Effects of Ginkgo leaf tablets on the pharmacokinetics of atovastatin in rats

Abstract Context: Ginkgo leaf tablets (GLT), an effective traditional Chinese multi-herbal formula, are often combined with atorvastatin calcium (AC) for treating coronary heart disease in clinic. Objective: This study investigated the effects of GLT on the pharmacokinetics of AC and the potential mechanism. Materials and methods: The pharmacokinetics of AC (oral administered at a dose of 1 mg/kg) with or without pre-treatment of GLT (oral administered at a dose of 80 mg/kg/day for 10 days) were investigated in male Sprague–Dawley rats. The effects of GLT on the metabolic stability of AC were also investigated using rat liver microsome incubation systems. Results: The results indicated that the Cmaxincreased from 36.84 ± 4.21 to 48.68 ± 6.35 ng/mL, and the AUC(0-t) increased from 135.82 ± 21.05 to 77.28 ± 12.92 ng h/mL, and t1/2 also increased from 2.62 ± 0.31 to 3.32 ± 0.57 h when GLT and AC were co-administered. The metabolic stability of AC was also increased (48.2 ± 6.7 vs. 36.7 ± 5.3 min) with the pre-treatment of GLT. Discussion: This study indicated that the main components in GLT could accelerate the metabolism of AC in rat liver microsomes and change the pharmacokinetic behaviours of AC. So these results showed that the herb-drug interaction between GLT and AC might occur, and the clinical efficacy could increase when they were co-administered. Therefore, the clinical dose of AC should be decreased when GLT and AC are co-administered.

Ginkgo leaf tablet (GLT) is an effective traditional Chinese multi-herbal formula, which is widely used in treating ischaemic cerebrovascular disease in the clinic (Lin et al. 2003;Chung et al. 2006;Yang et al. 2017). The main components of GLT are Ginkgo flavone glycosides, ginkgolides, and bilobalides (Liu et al. 2009;Guan et al. 2014;Rao et al. 2014). Atorvastatin calcium (AC) and GLT are often simultaneously used for the treatment of coronary heart disease in China clinics. However, the herbdrug interaction between GLT and AC are still unknown. Therefore, it is essential to investigate the effects of GLT on the pharmacokinetics of AC and its potential mechanism as adverse reactions such as toxicity and treatment failure might occur resulting from concurrent use of herbal drugs with over-thecounter drugs.
In this study, the potential herb-drug interactions of between GLT and AC were systematically investigated. The in vivo pharmacokinetics of AC in rats with or without pre-treatment with GLT were investigated using a sensitive and reliable LC-MS/MS method. The effects of GLT on the metabolic stability of AC were also studied using rat liver microsome incubation systems.
Acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid was purchased from Anaqua Chemicals Supply Inc. Limited (Houston, TX, USA). All other reagents and solvents were of analytical reagent grade.

Animals
This animal experimental protocol was approved by the Animal Ethics Committee of the Weifang Medical University (Weifang, China). Male Sprague-Dawley rats weighing 220-250 g were supplied by Sino-British Sippr/BK Lab Animal Ltd. (Shanghai, China). The rats were maintained in an air-conditioned animal quarters at 22 ± 2 C and 50 ± 10% relative humidity. Water and food (laboratory rodent chow, Shanghai, China) were allowed ad libitum. The animals were acclimatized to the facilities for 5 days, and then fasted with free access to water for 12 h prior to each experiment.

Instrumentation and conditions
The determination of AC was performed as previously reported by Sun et al. (2018). The analysis was performed on an Agilent 1290 series liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA). The chromatographic analysis of AC was performed on a Waters X-Bridge C18 column (3.0 Â 100 mm, i.d.; 3.5 lm, USA) at room temperature. The mobile phase was water (containing 0.1% formic acid) and acetonitrile (30:70, v:v) at a flow rate of 0.4 mL/min.
The mass scan mode was the positive MRM mode. The precursor ion and product ion were m/z 559.2 ! 440.2 for AC and m/z 419.9 ! 199.4 for the simvastatin, respectively. The collision energy for AC and simvastatin were 30 and 25 eV, respectively. The MS/MS conditions were optimized as follows: fragmentor, 140 V; capillary voltage, 4 kV; nozzle voltage, 500 V; nebulizer gas pressure (N 2 ), 40 psig; drying gas flow (N 2 ), 10 L/min; gas temperature, 350 C; sheath gas temperature, 400 C; and sheath gas flow, 11 L/min.

