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BRIEF REPORT/PHARMACOKINETICS |
From the Department of Neurology, Clinical Trials Coordination Center, University of Rochester Medical Center, Rochester, New York (Dr Constantinescu, Ms de Blieck, Dr Ravina, Dr Kieburtz); Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, New York (Dr McDermott); Department of Pharmacy Practice, University at Buffalo, SUNY Buffalo, New York (Dr DiCenzo, Dr Bednarczyk); London Health Sciences Centre, London, Ontario, Canada (Dr Hyson); and Weill Medical College/Cornell University, New York (Dr Beal, Dr Bogdanov, Ms Metakis, Dr Browne, Ms Lorenzo).
Address for correspondence: Karl Kieburtz, MD, MPH, 1351 Mt Hope Avenue, Suite 223, Rochester, NY 14620; e-mail: karl.kieburtz{at}ctcc.rochester.edu.
Key Words: coenzyme Q10 • bioavailability • oral formulations • bioequivalence
Coenzyme Q10 (CoQ10, or ubiquinone) is an endogenous enzyme cofactor, produced in all living human cells and naturally occurring in dietary sources. As a constituent of the proton/electron transport chain, it plays a role in the energy production within mitochondria. Its position in membranes also allows CoQ10 to act as a primary scavenger of free radicals, protecting membrane phospholipids from peroxidation and membrane proteins and mitochondrial DNA from oxidative damage. In addition, it regenerates other antioxidants such as tocopherol and ascorbate. CoQ10 may also stimulate cell growth and inhibit apoptosis.1,2 A decrease in CoQ10 levels occurs with genetic mutations, aging, cancer, and the intake of statins,1 and altered levels are seen in diabetes mellitus, cardiovascular disorders, Alzheimer's disease, and other neurodegenerative disorders.3
CoQ10 has been shown to have a potential impact in a wide array of clinical conditions.4,5 In vitro and in vivo data suggesting neuroprotective effects led to CoQ10 being investigated in a number of neurodegenerative conditions, such as Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, with promising although equivocal results.5-9
CoQ10 has a high molecular weight, is strongly lipophilic, and is almost insoluble in aqueous solution; it is absorbed slowly and incompletely from the small intestine, with a poor bioavailability in humans.10-13 Available data suggest, however, that the therapeutic effects of CoQ10 may be dosage dependent, with more benefit attained with higher dosages and at higher blood levels of CoQ10.6,14 Consequently, knowledge about the bioavailability of a CoQ10 formulation is important when evaluating its effects in clinical trials.
Currently, CoQ10 is classified as a dietary supplement and does not have to meet the same Food and Drug Administration (FDA) standards as prescription drugs. Consequently, there are numerous proprietary preparations of CoQ10 available on the market, with differing claims of bioavailability. Preparations include powder-filled capsules, powder-based tablets, soft gel capsules, fully solubilized soft gel capsules, chewable wafers, and intraoral sprays.15 In addition, in clinical research, CoQ10 has been given intravenously and intramuscularly.
One study showed that the bioavailability of CoQ10, measured by the area under the concentration-time curve (AUC), varied widely between different oral formulations.16 Thus, further efforts to characterize the bioavailability of different existing CoQ10 formulations are warranted. We therefore performed a randomized, double-blind, crossover trial to compare 4 different oral CoQ10 formulations with regard to CoQ10 bioavailability after a single 600-mg dose in healthy adults.
SUBJECTS AND METHODS
Subjects
Twenty-five healthy adult volunteers were enrolled at the Western New York Veterans Administration Medical Center (Buffalo, New York) from October 29, 2003, to February 18, 2004. Subjects with a history of unstable medical or psychiatric illness and with ongoing alcohol or drug abuse were excluded. None had been taking CoQ10 supplementation for at least 30 days prior to baseline. No subject used coumadin or drugs that adversely affect mitochondria (eg, HMG-CoA reductase inhibitors such as pravastatin and simvastatin).
