Effect of chronic unpredicted mild stress-induced depression on clopidogrel pharmacokinetics in rats

Chemicals and reagents

Clopidogrel standard sample (No: 20101201, 98.00%) was purchased from molnova Co., Ltd. (Shanghai, China), and ticlopidine (internal standard, IS; No: SLBD1982, ≥99%) was purchased from Sigma Trading Co., Ltd. Clopidogrel hydrogen sulphate tablets were obtained from Sanofi Pharmaceuticals Co., Ltd (Hangzhou, China). Substrates of S-mephenytoin, 4-hydroxymephenytoin, tolbutamide, and 4-hydroxytolbutamide substrates (≥95%) were purchased from Sinco Pharmachem (Middletown, DE, USA). Diazepam (internal standard, IS; 171225-201304) was purchased from the China Institute of Food and Drug (China). High-performance liquid chromatography (HPLC)-grade methanol and ethyl acetate were purchased from Merck (Kenilworth, NJ, USA). 5-Hydroxytryptamine (5-HT) ELISA Kit of rat was purchased from CUSABIO (Houston, TX, USA). TRIzol Reagent was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). The PrimeScript TM RT Master Mix (No:RR047A) and SYBR Premix Ex Taq™ (No: RR420A) were purchased from TaKaRa Bio Inc (Dalian, China).

Animals

Female SPF SD rats (5 weeks old, approximately 200 ± 20 g weight; Animal Quality Certificate: 2019-0004) were purchased from Hunan SLAC Laboratory Animal Co., Ltd. (Changsha, China). The rats were kept at the Laboratory Animal Center, Hunan Provincial People’s Hospital, Changsha, China (Animal Experiment License: SCXK 2020 - 260), in a well-ventilated and clean laboratory environment (temperature 22−25 °C, relative humidity 50%–70%). All rats had free access to obtain food and water for 1 week. Then rats were randomly divided into two groups (each group comprising nine rats): control and depression groups.

CUMS-induced depression model

The depression group was provided feed and water ad libitum in a cage alone for 12 weeks. The stressors included restraint (activity restriction in a bottle, 1 h), hot water swimming (45 °C, 5 min), cold water swimming (4 °C, 5 min), tail clipping (one cm apart from thetail, 1 min), cage stilting (45 °C, 24 h), horizontal shaking (10 min), damp padding (24 h), noise interference (10 min), and day/night inversion (24 h). Each stressor was randomly used 8–9 times (Wei et al., 2017), and the same stressor was not applied consecutively to avoid rat’s prediction. After the stress treatment, the rats were moved back. The control group rats were provided feed and water ad libitum for 12 weeks without any stressor. The experimental grouping and depression modeling are shown in flow chart (Fig. 1).

The sucrose preference test

The sucrose preference test was performed to explore depression-like behavior based on the rat’s natural preference for sweets taste (Duca, Swartz & Covasa, 2014). Before testing, all rats were kept in a single cage. Two bottles of 1% sucrose solution were provided to each cage of rats for adaptive training for 24 h. Thereafter, one bottle of 1% sucrose solution was replaced with ordinary drinking water, and the adaptive training was conducted for a further 24 h. Following this acclimation, the rats were deprived of water and feed for 24 h, and the formal test was started at 8:00 pm on the fourth day. Each cage of rats was provided with two bottles of pre-weighed solution (one bottle of 1% sucrose solution and one bottle of ordinary drinking water), without feed deprivation. After 12 h, they were weighed again to calculate the consumption from each bottle of solution using the following equation: ${\displaystyle \text{sugar water preference rate}\left(\text{\%}\right)=\left[1\text{\%}\text{sucrose water consumption}/\left(1\text{\%}\text{sucrose solution consumption}+\text{ordinary drinking water consumption}\right)\right]\times 100.}$

Open-field test (Katz, Roth & Carroll, 1981)

Before and after the model establishment, the open-field behavior of each rat was analyzed using a SuperMaze Video Analysis System (Shanghai XinRun Information Technology Co., Ltd., Shanghai, China). The apparatus comprised an opaque box (100 cm × 100 cm × 50 cm). The open-field area was divided into 33 × 33 cm² equal-sized squares. The average moving speed, total moving distance, freezing time, and central area entry times were determined as indices of locomotion activity and exploratory behavior. The test was conducted in a quiet room in the evening (5:00 pm–00:00 am) for 10 min.