Pharmacokinetic study
For pharmacokinetic study in vivo, 12 rats were equally randomized to two groups, six rats in each group, including AC-only group (1 mg/kg) (A) and AC (1 mg/kg) þ GLT (80 mg/kg/day for 10 days) group (B) Sun et al. 2018). Group B was pre-treated with GLT solutions by oral gavage at a dose of 80 mg/kg/day for 10 days before the administration of AC, and then AC were orally administered to rats by oral gavage at a dose of 1 mg/kg. Blood samples (0.25 mL) were collected into a heparinized tube via the oculi chorioideae vein before drug administration and at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h after drug administration. After centrifuge at 3500 rpm for 5 min, the supernatant was obtained and frozen at À80 C until analysis.

Inhibitory effects of GLT on the metabolic stability of AC in rat liver microsomes
Rat liver microsomes were used to determine the metabolic rate of AC. The assay conditions and reaction mixtures were similar as reported previously (Qi et al. 2013;Qin et al. 2014). The reaction mixture was incubated at 37 C for 5 min and then AC (100 lM) was added. The effects of GLT on the metabolic rate of AC was investigated by adding 350 lg/mL of GLT to rat liver microsomes and preincubating for 30 min at 37 C, and then AC (100 lM) was added. Aliquots of 30 lL were collected from reaction volumes at 0, 1, 3, 5, 15, 30, and 60 min and 60 lL ice-cold acetonitrile was added to terminate the reaction, and then the sample preparation method was the same as the plasma sample preparation method and determined by LC-MS/MS.

Statistical analysis
Experimental values are expressed as mean ± SD. Statistical analysis of results obtained from clinical study was performed using Student's paired t-test. Differences were considered statistically significant when p values were <0.05. Statistical analysis was conducted using Graph-Pad Prism version 3.0 for Windows (San Diego, CA, USA).

Pharmacokinetic study in vivo
As shown in Table 1 and Figure 1, the results indicated that the C max (48.68 ± 6.35 vs. 36.84 ± 4.21 ng/mL), AUC (0-t) (77.28 ± 12.92 vs. 135.82 ± 21.05 ng h/mL), and t 1/2 (3.32 ± 0.57 vs. 2.62 ± 0.31 h) increased significantly when GLT and AC were co-administered, which suggested that GLT might influence the pharmacokinetic behaviour of AC when they are co-administered. These results suggested that the herb-drug interaction between AC and GLT might occur when they are co-administered. Table 1. Pharmacokinetic parameters of AC in rats after oral administration of AC (1 mg/kg; n ¼ 6, Mean ± S.D.) with or without treatment of GLT (80 mg/kg/ day for 10 days).
Inhibitory effects of GLT on the metabolic stability of AC with rat liver microsomes As we know, the metabolism of AC was mainly modulated by CYP3A4 enzymes, and therefore, in this research, the effects of GLT on the metabolic stability of AC were further investigated in rat liver microsomes in vitro. The metabolic stability of AC was 36.7 ± 5.3 min, while the metabolic stability was decreased (48.2 ± 6.7 min) in the presence of GLT. The results indicated that the main components in GLT could inhibit the metabolism of AC with rat liver microsomes and change the pharmacokinetic behaviours of AC.

Discussion
The pharmacokinetic experiments showed that GLT could increase the system exposure of AC in rats when they are coadministered. As the system exposure of AC increased when coadministered with GLT, which suggested that the pharmacological activities of AC might be enhanced. Previous studies have also reported that the GLT could affect the pharmacokinetic profiles of amlodipine, cilostazol, fexofenadine, and clopidogrel when they are co-administered with GLT (Deng et al. 2016;Turkanovic et al. 2017). The main components in GLT are ginkgolides A, ginkgolides B, bilobalide, quercetin, and kaempferol, and several studies have indicated that the main components in GLT could inhibit the activity of CYP3A4 enzymes (Etheridge et al. 2007;Zadoyan et al. 2012;Palle & Neerati 2017).
To investigate its potential mechanism, the metabolism clearance was also investigated using rat liver microsome incubation systems, and the results revealed that GLT could inhibit its metabolism clearance in rat liver microsomes. AC is metabolized predominantly via CYP3A4 enzyme, and, therefore, co-administration of foods or drugs with influence on CYP3A4 enzymes may affect the pharmacokinetics of AC. Previous studies (Sun et al. 2018) have also reported that drugs or herbs could affect the pharmacokinetics of AC through affecting the activity of CYP3A4 enzyme.
Therefore, this study's results indicate that when the rats were pre-treated with GLT, the system exposure of AC would be increased significantly. The results suggest that the herb-drug interaction between GLT and AC might occur when they were co-administered. These changes could increase AC efficacy, so it was suggested that the dosage should be adjusted when GLT and AC are co-administered in the clinics.
In conclusion, the results indicated that GLT could influence the pharmacokinetic behaviour AC when they are co-administered. The main ingredients in GLT could inhibit the metabolism of AC in rat liver microsomes, which may be one of the reasons resulting in pharmacokinetic interactions when they were coadministered. Therefore, GLT could increase its clinical efficacy of AC when they are co-administered.