All subjects gave written informed consent to participate in the study.
Methods
This study was approved by the institutional review boards of the University of Rochester, New York and Western New York Veterans Administration Medical Center (Buffalo, New York).
Study procedures were conducted in the Western New York Veterans Administration Medical Center Clinical Research Center (CRC). Following provision of informed consent, subjects were screened at the CRC (screening visit). Screening procedures included intake of complete medical history and demographics, physical exam, and concomitant medication review. Screening safety labs included a complete blood count and standard chemistry profile, as well as a urine pregnancy test for women of childbearing potential. Subjects were instructed to fast beginning at midnight and return the following morning for a final review of study eligibility (baseline visit). Subjects were enrolled via a call to the University of Rochester Clinical Trials Coordination Center. During the enrollment call, the subject was assigned a subject identification number that corresponded to the randomized assignment to 1 of 4 possible sequences of administration of 4 CoQ10 formulations using a Williams square.17 Vital signs were assessed prior to administering CoQ10. After overnight fasting, each subject received a single oral 600-mg dose of the assigned CoQ10 formulation. At the time of dosing, the participants were offered a standardized breakfast, and a standardized lunch was served approximately 4 hours later (meal details available on request). Plasma concentrations of CoQ10 were assessed from serial blood draws performed 0.5 hours predose (baseline level) and at .5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24 to 30, and 48 to 54 hours postdose.
The subjects remained in the CRC during the baseline visit until the 12-hour postdose blood sample was collected. The last 2 blood draws were performed at separate visits (24 and 48 hours postdose). Subjects then underwent a 2-week washout period, with the same process being repeated for each of the other 3 formulations. Adverse events (voluntary complaints) as well as any changes in concomitant medications were recorded at each visit. The total length of the protocol was 60 days. At day 60, a telephone contact was conducted at which the participants were asked about adverse events.
Study Medication
Four different CoQ10 formulations were examined in this study: (1) plain chewable wafers containing 300 mg CoQ10 (Enzymatic Therapy, Inc; Green Bay, Wisconsin), hereafter referred to as plain chewable wafer; (2) chewable wafers containing 600 mg CoQ10 and 300 IU of vitamin E (Enzymatic Therapy, Inc), hereafter referred to as chewable wafer with vitamin E; (3) hard gelatin capsules containing 600 mg CoQ10 (Health Wright, Inc; Clackamas, Oregon), hereafter referred to as hard capsules; and (4) Mega Q-Gel "100" soft gel capsules containing 100 mg CoQ10 solubilized in an oil-based vehicle, together with 150 IU d-alpha tocopherol (Tishcon Corp; Westbury, New York), hereafter referred to as soft gel capsules.
To achieve the studied dose of 600 mg CoQ10, the following number of wafers/capsules had to be given as 1 single dose: 2 plain chewable wafers (600 mg CoQ10), 1 chewable wafer with vitamin E (600 mg CoQ10 and 300 IU of vitamin E), 1 hard capsule (600 mg CoQ10), and 6 soft gel capsules (600 mg CoQ10 and 900 IU of vitamin E).
