Abstract
Ubiquinol is the two-electron reduction product of ubiquinone (coenzyme Q10 or CoQ10) and functions as an antioxidant in both mitochondria and lipid membranes. In humans and most mammals, including dogs, the predominant form of coenzyme Q is coenzyme Q
The term coenzyme Q (CoQ) refers to a class of homologous benzoquinones that have been identified in all plants and animals, as well as in a majority of microorganisms (Budavari et al. 1996; Nohl, Gille, and Staniek 1998). Benzoquinone homologs consist of a redox active quinoid moiety, and a hydrophobic side chain comprising 6 to 10 isoprenoid units, depending on the species (Ibrahim et al. 2000; Matthews et al. 1998; Lenaz 2001). In humans and most mammals, including dogs, the predominant form of CoQ is coenzyme Q10 (CoQ10). CoQ10, which is also referred to as ubiquinone, consists of 10 isoprenoid units in the side chain (Ramasarma 1985). In rats and mice, the primary form is coenzyme Q9 (CoQ9), which contains 9 isoprenoid units. Low levels of CoQ10 have also been reported in rats and mice (Battino et al. 1992).
CoQ10 is located in the hydrophobic interior of nearly every cellular membrane, and to varying degrees in all tissues (Lass and Sohal 1999; Nohl, Gille, and Staniek 1998). Since its discovery in 1957 by Crane and colleagues (Bertelli and Ronca 1990), CoQ10 has been extensively studied for its key role in mitochondrial energy production, where it acts as both an electron carrier and proton translocator during cellular respiration and adenosine triphosphate (ATP) production (Hughes et al. 2002; Nohl et al. 2001; Nohl, Gille, and Staniek 1998).
Ubiquinol (the reduced form of CoQ10) is the two-electron reduction product of ubiquinone (the oxidized form of CoQ10) (Frei, Kim, and Ames 1990; Schoepp 1997; Pepping 1999). Like ubiquinone, ubiquinol has been shown to be an integral part of virtually all living cells, where it functions as an antioxidant in both mitochondria and lipid membranes in a number of biological and model systems (Forsmark-Andree et al. 1997; Noack, Kube, and Augustin 1994; Frei, Kim, and Ames 1990; Ernster and Forsmark-Andree 1993).
Ubiquinol is the only known lipid-soluble antioxidant that animals can synthesize de novo, and for which there exist mechanisms that can regenerate it from ubiquinone as a result of its antioxidant activity (Ernster and Dallner 1995). Although ubiquinol becomes oxidized as a result of its antioxidant function, a substantial amount of ubiquinol is maintained in its reduced state in the plasma membrane and endomembranes (Takahashi et al. 1993), as well as in lipoproteins (Stocker and Frei 1991).
The conversion between ubiquinone, the oxidized form used in energy production, and ubiquinol, the reduced form used as a lipid soluble antioxidant, is shown in Figure 1. It has been suggested that reduction of ubiquinone to ubiquinol by blood cells and the liver plays an important role in maintaining plasma concentrations (Stocker and Suarna 1993). Various electron transfer systems recycle ubiquinol from its oxidized form (Crane, Sun, and Sun 1993). In the plasma membrane, reduction of ubiquinone is achieved through the involvement of several CoQ reductases (e.g., DT-diaphorase and NADPH-CoQ reductase) that may be either integral membrane proteins or cytosolic enzymes (Arroyo et al. 2000). Stocker and Suarna (1993) also reported that natural ubiquinones are readily reduced after dietary uptake. Although it is generally accepted that oxidized CoQ10 is the final product of its biosynthetic pathway, some authors (Stocker and Suarna 1993; Schultz et al. 1996) have proposed that the de novo synthesis of the hydroquinone also contributes, at least in part, to the high levels of ubiquinol observed in vivo.
The reduction product ubiquinol is the most common form of CoQ10 in vivo (Frei, Kim, and Ames 1990). Ubiquinol represents more than 80% of the total ubiquinol + ubiquinone pool in human plasma, intestine, and liver (Edlund 1988; Okamoto et al. 1989; Åberg et al. 1992). In the plasma of healthy adults, ubiquinol accounts for approximately 95% of the total CoQ10 concentration, whereas ubiquinone accounts for only 5% (Yamashita and Yamamoto 1997); in human urine, ubiquinol accounted for approximately 59% of the total concentration (Okamoto et al. 1989). Åberg et al. (1992) also reported that high levels of the reduced form of CoQ10 (70% to 100%) were also observed in human tissues, including the liver, pancreas, and intestine. Only in the brain and lung was the majority (approximately 80%) in the oxidized CoQ10 state.
As noted previously, the primary form of CoQ in humans and dogs is CoQ10, whereasin rats and mice, the primary form is CoQ9. Takahashi et al. (1993) examined the concentrations of oxidized and reduced homologues in rat tissues and subcellular fractions to clarify their distribution and physiological role. Ubiquinol was present at higher concentrations than CoQ10 in plasma liver, heart, kidney, and spleen. A second difference was also seen in the degree of oxidized forms converted to reduced forms, as the degree of reduction in all rat tissues is less than in corresponding human tissues.
Similarly, CoQ9 and CoQ10 were detected in all blood cells isolated in rats (i.e., erythrocytes, ghost cells, endosomes, leukocytes, and platelets). Leukocytes and platelets, which have mitochondria, possessed higher concentrations of total CoQ9 and total CoQ10 than did erythrocytes, which do not have mitochondria. The reduced forms were below 10% of the total CoQ in erythrocytes and leukocytes, and they were not detected in platelets. With respect to subcellular distribution of oxidized and reduced CoQ homologues in rat liver and kidney, all fractions tested (i.e., nuclei, mitochondria, crude lysosomes, crude microsomes, cytosol, and plasma membranes) contained significant amounts of total CoQ homologues. The levels of the reduced CoQ forms reached 60% to 70% of those of the total CoQ homologues in a majority of subcellular fractions of the liver, and accounted for approximately 25% of those in kidney. Based on these findings, Takahashi et al. (1993) concluded that all rat tissues and subcellular fractions isolated from the liver and kidney contain significant amount of CoQ homologues. In addition, the authors noted that 70% to 80% of the total amounts of each homologue (i.e., CoQ9 and CoQ10) in the liver and plasma, as well as 20% to 30% of those in other tissues, exist as the reduced hydroquinone form.
Several authors have examined the relative concentrations of CoQ9 and CoQ10, as well as the ratio of reduced forms to oxidized forms, in mice (Podda et al. 1996; Tang et al. 2004).
Tissues involved in detoxification, such as the liver and kidney, were shown to have high levels of reduced forms, which may, as Podda et al. (1996) and Tang et al. (2004) suggested, protect them from radicals escaping the cytochrome P450 enzyme system.
In addition to de novo synthesis and regeneration, ubiquinol and ubiquinone are present in various food items including meats, fish, fruits, and vegetables (Kamei et al. 1986; Weber, Bysted, and Hølmer 1996; Mattila and Kumpulainen 2001; Passi et al. 2002; Kubo et al. 2008). Ubiquinone and ubiquinol are also available as dietary supplements. Ubiquinol has now been proposed for use as a new dietary supplement. Accordingly, as part of the process for assessing the safety of ubiquinol, a series of toxicological studies were conducted.
As previously noted, in humans and most mammals, including dogs, the predominant form of CoQ is CoQ10, whereas the primary form in rodents is CoQ9, which contains 9 isoprenoid units. Therefore, in order to assess the safety of ubiquinol more extensively, including investigation of any gender and species differences in toxicity, 13-week oral toxicity studies were conducted in rats and dogs. Results of these studies are reported here for the first time.
MATERIALS AND METHODS
Test Material
Kaneka produces ubiguinol via reduction of its CoQ10 normal fermentation product using food-grade materials.
Rat studies: The test material, ubiquinol (Kaneka QH; lot no. QH-P01, purity 98.71%), and reference material, ubiquinone (Kaneka Q10; lot no. U0009, purity 99.2%, for the initial study; lot no. S288, purity 99.8%, for the second study), were manufactured by Kaneka Corporation.
