detection was 4 ng/mL, which is defined as 2.5 standard deviations above the background mean. The software also calculates the coefficient of variation between the replicate samples. If the sample cytochrome c concentration fell above or below the dynamic range of the standard curve, the assay was repeated after appropriate dilution. Cytochrome c levels were expressed in log-transformed ng/mL. Cytochrome c levels were measured at the 0, 12, and 24 h time points. Cytochrome c was included as an exploratory outcome as a potential marker of mitochondrial injury [14].

We measured cell-free DNA, two markers of nuclear DNA, and two markers of mitochondrial DNA (mtDNA) at the 0, 12, and 24 h time points. In order to measure cell-free DNA in plasma, the plasma samples were centrifuged at 16,000 g for 10 min to remove any residual cells. The upper portion of the plasma was removed into a nuclease-free tube and stored at −80 °C prior to DNA extraction. Cell-free DNA in the plasma was isolated using a plasma/serum DNA isolation kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol. The amount of cell-free DNA was measured using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The quality of DNA was checked by the A260/280 ratio. The amount of mtDNA was measured with mtDNA-tRNA^leu and mtDNA-D loop genes, which are specifically present in the mitochondrial genome. Nuclear DNA was amplified using the single-copy beta-2-microglobulin (B2M) and RNase P nuclear genes. Primers and probes for real-time PCR were purchased from Life Technologies Corporation and utilized as previously described by Bai and Wong [15]. TaqMan real-time quantitative PCR assay was performed on a 7500 real time PCR system following the manufacturer’s protocol (Applied Biosystems, Life Technologies Corporation, CA, USA). DNA extracted from whole blood with a two-fold serial dilution was used to construct the calibration curve. All samples were measured in duplicate with the inter-assay coefficients of variability ranging from 1.6 to 5.5 %. Cell-free DNA was measured as a marker of cellular injury, as well as a surrogate for poor outcome [16‐18].

Descriptive statistics were used to summarize the study population. Data for continuous variables are presented as means with standard deviations (SD) or medians with quartiles, depending on the normality of the data. Categorical data are presented as counts with frequencies. Depending on the distribution of the data, t-tests or Wilcoxon rank sum tests were used to compare continuous data between the groups. Categorical data were compared using chi-squared or Fisher’s exact tests as appropriate.

To assess the difference in repeated continuous variables (CoQ10 levels, vascular and inflammatory biomarkers, cytochrome c levels, and DNA markers) between groups we used repeated measures models to account for within-subject correlation. Variables were log-transformed before statistical analysis. An appropriate covariance structure was chosen and the residuals were visually assessed for normality. Due to the exploratory nature of the current study we did not account for multiple comparisons. Statistical analyses were conducted with the use of SAS software, version 9.3 (SAS Institute Inc., Cary, NC, USA). All hypothesis tests were two-sided, with a significance level of P <0.05.