Sample Collection, Processing, and CoQ10 Analysis
Blood samples were drawn from an antecubital vein into glass EDTA vacuum tubes, centrifuged, and the plasma transferred to cryotubes for immediate freezing. Frozen samples were then transported to a research laboratory at Weill Medical College, Cornell University, where they were assayed for CoQ10 content under the direction of Dr M. Flint Beal, using an accurate and reproducible high-performance liquid chromatography (HPLC) method with electrochemical detection.18 Standards were prepared with CoQ10 purchased from Sigma (St Louis, Missouri) and were made in HPLC grade methanol containing 30 mg/L butylated hydroxyanisole. Standards were prepared with the appropriate amounts of the stock solutions in a 1:8 dilution of 30% human albumin in normal saline to approximate human plasma. The samples were analyzed on an HPLC system consisting of a Waters 515 HPLC pump, an Eppendorf TC-50 column temperature control module (set at 37°C), a Waters 717 plus Autosampler (temperature set at 4°C), ESA Coulochem III electrochemical detector, and a Dell Pentium 4 computer with Windows XP and Chromquest chromatography software. The column was ESA's MD 150 x 3.2. The mobile phase was 77% acetonitrile/methanol (50/50 v/v), 21% N-propanol, and 2% 1.0 M ammonium acetate buffer (pH 4.4) pumped at 0.8 mL/min. The Coulochem III detector was equipped with a model 5011 analytical cell; channel 1 was set at -600 mV, and results were collected from channel 2 set at +300 mV. A 5-point standard curve (in duplicate) was analyzed each time ranging from 0.200 to 2.000 µ/mL CoQ10. The response is linear over that range and has been extended to as high as 8.000 µg/mL. The lower limit of quantification for the assay was 1 ng. All other routine blood and urine tests were analyzed by the laboratory at the CRC.
Primary and Secondary Outcome Variables
The primary outcome variable was the area under the CoQ10 concentration-time curve from 0 to 48 hours (AUC0-48). This outcome was used to formally assess bioequivalence of the different CoQ10 formulations. Standard noncompartmental techniques were used to calculate pharmacokinetic outcomes using WinNonlin software, Version 4.1 (Pharsight Corp, Palo Alto, California). The area under the concentration-time curve was determined using the linear trapezoidal method. Secondary outcome variables estimated from this curve by visual inspection included maximum plasma concentration (Cmax) and time to maximum plasma concentration (tmax). The AUC extrapolated to infinity (AUC0-
) and the elimination half-life (t
) could not be adequately estimated because of the persistence of relatively high plasma concentrations of CoQ10 through 48 hours.
STATISTICAL ANALYSIS
Sample Size
Two formulations of CoQ10 were to be judged to be equivalent if the confidence interval for the geometric mean ratio of AUC0-48 was contained within the FDA standard of 0.80 and 1.25.18 The sample size determination was based on the use of the two 1-sided tests procedure and a significance level of 5% for each test.19,20 Under an assumption of a 20% coefficient of variation for the AUC0-48 measurement (after accounting for subject and period effects), a sample size of 24 subjects was determined to provide >90% power to reject the null hypothesis of nonequivalence when the formulations are actually bioequivalent (ie, to correctly declare bioequivalence of the 2 formulations).
The outcomes AUC0-48, Cmax, and tmax were log-transformed for purposes of analysis. For each outcome variable, an analysis of variance model was used to perform pairwise comparisons among the formulations; factors included in the model were subject, formulation, and period.17 The comparisons are expressed as geometric mean ratios, with a value of 1 indicating no difference between the treatment formulations. The geometric mean ratio is equivalent to the exponentiated difference between the formulations in terms of mean log-transformed outcome. Confidence intervals were computed for geometric mean ratios using the analysis of variance model, and these were used to judge whether 2 formulations were bioequivalent for the AUC0-48 outcome as described above. A confidence coefficient of 98.3% (instead of 90%) was used to adjust for the 6 pairwise comparisons performed among the formulations. Exploratory analyses were performed to determine the extent to which the bioavailability of CoQ10 differed between men and women. Student t tests were performed to compare the mean log-transformed AUC0-48 between men and women, separately for each formulation.
RESULTS
Subject Demographics and Discontinuations
Twenty-five subjects (15 men, 10 women) were randomized and enrolled in the study. Subjects were primarily Caucasian (21, 84%); 3 (12%) subjects were African American, and 1 (4%) was Asian. The mean age of subjects was 33.4 ± 10.5 years. Seven subjects (28%) reported current use of tobacco.
Twenty-four subjects (96%) completed the study protocol. One subject withdrew prematurely from the study shortly after randomization because of difficulty establishing venous access and was subsequently replaced.