Dog study: The test material, ubiquinol (Kaneka QH; lot no. QH-S08, purity 97.77%), and the reference-control article (Kaneka Q10; lot no. S244, purity 99.3%) were manufactured by Kaneka Corporation.
For each dose concentration, requisite amount of ubiquinol or ubiquinone was weighed and dissolved in corn oil vehicle (Nacali Tesque) warmed to approximately 60°C.
In the initial rat study, ubiquinol test solutions were prepared at concentrations of 60, 120, and 240 mg/ml, whereas in the follow-up study, ubiquinol test solutions with concentrations of 15, 30, 40, and 60 mg/ml were prepared. In both studies, the concentration of the ubiquinone test solution was 240 mg/ml. For dogs, ubiquinol test solutions were prepared at concentrations of 100, 200, and 400 mg/ml and a ubiquinone solution at a concentration of 400 mg/ml. Ubiquinol solutions were kept in a freezer set at − 20°C, whereas ubiquinone solutions were kept in a refrigerator at 4°C. At the time of administration, the solution was taken out of the freezer or refrigerator, and thawed by warming to approximately 60°C. For the administration to dogs, the requisite amount was put into ounce gelatine capsules (Torpac, NJ, USA) and the amount of dosing solution to be filled in a capsule was approximately 8 ml. The preparation of dosing solutions was done once or twice a week, and solutions were used within 7 days of preparation.
All test material solutions were analyzed to confirm test article stability and concentration using standard sampling and analytical techniques.
Study Organization
The studies were performed at Bozo Research Center (3–11, Hanegi 1-chome, Setagayaku, Tokyo 156-0042, Japan). With the exception of the determination of total CoQ10 in liver in the initial rat study, the studies were conducted in accordance with relevant Good Laboratory Practice (GLP) Standards (The Ordinance on Standard for Conduct of Non-Clinical Studies on Safety of Drugs, Ordinance No. 21 of the Ministry of Health and Welfare, Japan, March 26, 1997) and Toxicity Study and Animal Welfare Guidelines (Law Concerning the Protection of Animals, Law. No. 105, October 1, 1973, Revised on December 22, 1999), and in compliance with “Guidelines for Toxicity Studies Required for Applications for Approval to Manufacture (Import) Drugs” (Notification I, Article No. 24 of the Pharmaceutical Affairs Bureau, Japanese Ministry of Health and Welfare, September 11, 1989).
Animals and Maintenance
Rats
For the initial 13-week study, male and female Sprague-Dawley strain SPF [Crj:CD(SD)IGS] rats were received from Atsugi Breeding Center, Charles River Japan, at the age of 4 weeks. Male and female animals were acclimated for 14 and 15 days, respectively. Seventy-seven animals of each sex (50 animals of each sex for the main group and 27 animals of each sex for the toxicokinetic satellite groups for total CoQ10 concentration in plasma) showing normal body weight gain without abnormalities in ophthalmological examination and general condition were selected and subjected to the study at 6 weeks of age. In the follow-up study, female Sprague-Dawley strain SPF [Crj:CD(SD)IGS] rats were obtained from Atsugi Breeding Center, Charles River Japan, at the age of 5 weeks. The animals were quarantined/acclimated for 9 days, and 78 animals (60 animals for the main groups, and 18 animals for the toxicokinetic satellites group for total CoQ10 concentration in plasma) with normal body weight gain and without abnormalities in general condition or ophthalmological examination were selected and subjected to the study at 6 weeks of age.
After the selection of animals based on their body weight gain during the quarantine/acclimation period, animals in each study were stratified by body weight on the day of grouping (2 days before the start of administration) and assigned to groups so that group mean body weight was comparable among groups. Animals were assigned to groups by a combination of the block placement method and random sampling method using a computer (requisite number of groups was composed by the block placement method and study groups and individual animal numbers were assigned at random). Animals remaining after grouping were subjected to collection of blank plasma for CoQ10 concentration in plasma.
Rats were housed individually in bracket-type metallic wire-mesh cages (width 190 × depth 350 × height 170 mm; Lead Engineering) in animal rooms which were set to maintain the temperature at 23°C ±3°C, relative humidity at 50% ±20%, air ventilation at 10 to 15 times per hour, and artificial lighting (on/off for 12 h a day). The animals were allowed free access to pellet diet CRF-1 (Oriental Yeast). Drinking water (Gotemba City Water) was supplied ad libitum via water bottles.
Dogs
Seventeen beagle dogs (HRA Beagle; Covance Research Products, VA, USA) of each sex were purchased at 5 months of age. After 2 weeks of quarantine and 2 weeks of acclimation, 15 healthy male and 15 healthy female animals were selected on the day before the start of administration. Body weights of animals at the start of administration ranged from 7.7 to 9.6 kg in males and from 6.8 to 8.7 kg in females.
Dogs were housed individually in bracket-type metallic wire-mesh cages (Shinetsu Wiremesh) in an animal room which was set to maintain the temperature at 22°C ±4°C, relative humidity at 55% ±25%, the air ventilation at 13 to 15 times per hour, and artificial lighting for 12 h a day. The animals were given 300 g/day of DS-A pellet diet for dogs (Oriental Yeast) between 09:20 and 14:10 every day (within 5 min after dosing during the administration period) and residual food was removed between 08:03 and 09:33 in the next morning. Animals were allowed free access to tap water (Fujimi Water Union, via an automatic water supply system).
Administration of Test Material
Rats
In the initial rat study, male and female rats were treated with the test material at 300, 600, or 1200 mg/kg/day via oral gavage, because the oral route is the expected route of human consumption; treatment volume was 5 ml/kg body weight (bw). Dose levels were based on a previous 52-week toxicity study of ubiquinone in rats, where a no observed adverse effect level (NOAEL) of 1200 mg/kg/day was identified (Williams et al. 1999). Animals in the negative-control group received vehicle (corn oil) alone, whereas those in the reference-control group received ubiquinone at 1200 mg/kg.
In the second rat study, female rats only were treated with the test material at 75, 150, 200, or 300 mg/kg/day via oral gavage; treatment volume was 5 ml/kg bw. Animals in the negative-control group received vehicle (corn oil) alone, whereas those in the reference-control group received ubiquinone at 1200 mg/kg.
Dogs
Dose levels for the dog study were selected based on the results of the 13-week oral toxicity study in rats that is reported here and a 2-week preliminary toxicity study in dogs (data not published). The concentration of the test article in the plasma at 300 mg/kg during the 2-week study in dogs was approximately 1.5 to 3 times higher than the concentration obtained at the highest dose level of 1200 mg/kg in the 13-week rat study. In the dog study, in order to ensure blood concentration comparable with that obtained at 1200 mg/kg in rats and considering individual variations, the high-dose level was set at 600 mg/kg and 300 and 150 mg/kg were selected as the mid- and low-dose levels, respectively, using a common ratio of 2. The dose level of ubiquinone was set at 600 mg/kg, the same dose level as for the high level of ubiquinol.
Male and female beagle dogs were treated with the test material at 150, 300, and 600 mg/kg/day via oral gavage, because the oral route is the expected route of human consumption. Dose volume was set at 1.5 ml/kg bw and requisite amount of the dosing solution was put in gelatine capsules immediately before dosing and administered to all dogs orally by gavage. Animals in the negative-control group received vehicle (corn oil) alone, whereas those in the reference-control group received ubiquinone at 600 mg/kg.
Dose groups and their respective treatments are summarized in Table 1. All animals were treated 7 times per week for 13 weeks.
Methods of Observation
The following methods of observation, measurement, and examination were used in the acclimation period and during the initial 13-week study in male and female rats and in the subsequent follow-up study in females and the 13-week study period in dogs.