Determination of serotonin (5-HT) level in rat brains

SD rats were euthanized by cervical dislocation after completing the pharmacokinetic experiment. Brain samples were collected from each rat and frozen in liquid nitrogen as described previously method (Wu et al., 2020b). When 5-HT was detected, the cortex of rats were first transferred to a refrigerator at −20 °C for freezing, and then thawed about at 20 °C. Subsequently, the left cerebral cortex (100 mg) was excised, cut into small pieces, and put into a tissue grinder, after which 1 mL 1 × phosphate-buffered saline (PBS) was added into the grinder to make a homogenate, which was then stored −20 °C overnight. After repeated freezing and thawing twice to destroy the cell membrane, the tissue homogenate was centrifuged at 5,000 × g at 2−8 °C for 5 min, and the supernatant was used for 5-HT determination. The 5 - HT levels were measured using ELISA (CUSABIO, US) according to the manufacturer’s instructions.

Dosage regimen and sample collection

All rats were fasted overnight but with free access to water, then administered 10mg/kg of clopidogrel orally (Wu et al., 2020a). Blood samples were collected via the eye canthusat at 0.07, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 12 h after isoflurane inhalation anesthesia. Unfortunately, three rats died of bleeding during the experiment. The blood samples were centrifuged at 3,000 × g for 5 min to obtain plasma, which was stored at −80 °C for further analysis.

Determination of clopidogrel concentration

The clopidogrel concentration in rats’ plasma were was determined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method (Xiao et al., 2019). A total of 50 µL plasma sample and 10 µL ticlopidine (5 ng/mL) were added to 0.2 mL ethyl acetate in a centrifuge tube, vortex-mixed for 3 min and centrifuged at 14,000 × g for 10 min. The supernatant was evaporated to dry. Next, the residue was redissolved in 100 µL methanol by vortex-mixing for 1 min and centrifuged at 16,000 × g for 1 min. Thereafter, 2.0 µL of the supernatant was injected into the HPLC-MS/MS system for analysis. The HPLC instrument equipped with verse-phasecolumn was connected to a triple quadrupole tandem MS with an electrospray interface. The system comprised an LC system (Nexera UHPLC/HPLC; Shimadzu, Kyoto, Japan) with a Triple Quad 6500 MS system (AB Sciex, Framingham, MA, USA). Chromatographic separation was achieved using an ACE Excel 5 SuperC18 column (SN: A191749; 150 × 2.0 mm; Advanced Chromatography Technologies Ltd, Aberdeen, UK). The mobile phase comprised methanol (A) and water (0.1% ammonium formate, B) with a flow rate of 0.3 mL/min. The gradient elution included 30%A (0–0.5 min), 5%A (0.5–1.5 min), 5%A (1.5–2.0 min), 30%A (2.0–2.5 min), and 30%A (2.5–4.0 min). The MS conditions were optimized as follows: a positive electrospray ionization (ESI⁺) mode, capillary at 5,500 V, an ion-spray gas temperature of 500 °C, a gas flow rate of 11 L/min, and nebulizer at 10 psi. The parameters of clopidogrel and Sit were as follows: fragmentary voltages of 70 V and 66 V, collision energy of 20 units and 23 units, respectively. The multiple-reaction monitoring mode was selected to quantify clopidogrel and Sit, for which the precursor-to-product ion transitions were 322.1 → 212.0 and 264.3 → 154.1, respectively. The MassHunter Workstation software (Version B.06.00; Agilent Technologies, Santa Clara, CA, USA) was used to collect and process data.

