In vivo pharmacokinetic study

Six-week-old (180 − 200 g) male Sprague Dawley (SD) rats were fed with water and were allowed to acclimatize in laboratory conditions for more than three days. Subsequently, healthy rats were selected and used for the in vivo experiment as an animal model. All experiments were conducted according to the guidelines approved by the Institutional Animal Care and Use Committee (BHCC-IACUC-135 2016-04). Doses of CoQ₁₀ (60 mg/kg) were prepared for both the CoQ₁₀ suspension (0.1% sodium CMC) and the optimized SMEDDS formulation, and they were administered via oral gavage, twice a day, at 12-h intervals, for 4 days. Rats were fasted approximately for 10 h prior to blood collection. After the administration of the last dose of CoQ₁₀, blood was collected hourly. During the procedure, animals were anesthetized 10 min prior to the blood collection by an intraperitoneal injection of a mixture of Zoletil^® 50 (50 mg/mL) and Rompun^® (23.32 mg/mL). Blood samples were collected from the tail vein at 0, 1, 4, 8, 12, and 24 h after the last CoQ₁₀ dose; blood was collected in a heparinized tube by using a syringe. To study the effect of SMEDDS on CoQ₁₀ distribution characteristics in the brain tissue, a comparison was made between the optimized SMEDDS formulation and the CoQ₁₀ suspension. The brain distribution study was conducted on the different sets of male SD rats and divided into two groups: CoQ₁₀ suspension and optimized SMEDDS formulation group. Both groups were subdivided into two subgroups (n = 5): 4 h and 8 h group. Brain tissues were collected at 4 h and 8 h after the last dose of CoQ₁₀ administration and extent of CoQ₁₀ distribution in brain was analyzed.

For the preparation of the calibration curve, 20 µL of the standard CoQ₁₀ solution (CoQ₁₀ in hexane) was added to 200 µL of drug-free plasma samples, vortexed, and collected in a glass tube. Subsequently, 1 mL of 1-propanol was added to the solution and the latter was vortexed for 5 min, and then centrifuged at 3,000 × g for 5 min, for the precipitation of proteins. A rotary centrifugal vacuum evaporator was used in order to collect the supernatant and evaporate it. The residue was redistributed in 100 µL of 1-propanol, and analyzed by using HPLC (as described in the solubility study). After the last scheduled oral administration of the CoQ₁₀ suspension and the CoQ₁₀-loaded optimized SMEDDS to rats, 400 µL of blood samples were collected from the tail vein of each rat at 0, 1, 4, 8, 12, and 24 h. The collected blood samples were then centrifuged at 3,000 × g, for 15 min, at 4 °C, in order to prepare plasma samples, and these plasma samples were then stored at −70 °C until further analysis.

Rats brains were collected by opening the skull after cervical dislocation at 4 h and 8 h after the last CoQ₁₀ dose. After weighing, brain tissues were stored at −20 °C until the undertaking of the extraction experiment. Subsequently, 1 mL of distilled water was added to the brain tissue and homogenization was performed by using a homogenizer. After adding 900 μL of tetrahydrofuran to 100 μL of brain tissue homogenate of each experimental group sample, vortex-mixing was performed for 5 min, and centrifugation took place at 4 °C and 13,000 rpm, for 10 min, in order to precipitate the protein. The supernatant was then transferred to a new tube, diluted 10-fold, filtered, and analyzed by using liquid chromatography with tandem mass spectrometry (LC-MS/MS; Agilent 6410 Triple quad, Agilent Technologies, California, USA).

In this paper we developed and validated LC-MS/MS method, based on a liquid-liquid extraction procedure. The method was optimized using beta-carotene as internal standard. LC-MS/MS was used for the CoQ₁₀ analysis in the obtained rat blood plasma samples and brain homogenates. A 4.6 × 250 mm (5 µm particle size) Agilent Technologies Eclipse XDB-C₁₈ column was used. The column temperature was maintained at 35 °C, while the flow rate was set at 1.0 mL/min. The analysis wavelength was adjusted at 274 nm (UV) and the sample injection volume was 20 µL. Isocratic mobile phase containing acetonitrile and tetrahydrofuran (65:35, [v/v]) was used, and the retention time obtained was in the range of 6.2 − 6.4 min.

The plasma drug concentration, peak plasma drug concentration (C_max), and the time to reach the peak plasma concentration (T_max), were obtained directly from the blood concentration versus time profile. The area under the curve (AUC) was calculated by using a trapezoidal rule.

To visually conform the brain distribution characteristics of CoQ₁₀, SD rats were divided in two groups, a cyanine 5.5 SMEDDS group and a cyanine 5.5 suspension group. Two hundred µL of cyanine 5.5 loaded SMEDDS and suspension at a dose equivalent to 0.2 mg/kg were delivered to the rats orally. Optical imaging was performed using a VISQUE Invivo ART100 imaging system (Anyang, South Korea). Finally, near-infrared fluorescence (NIR) images were obtained with a cyanine 5.5 filter channel.