Clinical Observations, Body Weight, and Ophthalmoscopy
Rats
All animals were examined daily for general behavior, signs of toxicity and mortality during the administration period. Body weights were measured three times during week 1 of administration (days 1, 4, and 7) and twice a week every 3 or 4 days thereafter. At the scheduled sacrifice, final body weights were also determined following overnight (ca. 16 h) fast. Ophthalmological examinations were performed during the quarantine/acclimation period and in week 13 (between days 85 and 89) of administration. Only 6 of 10 animals per group were examined during week 13 of administration in the follow-up study. The anterior portion of the eyeballs, intermediate optic media (cornea, aqueous chamber, lens, vitreous body, etc.), and fundus oculi were examined with an ophthalmoscope (BXα model, Neitz Insturments) following the application of Mydrin P, a mydratic agent (Santen Pharmaceutical, lot nos. MP0669 and MP0732).
Dogs
All animals were observed, palpated, and ausculated for general condition once daily during the week prior the start of administration, and at least twice daily during the administration period. Body weights were measured once a week before administration, at the start of dosing, once weekly before dose administration, and at scheduled sacrifice. On the day of necropsy, animals were weighed after fasting for at least 16 h from the previous day in order to calculate relative organ weights. Ophthalmological examinations were performed 2 weeks prior to the start of dosing and at weeks 6 and 12 of administration. Macroscopic observation of the external appearance of the eyes, such as eyelid and light reflex, were performed using a penlight. Observation of the anterior portion of the eyeballs, intermediate optic media (cornea, aqueous chamber, lens, vitreous body, etc.), and fundus oculi were examined with a binocular indirect ophthalmoscope (Omega 200; Heine OPTOTECNIK, Germany) following the application of Mydrin P, a mydratic agent (Santen Pharmaceutical).
Electrocardiography
Electrocardiography evaluations were performed on dogs only, with an initial evaluation 1 week before the start of dosing and subsequent evaluations at weeks 7 and 13 of administration. Heart rate, PR, QT, and QRS intervals, QTC, QT interval(s)/[RR interval(s)]1/2× 1000, and QRS electrical axis were measured in unanaesthetized animals using an electrocardiograph (LABO-SYSTEM ZM-5012; Fukuda ME).
Food Consumption
Rats
Cumulative food consumption was measured three times (days 1, 4, and 7) during week 1 of administration and twice a week every 3 to 4 days thereafter, and mean daily food consumption values per rat were calculated. Water was given to animals using water bottles while they were placed in cages with urine collectors, and 1-day consumption from the previous day was measured.
Dogs
For all animals from week 1 before the start of administration and throughout the 13-week administration period, 1-day food consumption was calculated each day from the difference between the amount of supplied food and the amount of residual food. Weekly mean food consumption was calculated from cumulative food consumption for 1 week.
Hematology, Clinical Chemistry, and Urinalysis
Hematological examinations were performed on rats the day of scheduled necropsy following the end of the administration period. Hematological examinations were performed on all dogs in weeks − 2 and − 1 (before the start of administration) and in weeks 7 and 13 of administration.
Rats were subjected to laparotomy under ether anesthesia after fasting for approximately 16 to 20 h from the previous day, and blood samples were drawn from the abdominal aorta under anesthesia, whereas approximately 2 ml of blood was collected via the cephalic vein of dogs after fast (approximately 18 h). K2EDTA was used as the anticoagulant for analysis of red blood cell count (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte percentage, platelet count, white blood cell count (WBC), and differential leukocyte count while sodium citrate was used for prothrombin time (PT), activation partial thromboplastin time (APTT), and fibrinogen. Hematological parameters were determined using standard methods and determined using a Coulter Counter T890 (Beckman Coulter) and Coagulomoeter ACL 100 (Instrumentation Laboratory).
Clinical chemistry assessment was performed for all animals using sera or plasma obtained by centrifugation of the blood samples collected at the same time as those for hematological examination in test tubes containing a coagulation promoting agent (Venoject II-Autosep, Terumo) or heparinized test tubes. Parameters were estimated using the Clinical Laboratory System TBA-120 FR (Toshiba) or Automatic Electrophoresis Cliniscan SA-V (Helena).
Fresh urine and cumulative urine were collected from all animals in weeks − 2 and − 1 before the start of administration and in weeks 7 and 13 of administration in dogs, whereas urinalysis was performed in week 13 of administration (between day 87 and 89) in rats. All animals were placed individually in cages equipped with a urine collector under deprivation of food but with free access to water, and 4-h urine samples were collected. Subsequently, 16-h (dog) and 20-h (rat) urine samples were collected while animals were allowed free access to food and water. Urine volume was calculated using the total values for all samples.
In rats, urine pH, protein, ketones, glucose, occult blood, bilirubin, and urobilinogen were determined from the 4-h urine using Aution Mini AM-4290 (Arkray). Color and sediment were determined via macroscopic examination of the 4-h samples. Osmolality was determined via cryoscopy (automatic osmometer; AUTO & STAT OM-6030, Arkray). Sodium and potassium were determined using an ion-selective electrode method, whereas chloride was determined via coulometric titration (Automatic Electrolyte Analyzer PVA-αII; Analytical Instruments).
Urine pH, protein, ketones, glucose, occult blood, bilirubin, and urobilinogen were determined from dog samples using mutlsitix paper test (Bayer-Medical). Osmolaltiy was determined using Osmometer Osmostat OM-6040 (Arkray), whereas sodium, potassium, and chloride were determined via the ion-selective electrode method using the Clinical Laboratory System TBA-120 FR (Toshiba)
Pathology
All animals were fasted before necropsy and sacrificed. Pathological examination was done systematically by external observation and internal examination of organs and tissues as soon as possible after sacrifice.
The absolute weights and relative weights of several organs were measured and calculated. For the paired organs, organ weight was measured separately; however, evaluation was done on the total values of the right and left organs.
For all animals, a comprehensive array of organs and tissues were removed and preserved and subjected to histopathological examination.
Determination of Total CoQ10 Concentration in Plasma
Blood was sampled from three males and/or three females per point in each group (for ubiquinol and ubiquinone groups with six satellite animals in the rat study, blood was sampled alternately from three animals of each sex per point).
Blood was sampled at 1 (initial study only), 4, 8, and 24 h after dosing on the starting day of administration and in week 13 (day 86, 90, or 91) of administration in rats. In dogs, blood samples were obtained from all animals before and at 1, 4, 8, and 24 h after dosing on the starting day of administration (day 1 of administration) and in weeks 7 and 13 of administration.
The concentrations of ubiquinol and ubiquinone in plasma were determined by high-performance liquid chromatography (HPLC). Ubiquinol in plasma was oxidized and determined as ubiquinone. Because ubiquinone is partially reduced to ubiquinol in blood, the concentration in plasma was determined as ubiquinone after oxidizing ubiquinol. As the concentrations of endogenous ubiquinol and ubiquinone in beagle dog plasma were not less than 0.1 μg/ml, the lower limit of quantitation, measured concentrations were corrected using endogenous total CoQ10 level determined in intact animals. The outline of the method of determination is shown below:
To 100 μl of test sample, 10 μl of internal standard (coenzyme Q7, 50 μl/ml in ethanol), 10 μl of ethanol, and 10 μl of 1 % (w/v) ferric chloride solution, 2 ml of methanol, and 0.5 ml of purified water were added and mixed well. Hexane (3 ml) was added and then the mixture was extracted by shaking for 5 min, centrifuged (1500 × g, 2500 rpm, set at 4°C, 5 min), and the organic layer was transferred to a pointed centrifuge tube. Extraction for 5 min, followed by centrifugation, was repeated with another 3-ml portion of hexane and the organic layer was added to the pointed centrifuge tube. It was then evaporated to dryness in a steam of nitrogen set at 40°C. The residue was dissolved in ethanol (150 μl), and then 50 μl of the solution was injected into the HPLC system with an analytical column (4.6 mm i.d. × 150 mm, 5 μm YMC-Pack ODS A302; YMC, Kyoto, Japan) and ultraviolet absorbance detection (275 nm). The mobile phase consisted of methanol/hexane (90:10, v/v) at a flow rate of 1.0 ml/min. The concentrations of total CoQ10 were obtained by comparison of the peak area ratios of CoQ10 to the internal standard for test samples and those for the standard solutions of known concentrations. The calibration curves were obtained by weighted (1/concentration) least squares linear regression analysis.