Determination of CYP2C19 and CYP2C9 activity in liver microsomes

The control and CUMS-induced depression rats were fasted for 12 h and killed by cervical dislocation before the liver was excised, rinsed with ice-cold normal saline (0.9% NaCl, w/v), weighed to 1 g, and homogenized with 4.0 mL in a 0.05 M Tris-HCl buffer (pH 7.4). The homogenate was centrifuged at 10,000 × g at 4 °C for 20 min, and the supernatant was further centrifuged at 105,000 × g at 4 °C for 60 min. Afterward, the helvus sediment was reconstituted with 1.0 mL Tris-HCl buffer and stored at −80 °C for further analysis. The total protein concentration of liver microsomal enzymes was determined using a Bicinchoninic Acid Protein Assay Kit. In addition, the CYP450 activity of the liver microsomes was determined using a cocktail of probe drugs in which the activities of CYP enzymes were assessed by determining probe drug production. Tolbutamide (TOL) or mephenytoin (MPT) were metabolized to 4-OH-TOL or 4-OH-MPT by CYP2C9 or CYP2C19, respectively, and these products were determined by HPLC-MS (Lasker et al., 1998).

The liver microsome working solution was thawed at 4 °C, then absorbed 100 µL, probe substrate 100 µL, phosphate buffer 250 µL in 5 mL brown centrifuge tube, vortex oscillation for 1 min, incubation in 37 °C water bath for 5 min, adding NADPH 50 µL to start the reaction, and incubation in 37 °C water bath for 20 min. After the reaction, the incubation system was placed in an ice bath, then 1.4 mL of ethyl glacial acetate and 20 µL internal standard (diazepam; 5 µg.mL⁻¹) were added, subjected to vortex oscillation for 2 min, and centrifuged at 3,500 × g at 4 °C for 10 min. Thereafter, the supernatant was sucked into a 1.5 mL brown centrifuge tube and placed in a vacuum drying oven (80 °C, 0.9–1.0 MPa) for evaporation. Next, the supernatant was redissolved with 200 µL methanol, vortex oscillated for 1 min, centrifuged at 14,000 × g for 2 min, and 5 µL of the supernatant were sucked into an injection bottle for analysis. The whole experiment was conducted in darkness.

Chromatographic separation was achieved using an ACE Excel 5 SuperC18 column (2.0 mm × 150 mm, 1.8 µm, SN: A191749; Advanced Chromatography Technologies Ltd.). The mobile phase contained 0.1% formic acid water (A) - methanol (B) with a flow rate of 0.3 mL/min. The gradient elution included 50% A (0–2.4 min), 5% A (2.4–3.2 min), and 50% A (3.2–4.5 min). The optimized MS conditions were as follows: ESI⁺ mode, a source injection voltage of 5,500 V, collision gas of 10 psi, inlet voltage of 10 V, temperature of 500 °C, curtain gas of 30 psi, and multi-reaction detection scanning (MRM). The parameters of 4-OH-TOL and 4-OH-MPT included a fragmentary voltage of 100 V and collision energies of 24 and 23 units, respectively. The monitoring ions were set as m/z 287.3 →170.8 for 4-OH-TOL and 235.2 →150.2 for 4-OH-MPT.

mRNA expression of Cytochrome P450 by RT-qPCR

Rat livers were collected after the pharmacokinetic experiment, washed with 0.9% NaCl solution, and stored at −80 °C until further use. Total RNA in rats’ livers was extracted using TRIzol Reagent (Sigma Trading Co., Ltd.). The RNA concentration was determined using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at an absorbance of 260 nm. Total RNA was transcribed to cDNA using a PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio Inc.), and RT-qPCR was performed using an ABI 7500 Real-time PCR system with SYBR Green Quantitative PCR Master Mix. To normalize the mRNA expression, the housekeeping gene β-actin was used as an external standard. The primer sequences used were shown in Table 1. The relative amount of each mRNA was calculated using the^2−ΔΔCt method (Wei et al., 2017).