Bioequivalence Comparisons
Comparison of baseline CoQ10 concentrations before each dose of the CoQ10 formulation indicated that plasma concentrations were within the normal (endogenous) range. There was no evidence of carryover effects from 1 treatment period to the next as the concentrations had returned to baseline levels before each dosing. The mean baseline levels (µg/mL) prior to each treatment were 0.74 ± 0.29 (plain chewable wafers), 0.71 ± 0.27 (chewable wafers with vitamin E), 0.73 ± 0.28 (soft gel capsules), and 0.72 ± 0.27 (hard capsules).
Plasma concentration-time curves for the 4 CoQ10 preparations are shown in Figure 1. The mean AUC0-48 values (µg/mL x hours) were as follows: 57.9 ± 19.3 (plain chewable wafers), 54.4 ± 17.2 (chewable wafers with vitamin E), 56.9 ± 19.6 (soft gel capsules), and 53.3 ± 21.9 (hard capsules). The mean Cmax values (µg/mL) were as follows: 1.51 ± 0.46 (plain chewable wafers), 1.34 ± 0.41 (chewable wafers with vitamin E), 1.42 ± 0.47 (soft gel capsules), and 1.38 ± 0.57 (hard capsules). The mean tmax values (hours) were as follows: 17.4 ± 11.2 (plain chewable wafers), 20.9 ± 12.7 (chewable wafers with vitamin E), 13.3 ± 8.8 (soft gel capsules), and 14.3 ± 11.7 (hard capsules). The curves were similar for the 4 formulations; the plain chewable wafer and soft gel capsule formulations appeared to have slightly better bioavailability overall. At 48 hours, the plasma concentrations for the 4 formulations were similar and remained well above baseline levels (Figure 1).
|
Formal comparisons among the formulations regarding AUC0-48 revealed that the 98.3% confidence intervals for the geometric mean AUC ratios always were contained within the standard limits of 0.80 and 1.25 for each pairwise comparison except for one; the hard capsules versus plain chewable wafers comparison included 0.80 in the 98.3% confidence interval (Table I).
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Formal comparisons with respect to Cmax showed higher values for the plain chewable wafer formulation compared to the chewable wafer with vitamin E and hard capsule formulations, but the geometric mean ratios were not statistically significant (Table I).
For tmax, the chewable wafer with vitamin E formulation had a higher geometric mean than the hard capsule (P = .008) and soft gel capsule (P = .007) formulations (Table I). The plain chewable wafer formulation similarly had a higher geometric mean tmax than the hard capsule and soft gel capsule formulations, but these comparisons did not reach statistical significance (Table I).
The bioavailability of CoQ10 was compared in men and women separately for each formulation using log-transformed AUC0-48, with mean ± standard deviation values as follows: plain chewable wafer, 4.19 ± 0.32 (women) vs 3.90 ± 0.32 (men), P = .03; chewable wafer with vitamin E, 4.07 ± 0.25 (women) vs 3.88 ± 0.35 (men), P = .16; soft gel capsules, 4.08 ± 0.31 (women) vs 3.91 ± 0.43 (men), P = .29; hard capsules, 4.03 ± 0.34 (women) vs 3.80 ± 0.49 (men), P = .23. Thus, although the bioavailability of CoQ10 (in terms of log-transformed AUC0-48) appeared to be slightly higher in women, the differences were generally not statistically significant.
Safety and Tolerability
No serious or severe adverse events occurred during the conduct of the trial. Nine mild adverse events were documented, 8 thought to be unrelated (1 fall, 1 urinary tract infection, 2 occurrences of back pain, 1 sinusitis, 2 headaches, 1 migraine) and 1 unlikely related (pruritus) to the study medication. Subjects recovered without any action being taken. In particular, no gastrointestinal adverse events were observed. No significant changes occurred throughout the trial with regard to vital signs with any of the CoQ10 formulations.
DISCUSSION
Given the interest in studying the effects of CoQ10 in a variety of diseases, information on the bioavailability of competing proprietary formulations can be of value in selecting a formulation for use in a clinical trial. We found no important differences among the 4 oral CoQ10 preparations that we studied with respect to bioavailability of a single 600-mg dose in healthy adults, although we were not able to establish the bioequivalence of the plain chewable wafer and hard capsule formulations.