During the first rat study in males and females, reanalysis of some samples was performed to confirm the validity of the drug concentration profile in the first analysis. The values obtained in the reanalysis were comparable to the initial values.
Parameters Calculated
Measured values of individual animals were indicated together with group mean and standard deviation that were calculated for each point. In addition, the maximum concentration (C max), the time to reach the maximum concentration (T max), and the area under the concentration versus time curve (AUC0–24h) were calculated.
Determination of total CoQ10 Concentration in Liver
Rats
In the initial study, after the final blood sampling in the satellite groups, livers were removed from three rats of each sex and stored in a freezer set at − 20°C. The frozen samples were sent to Life Science Research Laboratories, Kaneka Corporation, and were analyzed under non-GLP conditions. A requisite amount of liver was weighed and subsequently homogenized in a 50-fold weight of purified water. The homogenate was diluted 40 times with purified water to prepare test samples. To 500 μl of test sample, 20 μl of internal standard (coenzyme Q7, 5 mg/ml in ethanol), 500 μl of 0.1 M sodium dodecyl sulfate, and 1% (w/v) ferric chloride solution were added and mixed well. Methanol (4 ml) and hexane (6 ml) were added and then the mixture was extracted by shaking (5 min), centrifuged (1870 × g, 4°C, 10 min), and organic layer was obtained. The remaining aqueous layer was further extracted with 6 ml of hexane in similar fashion. The hexane layers were combined and evaporated to dryness, and the residue was reconstituted in 200 μl of ethanol, and then 50 μl of the solution was injected into HPLC system with an analytical column (4.6 mm i.d. × 250 mm, 5 μm; YMC-Pack ODS-A303; YMC) and ultraviolet absorbance detection (275 nm). The mobile phase consisted of methanol/hexane (88:12, v/v) at a flow rate of 1.0 ml/min. The concentrations of total CoQ10 were obtained by comparison of the peak area ratios of CoQ10 to the internal standard for test samples and those for the standard solutions of known concentrations. The calibration curves were obtained by weighted (1/concentration) least squares linear regression analysis.
In the follow-up study, approximately 0.5 g of liver per animal was collected at necropsy from females of the main group at each dose level. The outline of the method of determination is identical to that employed for the initial study.
Dogs
At necropsy, portions of the left lateral lobe of the liver (at least 1 g per animal) were collected. A requisite amount of liver was weighed and subsequently homogenized with a 50-fold weight of purified water. The homogenate was diluted 40 times with purified water to prepare test samples. The homogenate and test samples were stored in a freezer seat at − 20°C until use. The concentrations of ubiquinol and ubiquinone in the liver were determined by HPLC. Ubiquinol in the liver was oxidized and determined as ubiquinone. Because ubiquinone is partially reduced to ubiquinol in the liver, the concentration in plasma was determined as ubiquinone after oxidizing ubiquinol. As endogenous ubiquinol and ubiquinone exist in the dog liver, measured concentrations were corrected using endogenous total CoQ10 level determined in intact animals.
The outline of the method of determination is identical to that employed for determination of ubiquinol and ubiquinone concentration in plasma with the following exceptions: To test sample (250 μl), internal standard solution (10 μl), ethanol (50 μl), and 0.1 mol/L sodium dodecylsulfate (250 μl) were added and mixed for 10 s. Then 1 w/v% ferric chloride solution (10 μl) and ethanol (2 ml) were added to the mixture and mixed for 10 s. Following the addition of 3 ml of hexane, the mixture was extracted for shaking for 5 min and centrifuged at 1500 ×g, 2500 rpm, and 4°C, for 10 min. Pretreatment of the samples then continued as described above for plasma. An analytical column of YMC-Pack ODS-A303 (4.6 mm i.d. × 150 mm, 5 μm; YMC) and mobile phase consisted of methanol/hexane (88/12, v/v) were used.
Statistical Analysis
Numerical data (body weight, food consumption, data obtained in electrocardiograms [except electrical axis], urinalysis [urine volume, osmolaltiy, and urine electrolytes], hematology, blood chemistry, and organ weight) were subjected to calculation of mean with standard deviation, and analysis for homogeneity of variance in each group by Bartlett’s test (level of significance: 1%, two-tailed). For homogeneous data, the group mean differences between the control group and each treated group were analyzed by Dunnett’s test (levels of significance: 5% and 1%, two-tailed). For heterogeneous data, the mean rank differences between the control and each treated group were analyzed by a Dunnett-type mean rank test (levels of significance: 5% and 1%, two-tailed).
RESULTS
To simplify the presentation of tabulated data, results of the initial and follow-up studies in rats will be combined and referred to as trials 1 and 2, respectively.
All test material solutions were analyzed to confirm test article stability and concentration using standard sampling and analytical techniques.
Clinical Observations
Rats
No deaths were observed in either the control and treated groups during either trial 1 or 2. Likewise, no significant changes related to the test material were observed in the general condition of the animals. No abnormal findings were observed upon ophthalmic examinations.
Dogs
No deaths were observed in either sex of control and treated groups during the course of the experiment. Fecal abnormalities and vomiting were observed throughout the administration period. Incidences are indicated in Table 2.
In the ubiquinol groups, soft or mucous feces were observed during the administration period 7 times in one of three males in the 150 mg/kg group, once each in two of three males and twice in one of three females in the 300 mg/kg group, and 1 or 33 times in all males and one or two times in all females in the 600 mg/kg group. The incidence was high in one of three males in the 600 mg/kg group. In the ubiquinone group, soft feces were observed during the administration period 3 times each in two of three males and 3 or 29 times in two of three females. Soft, mucous, or watery feces were also observed 10 times in one of three males and once in one of three females in the control group.
Feces containing test article–like or reference-control article–like material were observed throughout the administration period in the ubiquinol groups and ubiquinone groups. They were observed 1 or 5 times in two of three males in the ubiquinol 150 mg/kg group, whereas they were observed frequently throughout the administration period in all males and females in the ubiquinol 300 and 600 mg/kg groups and ubiquinone group.
In the ubiquinol groups, vomiting of foamy fluid or ingesta was observed at all dose levels and vomitus containing test article–like material was observed sporadically in the ubiquinol 300 and 600 mg/kg dose groups. The incidence of vomiting was 3 or 5 times in two of three females in the 150 mg/kg group, 2 or 4 times in the in two of three males and 3 or 12 times in two of three females in the 300 mg/kg group, and 2 or 3 times in two of three males and 3 or 7 times in all females in the 600 mg/kg group. Vomiting of foamy fluid or ingesta was also observed in the control group 1 or 4 times in one of three males and two of three females. The incidence had wide inter-individual variation and was not dose dependent. Therefore, these changes were judged to be unrelated to treatment with ubiquinol. In addition, there was no correlation between incidence of vomiting and plasma total CoQ10 concentration based on individual data.
In the ubiquinone group, vomiting of foamy fluid or ingesta was observed throughout the administration period once in one of three males and 1 or 9 times in two of three females, and vomitus containing reference-control article-like substance was observed once in one of three females.
Estrus hemorrhage was observed in one of three females each in the control group, ubiquinol 300 mg/kg group, and ubiquinone group from weeks 9 or 11 of administration. In one of three females in the control group, no feces were observed from the previous day (in week 4 of administration).
Body Weight and Food Consumption
Rats
No significant changes related to the test material were observed in the body weight (data not shown).