Statistical analysis

Statistical and pharmacokinetics parameter analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA) and DAS 3.0 software (Shanghai University of Traditional Chinese Medicine, Shanghai, China). Differences between the two groups were analyzed using an independent samples t-Test. Data are expressed as the mean ± standard deviation (SD). ^∗p < 0.05 or < 0.01 was considered statistically significant.

Validation of depression model

The sucrose preference test, behavior scores of open-field test, and 5-HT levels were shown in Fig. 2. The sucrose preference and behavior scores were the same between the two groups before the depression model establishment, but changed significantly after the depression model establishment (^∗∗p <0.01, Fig. 2). The sucrose preference, average moving speed, total moving distance, and central area entry times decreased from 81.71 ± 10.73 to 68.16 ± 9.33%, from 104.21 ± 38.69 to 7.32 ± 4.05 mm/s, from 3,137.46 ± 1,166.84 to 438.95 ± 243.13 cm, and from 6.89 ± 4.49 to 0.22 ± 0.67, (^∗∗p <0.01) in the depression group, respectively. However, the freezing time increased from 271.39 ± 50.74 to 523.53 ± 31.50s in the depression group. There were no change for these in the control group (p > 0.05). After the model establishment, the cortex 5-HT levels of rats were also reduced significantly (4.52 ± 0.99 vs. 2.59 ± 0.31 ng/mL) compared with the two groups.

Clopidogrel assay and pharmacokinetic parameters

Clopidogrel and IS retention times were 2.74 min and 1.21 min, respectively (Fig. 3). No significant interference or ion suppression peaks were observed in the retention regions of both compounds. The correlation coefficient of calibration curves showed linearity (R²>0.99) by weight of 1/x² over the concentration range of 0.35–49.93 ng/mL. The lowest limit of quantitation (LLOQ) was 0.35 ng/mL, and the matrix effects were approximately 96%. Intra-day accuracy and precision were 99.79%-106.46% and 2.18%−6.34%, respectively; for inter-day, these were 97.46%-106.25% and 2.54%−8.10%, respectively (Table 2). The concentration–time parameters were calculated using the non-compartmental analysis method (Fig. 4 and Table 3). The area under curve (AUC_0−∞), peak time (T_max), peak concentration (C_max), and mean residence time (MRT_0−t) in the depression group decreased by 33.13% (from 82.56 ± 22.22 to 55.21 ± 21.91), 10.52% (from 0.38 ± 0.24 to 0.34 ± 0.17), 17.71% (from 12.42 ± 7.17 to 10.22 ± 4.63) and 11.89% (from 11.27 ± 3.03 to 9.93 ± 2.09), respectively, but only the area under curve (AUC_0−∞) had a notable significance (^∗p < 0.05). The distribution volume (Vz/F; 1841.58 → 3718.20 L kg ⁻¹) and clearance (CLz/F; 128.31 → 219.87 L h kg ⁻¹) increased significantly (^∗p < 0.01). No statistically significant difference was observed in half-time (T_1/2) between the two groups.

Determination of CYP450 activity and relative expression

The retention times of 4-OH-TOL and 4-OH-MPT were 3.38 min and 1.81 min, respectively. The CYP2C19 and CYP2C9 activities of liver microsomes in the depression group were significantly increased than those in the control group (Fig. 5). The activity of CYP2C19 in depression group increased approximately 3-fold from 0.025 ± 0.004 to 0.072 ± 0.005 ng/(min mg protein) (^∗∗∗p < 0.001). The CYP2C9 activity also varied significantly, increasing from 0.948 ± 0.112 to 1.528 ± 0.151 ng/(min mg protein) (^∗∗p < 0.01).

The expression of CYP450 in rat livers was analyzed using RT-qPCR. The relative expression of CYP2C11 mRNA increased by 22.60% in CUMS group, while that of CYP2C13 mRNA decreased by 27.43%. CYP1A2 increased by 34.39% (^∗∗p = 0.01) in CUMS group compared with control group. CYP3A1 and CYP2C12 mRNA relative expression increased slightly between the CUMS group, but did not reach the significance. CES1 mRNA relative expression did not change in CUMS group.