Although brain CoQ10 levels have been shown to increase in rodents after oral supplementation,18 the bioavailability of CoQ10 in the human brain has not been sufficiently studied. Consequently, our results pertain to the bioavailability of CoQ10 in plasma and cannot be extrapolated to the bioavailability of CoQ10 in the brain.
In this study, a "2-peak pattern" was seen for the CoQ10 plasma concentration curves, with the first peak at approximately 6 hours after baseline, followed by a second peak about 18 hours later. This phenomenon has been described previously and is thought to be caused by redistribution and enterohepatic recycling of CoQ10.21,22 Because of this pattern, it is somewhat difficult to interpret the results of the comparisons of tmax among the different CoQ10 formulations.
One previous study reported a higher AUC in men than in women after a single oral dose of CoQ10.22 Our findings did not support these results, and the bioavailability of CoQ10 tended to be higher in women, although the gender differences were not statistically significant. Our sample sizes were small, however, and true gender differences in either direction cannot be ruled out by our study.
The 4 CoQ10 formulations were given sequentially, with 2-week washout periods between doses. This proved to be sufficient to eliminate any potential carryover effects; the predose CoQ10 levels were basically unchanged throughout the trial.
These results suggest that formulation (chewable wafer, hard capsule, soft capsule) does not have an important impact on bioavailability, although we were not able to establish the bioequivalence of the plain chewable wafer and hard capsule formulations. Even though some commercial preparations include a lipid vehicle, such as vitamin E, in an attempt to improve the bioavailability of CoQ10, our findings do not suggest any important impact of vitamin E on the bioavailability of CoQ10. Hence, assuming no changes in formulation by manufacturers, all these formulations are appropriate for further research, and choice of formulation can be driven by factors such as ease of subject use, availability, and cost.
APPENDIX
Steering Committee: Karl Kieburtz, MD, MPH, principal investigator; Michael P. McDermott, PhD, biostatistician; Ira Shoulson, MD; Robert Ferrante, MS, PhD; Bernard Ravina, MD, MSCE; H. Christopher Hyson, MD (medical monitor, ex-officio); Elisabeth A. de Blieck, MPA, CCRC (project manager, ex-officio).
Consultant: Robert DiCenzo, PharmD
Site Personnel (Investigator/Coordinator, Staff, Pharmacy): Edward Bednarczyk, PharmD, site investigator; Ellana Eberhardt, RN, study coordinator; Jane Mastantuono, RN; Curtis E. Haas, PharmD; Thomas Kufel, MD
CoQ10 Research Lab: M. Flint Beal, MD; Mikhail Bogdanov, MD, PhD; Susan E. Browne, PhD; Beverly J. Lorenzo, BS; Linda J. Metakis, BA; and staff of Weill Medical College/Cornell University
Clinical Trials Coordination Center, University of Rochester, New York: Alicia Brocht, BA, lead data-base manager; Susan Daigneault, data control clerk; Elisabeth A. de Blieck, MPA, CCRC, project manager; Michelle Goldstein, MS, information analyst; Karen Hodgeman, CCRA, information analyst; H. Christopher Hyson, MD, FRCPC, medical monitor; Karl Kieburtz, MD, MPH, principal investigator and director, Clinical Trials Coordination Center
Biostatistics Center, University of Rochester, New York: Michael P. McDermott, PhD, biostatistician; Arthur Watts, BS, programmer
ACKNOWLEDGEMENTS
The authors acknowledge the Western New York Veterans Administration Medical Center Clinical Research Center (CRC). The authors thank the research participants for their participation in this study.
Financial disclosure: The authors thank the High Q Foundation for financial support for the study. They appreciate the in-kind donation of coenzyme Q10 from the following companies: Enzymatic Therapy, Health Wright Products, and Tishcon Corporation.
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