In trial 1, statistically significant higher food consumption values were observed in females in the 600 mg/kg/day ubiquinol group on day 31 of administration, in males in the 600 mg/kg/day ubiquinol group on day 91 of administration, and in females in the ubiquinone group on days 4 and 31 of administration. A significantly higher food consumption value was also observed in females in the ubiquinol 1200 mg/kg group on day 1 of administration, but this change occurred before the start of administration. Food consumption was not affected in trial 2. These slight differences were not considered to be a toxic response. There were no significant differences in water consumption between the control group and any ubiquinol or ubiquinone group in either trial (data not shown).
Dogs
No remarkable changes in body weight were observed during the administration period in the ubiquinol or ubiquinone groups. Likewise, no changes in food consumption were seen.
Ophthalmology (Dogs Only)
In the ubiquinol groups, focal corneal opacity, opacity of the anterior or posterior lens capsule, persistent hyaloid artery, and retinal fold were observed in males and females at all dose levels in week 2 before the start of administration. These changes were also observed in weeks 6 and 12 of administration. In the ubiquinone group, opacity of the posterior lens capsule was observed in one of three females in week 2 before the start of administration and weeks 6 and 12 of administration at similar severity. These changes are often observed spontaneously in experimental beagle dogs, and opacity of the posterior lens capsule was also observed in one of three males and two of three females in the control group. Therefore, these changes were judged to be unrelated to treatment with ubiquinol.
Hematology
Rats
Select hematology data are shown in Table 3. Statistically significant prolongations in PT and APTT were observed in males of the ubiquinol 1200 mg/kg group and the ubiquinone group, although they were slight changes that were within in-house historical control data (PT: 15.1 ±2.3, APTT: 19.6 ±1.9).
A significantly low value for the proportion of stab neutrophils in differential leukocyte was observed in females of the ubiquinol 1200 mg/kg group in trial 1; however, it was judged to be of no toxicological significance since it was not associated with inflammation. There were no other significant differences in any hematological parameter between the control and ubiquinol or ubiquinone groups.
Dogs
In the ubiquinol groups, a significantly low value in the proportion of eosinophils (p < .01) was observed in males in the 150 and 600 mg/kg groups, and a significantly low value in platelet count (p < .05) in females in the 300 mg/kg group in week 13 of administration. However, these changes were judged not to be test article related because these changes were not dose dependent, and values in individual animals were comparable to their preadministration values, or were within the range of background data of the test facility. In the ubiquinone group, a significantly high value in the proportion of band neutrophils (p < .05) was observed in males in week 7 of administration, but such a change was not observed in week 13 of administration. Otherwise, there were no remarkable changes in any animal in any group (data not shown).
Blood Chemistry
Rats
Select blood chemistry results are shown in Table 4. In trial 1, elevated levels of aspartate aminotransferase (AST [GOT]) activity in females of the ubiquinol 300 and 1200 mg/kg groups, elevated values of alanine aminotransferase (ALT [GPT]) activity in females in the ubiquinol 300 mg/kg and above groups, and elevated values in lactate dehydrogenase (LDH) activity in females of the ubiquinol 300 and 600 mg/kg groups were observed. Such changes suggest adverse liver effects. Although such changes may suggest potential adverse effects on the liver, the changes observed in these trials were slight (approximately twofold) and may have been in normal physiological response to ubiquinol loading. Significantly lower values of A/G ratio in females of the ubiquinol 300 and 1200 mg/kg groups, and a significantly higher value in the proportion of β-globulin in protein fractions in females in the ubiquinol 1200 mg/kg group were observed. In addition, a significantly higher value in the proportion of γ-globulin in males in the ubiquinol 300 mg/kg group was observed. Because there were no abnormalities in total protein or albumin, however, these later effects were judged to be of no toxicological significance.
In trial 2, a significantly high value in AST (GOT) activity was observed in females of the ubiquinol 300 mg/kg group, suggesting effects on the liver, a significantly lower value in total protein was seen in the ubiquinol 200 and 300 mg/kg groups and ubiquinone group, and a significantly lower value in albumin was seen in the ubiquinol groups at the 150 mg/kg level and above and in the ubiquinone group. In addition, a significantly lower value in A/G ratio was observed in the ubiquinol 200 mg/kg group and the ubiquinone group, a significantly lower value in the proportion of albumin in protein fractions was seen in the ubiquinol 200 mg/kg group and ubiquinone group, a significantly higher value in the proportion of β-globulin was seen in the ubiquinol 200 mg/kg and above group and the ubiquinone groups, and a significantly higher value in the proportion of γ-globulin was seen in the ubiquinone group. These changes were all believed to be superficial variation due to decreased production of albumin. However, they were thought to be incidental variation judging from results of both trials 1 and 2 because the degree of these changes in individual rats were not dose related, similar levels were observed in the vehicle control group, and they were not observed in trial 1.
Dogs
In the ubiquinol groups, a statistically significant low value in potassium (p < .05) was observed in females in the 150 and 300 mg/kg groups in week 7 of administration, and a significantly low value in total protein (p < .05) in females were observed in the 150 mg/kg group in weeks 7 and 13 of administration. However, values in individual animals were comparable to their pre-administration values and within the background data of the test facility, and they were not dose dependent. Therefore these changes were considered not to be test article related. A statistically significant low value in A/G ratio (p < .05, .01) was observed in males in the 600 mg/kg group in weeks 7 and 13 of administration. However, neither change was judged not to be test article related because a similar low value (p < .05) was observed in week 2 before the start of administration and it was within the range of background data of the test facility. High values in AST, ALT, and LDH were observed in one of three females in the 300 mg/kg group in week 13 of administration, and ALT was significantly higher (p < .05) in this group than in the control group. However, they were judged to be incidental because there was no dose-relationship and pathological examination revealed no related changes. In the ubiquinone group, a low value in organic phosphorus was observed in females in week 13 of administration, but such as change was not observed in males. There were no other remarkable changes in any animal in any group (data not shown).
Urinalysis
Rats
There were no abnormalities in qualitative items or urinary sediment in males or females in the control group, ubiquinol groups, or ubiquinone groups in either trial 1 or 2. Likewise, there were no significant differences in urine volume, osmolality, or electrolytes in urine between the control group and any ubiquinol or ubiquinone group (data not shown).
Dogs
In the ubiquinol group, no remarkable changes were observed in qualitative or quantitative examination in any animal in any group. In the ubiquinone group, qualitative examination revealed marked severe urine occult blood reaction, moderate urine protein reaction, and moderate occurrence of red blood cells in sediment in one of three females in week 13 of administration. Because estrus hemorrhage was observed in this animal around the same time, these changes were judged to be related to estrus. Quantitative examination revealed a high value in potassium (p< .05) in females in week 7 of administration, but it was comparable to the pre-administration value (data not shown).
Electrocardiography (Dogs Only)
As shown in Tables 5 and 6, low values in heart rate (lower than in week − 1 of administration by 25% or more) were observed in males and females in week 13 of administration in the ubiquinol groups. Similar changes were observed in one of three females each in the 300 and 600 mg/kg groups in week 7 of administration. The degree of the decrease in heart rate was not dose dependent. Prolongation of the PR interval (first-degree atrioventricular block) was observed in one of three females in the 300 mg/kg group in week 13 of administration (PR value: 151 ms). No such effects were seen in the other two animals at this dose level or in animals in the 150 or 600 mg/kg dose groups. In the ubiquinone group, similar low values in heart rate were observed in one of three males and two of three females in week 7 or 13 of administration. Otherwise, there were no remarkable changes such as waveform abnormalities in any animal in any group.
Gross Pathological Findings
Rats
In trial 1, changes thought to be attributable to the administration of ubiquinol and ubiquinone were observed in the lung. A yellow focus was observed in one female each in the ubiquinol 300, 600, and 1200 mg/kg groups, and two males and three females in the ubiquinone group. A yellow focus of lung was thought to be test article aspirated into lung during administration and not to be toxic change as described in histopathological finding. A cyst was observed in the right kidney of one male in the ubiquinone group and dilation of the right renal pelvis was observed in one control female. A white focus in the limiting ridge of the stomach was observed in one female each in the control and the ubiquinone group. Changes to the kidney and stomach were judged to be unrelated to the administration of ubiquinone based on their incidence.
In trial 2, a yellow focus of the lung was observed in one female in the 150 mg/kg ubiquinol group, and two females each in the ubiquinol 300 mg/kg group and ubiquinone group, respectively.
Dogs
In the ubiquinol groups, yellow discoloration of the liver was observed in one of three males in the 600 mg/kg group. Otherwise, a dark red focus (mucosal, one, 20 × 5 mm) was observed in the duodenum of one of three females in the 150 mg/kg group; however, it was observed only in one animal in the low-dose group and was judged to be unrelated to treatment with ubiquinol. In the ubiquinone group, enlargement of the liver was observed in one of three males and one of three females, and a dark red focus (one, 3 × 2 mm) of the heart was observed in one of three males.
Organ Weight
Rats
Select absolute and relative organ weight data for males and females are summarized in Table 7. Changes thought to be attributable to the administration of ubiquinol and ubiquinone were observed in the liver and spleen. A tendency toward high value in the absolute liver weight and a significantly high value or a tendency toward high value in the relative liver weight were observed in females in all ubiquinol groups of trial 1. A significantly low relative liver weight was observed in males in the ubiquinol 300 mg/kg group and the ubiquinone group. Significantly high absolute and relative spleen weights were observed in females in the 600 and 1200 mg/kg ubiquinol groups. In the follow-up study, significant increases in relative salivary gland and adrenal gland weight was observed in the 75 mg/kg ubiquinol group. Absolute ovary weight was significantly reduced in the 300 mg/kg ubiquinol group. Absolute and relative uterus weights were significantly reduced in the 150 mg/kg group, whereas the absolute weight of the uterus was also significantly reduced in the ubiquinone group. However, all changes of organ weight were judged to be unrelated to the administration of ubiquinol because there was no dose response in these changes and no histopathological changes in these tissues.
Dogs
In the ubiquinol groups, significantly lower values in the absolute and relative weights of the thymus (p < .05, .01) were observed in females at all dose levels, a significantly higher value in the relative weight of the thymus (p < .05) in males in the 300 mg/kg group, a significantly higher value in the absolute weight of the thyroid (left) (p < .05, .01) in females in the 300 and 600 mg/kg groups, and a significantly higher value in the relative weight (p < .05) in females in the 600 mg/kg group, but they were unrelated to dose, unilateral change and observed only in either sex. Furthermore, no histopathological changes related to the increase of thyroid weight was observed in females. In fact, C-cell hyperplasia in the thyroid was observed in only one female in the 600 mg/kg group, the same incidence as in control group. Therefore, these changes in organ weights were judged to be unrelated to treatment with ubiquinol.
In the ubiquinone group, significantly lower values in the absolute and relative weights of the thymus (p< .05, .01), and significantly higher values in the absolute weights of thyroid (left) (p < .01) were observed in females, but they were observed only in females. A significantly higher value in the absolute weight of the liver was observed in one of three males and one of three females, but no changes were observed in the relative weight.
High values in the weights of the ovary and uterus were observed in one of three females each in the control group, ubiquinol 300 mg/kg group, and ubiquinone group, but they were judged to be related to estrus (data not shown).
Histopathological Findings
Rats
A summary of the incidence of histopathological findings in females rats from trials 1 and 2 is presented in Table 8.
In trial 1, changes thought to be attributable to the administration of ubiquinol and ubiquinone were observed in the liver, spleen, mesenteric lymph node, and lung (including bronchus).
Liver. Slight to mild microgranuloma was observed in the livers of four females in the ubiquinol group at 300 mg/kg, three at 600 mg/kg, and six at 1200 mg/kg. Slight to mild microgranuloma was also present in the livers of three females in the ubiquinone group (Figure 2).
Slight vacuolation of Kuppfer cells in the livers was present in one female in the ubiquinol group at 600 mg/kg and in four females at 1200 mg/kg. Slight fine vacuolation was seen in hepatocytes in seven females each in the ubiquinol group at 300 and 600 mg/kg groups, in nine females at 1200 mg/kg, and in five females in the ubiquinone group. Mild accumulation of macrophages in the liver was seen in one female each at 300 and 1200 mg/kg (data not shown). Slight or mild focal necrosis in the liver was observed in one female each in the 300, 600, and 1200 mg/kg ubiquinol group and the ubiquinone group (Figure 3).
Oil red O staining showed slight or mild positive reactions at the sites of microgranuloma, vacuolation of Kupffer cells, fine vacuolation of hepatocytes, and accumulation of macrophages in the livers of females. Therefore, it is likely that the vacuolation in the liver were lipid accumulation. No histopathological effects were noted in the livers of males.
Spleen. Slight to moderate accumulation of macrophages was seen in the spleens of 3 females in the ubiquinol group at 300 mg/kg, in 9 females at 600 mg/kg, and in 10 females at 1200 mg/kg. Mild focal necrosis was observed in one female in the ubiquinol group at 300 mg/kg.
Mesenteric Lymph Node. Slight or mild accumulation of macrophages was observed in the mesenteric lymph node of one female in the ubiquinol group at 300 mg/kg, in two females at 600 mg/kg, and in six females at 1200 mg/kg.
Lung. Slight or mild crystalline-shaped spaces were observed in the lungs of one female each in the 300 and 600 mg/kg ubiquinol groups, and in two males and two females in the ubiquinone group. This finding suggested the characteristic of crystalline material dissolved during tissue processing, and the material was probably test article aspirated into lung during dosing. Slight or mild accumulation of foam cells was seen in alveoli in the ubiquinol groups in one female at 300 mg/kg, in one male and one female at 600 mg/kg, and in one female at 1200 mg/kg, and in two males and three females in the ubiquinone group (data for lung not shown). These changes were judged to be due to aspiration of the test article and not toxic changes because there were no changes in alveolar epithelium.
Although some other changes were also observed, they were judged to be unrelated to the administration of ubiquinol because the incidence and degree were observed spontaneously.
In trial 2, changes thought to be attributable to the administration of ubiquinol and ubiquinone were observed in the liver, spleen, mesenteric lymph node, and lung (including bronchus).
Fine vacuolation in hepatocytes was observed in three females in the ubiquinol group at 200 mg/kg, in four females at 300 mg/kg ubiquinol, and in three females in the ubiquinone group. Slight or mild microgranuloma was observed in the livers of three females each in the ubiquinol 300 mg/kg group and the ubiquinone group, and slight focal necrosis was seen in one female each in the 300 mg/kg ubiquinol group and the ubiquinone group. Mild accumulation of macrophages was observed in the spleen of one female in the 300 mg/kg ubiquinol group. Slight accumulation of macrophages was seen in the mesenteric lymph nodes of three females each in the 300 mg/kg ubiquinol group and the ubiquinone group. The mild presence of crystalline-shaped space in the lung was observed in one female in the ubiquinone group. Mild accumulation of foam cells in alveoli was seen in the ubiquinol groups in one female at 150 mg/kg, three females at 300 mg/kg, and two females in the ubiquinone group. Slight cell infiltration in alveoli was noted in one female each in the 300 mg/kg ubiquinol and ubiquinone groups (data for lung not shown).
Although some other changes were also observed, they were judged to be unrelated to the administration of ubiquinol because the incidence and degree were observed spontaneously.
Dogs
No treatment-related effects were observed in any animal in the ubiquinol groups or ubiquinone group. Slight or mild changes were observed in various organs/tissues from control and treated animals. These changes were judged to be incidental based on their pathological nature or incidence of occurrence (data not shown).
Total CoQ10 Concentrations in Plasma
Rats
Results in males and females are shown in Tables 9 and 10, respectively.
On day 1 of administration in trial 1, the concentration of total CoQ10 in plasma was increased quickly after dosing in both males and females in the ubiquinol groups, showing largely dose related increases. The C max and AUC0–24 h were 1.21 μg/ml and 20.38 μg·h/ml, 1.92 μg/ml and 33.20 μg·h/ml, and 3.59 μg/ml and 47.53 μg·h/mL in males given 300, 600, and 1200 mg/kg ubiquinol, respectively. In females, the C max and AUC0–24 h values were 1.89 μg/ml and 30.92 μg·h/ml, 2.30 μg/ml and 29.57 μg·h/ml, and 3.18 μg/ml and 55.13 μg·h/ml, respectively, in the 300, 600, and 1200 mg/kg groups. T max was 4 or 8 h after dosing in males and 4 h after dosing in females. In the ubiquinone group, C max and AUC0–24 h were 1.31 μg/ml and 22.15 μg·h/ml in males and 1.19 μg/ml and 23.31 μg·h/ml in females. T max was 1 h after dosing in males and 4 h after dosing in females.
Increases in plasma concentrations of total CoQ10 were also largely dose related in week 13 of administration, and the concentration of CoQ10 in plasma again increased quickly after dosing with ubiquinol in both males and females. The C max and AUC0–24 h were 2.24 μg/ml and 38.04 μg·h/ml, 3.38 μg/ml and 51.15 μg·h/ml, and 5.41 μg/ml and 57.57 μg·h/ml in males given 300, 600, and 1200 mg/kg ubiquinol, respectively. In females, the C max and AUC0–24 h values were 2.98 μg/ml and 50.40 μg·h/ml, 4.61 μg/ml and 66.18 μg·h/ml, and 6.09 μg/ml and 73.65 μg·h/ml, respectively, in the 300, 600, and 1200 mg/kg groups. T max was 4 h after dosing in both males and females. In the ubiquinone group, C max and AUC0–24 h were 4.86 μg/ml and 75.44 μg·h/ml in males and 4.67 μg/ml and 73.74 μg·h/ml in females. T max was 4 h after dosing in both males and females.
Likewise, the concentration of total CoQ10 in plasma was increased quickly after dosing on day 1 of administration in trial 2. The C max and AUC0–24 h were 1.10 μg/ml and 15.16 μg·h/ml, 1.21 μg/ml and 15.99 μg·h/ml, 1.61 μg/ml and 20.81 μg·h/mL, and 1.41 μg/ml and 23.09 μg·h/mL in rats given 75, 150, 200, and 300 mg/kg ubiquinol, respectively. T max was 4 or 5 h after dosing. In the ubiquinone group, C max and AUC0–24 h were 1.91 μg/ml and 28.35 μg·h/mL, and T max was 7 h after dosing.
In week 13 of administration, the concentration of total CoQ10 in plasma again increased quickly after dosing with ubiquinol. The C max and AUC0–24 h were 2.54 μg/ml and 40.43 μg·h/ml at 75 mg/kg, 3.54 μg/ml and 48.72 μg·h/ml at 150 mg/kg, 3.63 μg/ml and 47.08 μg·h/ml at 200 mg/kg, and 5.57 μg/ml and 64.91 μg·h/ml at 300 mg/kg. T max was 4 h after dosing. In the ubiquinone group, C max and AUC0–24 h were 4.38 μg/ml and 62.92 μg·h/ml, and T max was 4 h after dosing.
Dogs
Pharmacokinetic data are summarized in Tables 11 to 13.
On the starting day of administration, the concentrations of total CoQ10 in plasma in the ubiquinol groups rose quickly after dosing. T max was 1 h after dosing in 1/3 females in the 150 mg/kg group, whereas it was 4 or 8 h after dosing in the other animals. C max was 3.54/4.51, 5.20/5.23, and 6.79/6.19 μg/ml in males/females in the 150, 300, and 600 mg/kg groups, respectively. The concentration of total CoQ10 in plasma decreased thereafter, but the total CoQ10 was remaining in plasma 24 h after dosing. AUC0–24 h was respectively 65.85/66.60, 94.97/89.99, and 129.81/102.44 μg·h/ml, which increased with the increase in the dose level. The concentrations of total CoQ10 in plasma in the ubiquinone group increased similarity to those in the ubiquinol groups, and T max was 8 h after dosing, C max was 4.35 and 3.58 μg/ml, and AUC0–24 h 86.46/74.27 μg·h/ml in males/females.
In week 7 of administration, total CoQ10 was detected before dosing in plasma in the ubiquinol groups and its concentration increased quickly after dosing with individual variations. T max was 4 to 8 h after dosing. C max was 9.71/8.02, 8.58/8.87, and 11.90/7.60 μg/ml. The concentrations of total CoQ10 in plasma decreased thereafter, and the concentrations 24 h after dosing were comparable to those before dosing. AUC0–24 h was respectively 175.50/139.87, 174.12/184.17, and 213.02/146.20 μg·h/ml. Neither C max nor AUC increased with the increase in dose level. The concentrations of total CoQ10 in plasma in the ubiquinone group increased similarly to those in the ubiquinol groups, and T max was 4 or 8 h after dosing, C max was 8.03/5.96 μg/ml, and AUC0–24 h 164.87/126.72 μg·h/ml.
In week 13 of administration, the concentrations of total CoQ10 in plasma were similar to those in week 7 of administration in the ubiquinol groups. T max was before dosing in 1/3 females in the 300 mg/kg group, whereas it was 4 or 8 h after dosing in the other animals. C max was 8.20/7.17, 9.57/8.80, and 8.55/5.76 μg/ml, and AUC0–24 h was respectively 167.86/144.18, 181.77/185.27, and 164.41/119.03 μg·h/ml. Neither C max nor AUC0–24 h increased with the increase in the dose level. The concentrations of total CoQ10 in plasma in the ubiquinone group increased similarly to those in the ubiquinol groups, and T max was 1 or 8 h after dosing, C max was 6.06/5.63 μg/m, and AUC0–24 h was 122.41/108.68 μg·h/ml.
Total CoQ10 Concentrations in Liver
Rats
As shown in Table 14, the liver total CoQ10 concentrations in female rats in trial 1 after administration of 300, 600, and 1200 mg/kg of ubiquinol were 8.49, 12.77, and 16.10 mg/g, respectively, 4 to 6 times higher than those in males (1.38, 3.30, and 3.38 mg/g, respectively). The same sex difference was noted in animals treated with 1200 mg/kg of ubiquinone, with total CoQ10 concentrations of 1.36 and 5.09 mg/g measured in the livers of males and females, respectively. These concentrations were lower than those measured in animals in the low-dose ubiquinol group.
In the ubiquinol groups in trial 2, the concentration of total CoQ10 in the liver was 2.21 mg/g at 75 mg/kg, 6.09 mg/g at 150 mg/kg, 10.10 mg/g at 200 mg/kg, and 11.65 mg/g at 300 mg/kg. In the ubiquinone group, the concentration of total CoQ10 in the liver was 7.70 mg/g.
Dogs
Data are summarized in Table 15. The concentrations of total CoQ10 in the liver in males/females in the control group were 24.740/24.935 μg/g wet weight, and 1.386/1.356, 2.165/2.575, and 6.865/2.283 mg/g wet weight in the ubiquinol groups at 150, 300, and 600 mg/kg, respectively. In the ubiquinone groups, the concentrations of total CoQ10 in the liver in males/females were 1.090/0.645 mg/g wet weight.
DISCUSSION
The present studies were conducted to investigate and compare the potential subchronic toxicity of ubiquinol administered by gavage to Sprague-Dawley rats and beagle dogs for 13 weeks. In the initial rat study, males and females were given ubiquinol at doses of 0, 300, 600, or 1200 mg/kg or ubiquinone at 1200 mg/kg by gavage for 13 weeks. This was followed by the second study, where females were given with doses of 75, 150, 200, or 300 mg/kg/day in order to determine a NOAEL. In the dog study, the test material was administered to males and females at dose levels of 150, 300, and 600 mg/kg, and ubiquinone was included at 600 mg/kg.
Administration of ubiquinol to rats was associated with prolongations of prothrombin time and partial activated thromboplastin time in males 1200 mg/kg. Such changes were not observed at the 600 mg/kg dose level. Because the chemical nature of the test article is similar to vitamin K, these observations may be the result of antiagonistic activity against vitamin K in the blood coagulation system (Combs, Porter, and Falkers 1976; Saupe et al. 1994); however, because there were no changes suggesting hemorrhages in the other examinations, including pathological examination, they were considered to be of little toxicological significance.
Pathological examinations revealed test article–related effects on the liver, spleen, and mesenteric lymph node in female rats but not in male rats. In the liver, upon histopathological examination, fine vacuolation of hepatocytes was observed in the ubiquinol groups at 200 mg/kg and above. These changes were judged to be of no toxicological significance because they were not considered to induce cytotoxic changes. Microgranuloma and focal necrosis with accumulation of macrophages were observed in the ubiquinol groups at 300 mg/kg and above. These findings were accompanied by increases in blood chemistry enzymes (AST, ALT, and LDH), which suggested potential hepatotoxicity. Microgranuloma, characterized by the presence of foamy macrophages in this study, and focal necrosis were judged to be only adverse effects induced by test article based on their incidence and pathological characteristics, although they were considered to be not direct hepatic damage because no single hepatocyte necrosis was observed, and microgranuloma are occasionally observed as a spontaneous change in this strain of rats. These changes observed in liver were thought to be due to uptake of the administered ubiquinol by the liver, as an adaptive response to xenobiotics, and the microgranulomas and focal necrosis were considered the results of excessive uptake of ubiquinol, which exceeded the capacity for adaptive response. These conclusions were based on the finding of localization to the liver of concentrated CoQ10 dissolved in lipoproteins, as suggested by the positive reaction of the liver cells to oil red O staining for lipids on histological examination and by extremely high concentrations of total CoQ10 in the liver, more than 8 mg/g detected on HPLC analysis, together with the previous finding by Mohr et al. (1992) and Tomasseti et al. (1999) that ubiquinol is extremely lipophilic and readily distributes into lipoproteins. Furthermore, in females, spleen weight was increased in the ubiquinol 600 mg/kg and above groups, and histological accumulation of macrophages was observed in the spleen and mesenteric lymph node in each ubiquinol group, which was thought to be secondary to the uptake of ubiquinol and judged to be not adverse effects because these events did not appear to be associated with tissue damage or organ dysfunction similarly to the liver. In females in the 300 mg/kg group, histological examination revealed focal necrosis in the spleen, but it was judged to be incidental because it was not dose related. As a conclusive result of two rat trials, slight prolongation of PT and APTT were observed in male 1200mg/kg, and microgranuloma and focal necrosis, which were judged to be only adverse effects, was observed in female 300mg/kg and above. Therefore, the NOAEL for ubiquinol under the conditions of this study was conservatively estimated to be 600 mg/kg/day for males and 200 mg/kg/day for females on rats.
Although low heart rates were observed in individual dogs, the degree of decrease was not dose dependent, and the mean heart rate values seen in male or female dogs were not significantly different from the values observed in the control group and all individual values were within the range of in-house historical control values. Furthermore, there were no other remarkable changes in other electrocardiography (ECG) parameters such as waveform abnormalities, nor were there abnormalities in blood chemistry, and histopathology indicative of cardiac effects. Therefore they were judged not to be test article related. Prolongation of PR interval (first-degree atrioventricular block) was observed in a single female in the 300 mg/kg group at week 13 of administration, although the PR intervals of this animal at weeks 1 and 7, as well as those of other dogs in this group at all measurement points, were within historical in-house control values. This finding was considered to be an isolated observation unrelated to dosing because it was observed only one animal in the middle-dose group and there was no similar tendency in the 150 or 600 mg/kg group. Furthermore, pathological examination revealed no related changes, and prolongation of PR interval at this degree is occasionally observed in untreated beagle dogs in this test facility.
In pathological examinations in the dog study, yellow discoloration in the liver was observed at necropsy in one of three males in the ubiquinol 600 mg/kg group. Ubiquinol is the reduced form of CoQ10 (ubiquinone), which exists in various organs such as the liver, kidney, heart, and brain, and in plasma (Åberg et al. 1992; Kontush et al. 1999; Reahal and Wrigglesworth 1992). Because the concentration of total CoQ10 in the liver was higher in two of three males, including this animal, than in the other animals, the finding was thought to be related to an intake of excessive amount of ubiquinol into the liver. However, because there were neither morphological nor functional effects on the liver, it was judged to be of little toxicological significance. Histopathological examination revealed no effects attributable to administration of ubiquinol or ubiquinone in any organs examined.
The results of total CoQ10 concentrations in plasma and liver in dogs were different from those in rats. Liver total CoQ10 concentrations in female rats after administration of 300, 600, and 1200 mg/kg of ubiquinol were 8.46, 12.77, and 16.10 mg/g, respectively, and were 4 to 6 times higher than those in males. There was a small sex difference in AUC0–24h values for plasma total CoQ10 concentrations (ranging from 20.38 to 47.53 μg·h/ml and from 38.04 to 57.57 μg·h/ml in males, and from 29.57 to 55.13 μg·h/ml and from 50.4 to 73.05 μg·h/ml in females in day 1 and in week 13, respectively) in males and females. On the other hand, in dogs, there was no sex difference in the total CoQ10 concentrations in liver at 150 and 300 mg/kg (ranging from 1.368 to 2.165 g and from 1.356 to 2.575 in males and females, respectively), whereas those at 600 mg/kg in males was about three times higher than those of in females. Likewise, AUC0–24h for concentrations of total CoQ10 in plasma after administration of ubiquinol at 150 and 300 mg/kg at all sampling points (ranging from 65.85 to 181.77 μg·h/ml and from 66.6 to 185.27 μg·h/ml in males and females, respectively) showed no sex difference, whereas those obtained in males dosed with ubiquinol at 600 mg/kg were similar to or slightly higher than those in females.
These species difference in total CoQ10 concentrations in plasma and liver may explain one of the reasons for the difference in the effects of ubiquinol on liver in rats and dogs.
Given the species-dependent differences that exist with respect to coenzyme Q homologs, it is conceivable that rats in general exhibit a higher sensitivity to liver accumulation of CoQ10. Furthermore, higher sensitivity of female rats to accumulation of CoQ10 in the liver compared to male rats may be the result of gender-dependent differences in hepatic metabolism. Czerniak (2001) reported that a majority of the known gender-related differences in toxicity of compounds in rats are due to variations in the expression of hepatic enzymes. In fact, rat liver was shown to contain approximately a dozen gender-dependent isoforms of cytochrome P450 (Pampori and Shapiro 1999); for example, the gender-specific cytochrome P450s CYP2C11, CYP2C13, and CYP3A2 are expressed in males, whereas CYP2C12 is expressed in females (Czerniak 2001). In addition, it has been suggested that coenzyme Q homologues are degraded by oxidation (Imada et al. 1970), which is generally catalyzed by cytochrome P450 enzymes (Coon 2005). In this study, CoQ10 accumulation in the liver of male rats was much less (1/6 to1/4) than in females. These data suggest that CoQ10 accumulation in the livers of female rats at least partly contributed to a gender and species-specific hepatic lesion.
Indeed, no such changes were observed following the administration of ubiquinol to beagle dogs for 13 weeks. As a result, the NOAEL was considered to be 600 mg/kg/day, the highest dose tested.
In humans and most mammals, including dogs, the predominant form of coenzyme Q is CoQ10, which consists of 10 isoprenoid units in the side chain (Ramasarma 1985). The dog is considered to be a species more similar to humans than the rat with respect to CoQ10 status and thus considered more relevant to assessing safety of ubiquinol in humans.
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Conflict of Interest Statement. The authors are presently employed by Kaneka Corporation, which manufactures the ubiquinol product (Kaneka QHTM) used in the study and is the sponsor and sole source of funding for the present study.