| BackgroundCardiovascular disease (CVD) is the most leading disease of death worldwide. Atherosclerosis (AS) is the main pathological basis of cardiovascular disease and it is a kind of chronic and progressional disease which can change the structure and function of vital organs such as heart, brain and kidney. Statins are currently the first-line drugs to prevent the coronary heart disease and atherosclerosis caused by high blood cholesterol. A number of statins are available on market, namely, atorvastatin, simvastatin, fluvastatin, pravastatin, lovastatin, and rosuvastatin. Atorvastatin was considered safest due to its least adverse reactions and the best efficacy. Studies have shown that only atorvastatin can significantly reduce the mortality of acute coronary syndrome (ACS).Being a 3-hydroxy-3-methylglutarylcoenzymeAreductase (HMG-CoA) inhibitor, atorvastatin is used in the form of calcium salt. It was registered in Germany and the UK in February 1997, firstly approved in the UK market in April 1997, and approved in the China market in September 1999.Wuzhi capsule (WZC) is made of ethanol herbal extract of Schisandra sphenanthera (Nan-Wuweizi), has been widely used to improve liver function in patients with chronic hepatitis and liver dysfunction. It has been reported that the components in Schisandra could affect the pharmacokinetic profiling of some drugs, particularly those CYP3A4 substrates. Since atorvastatin is also mainly metabolized by CYP3A4, it may add the risk of adverse reaction when atorvastatin is co-administered with Wuzhi capsule.Objectives(1) To set up a high performance liquid chromatography (HPLC) method to measure the concentration of Atorvastatin in rat plasma.(2) To investigate the effect of WZC on the pharmacokinetic profiling of Atorvastatin in rats.(3) To investigate the inhibitory effects on WZC and its main components on the metabolism of atorvastatin in vitro using rat liver microsome.Methods(1) An HPLC method for the determination of atorvastatin in vitro and in vivo samples. Chromatographic condition:an Intersil C18 column (250 X 4.6mm,5μm) was used. The mobile phase was composed of ammonium acetate (0.02M; pH4) and acetonitrile (1:1, v:v). The flow rate was 1.0 mL/min. The UV wavelength was at 246 nm and the column temperature was 30℃. The method validation included the specificity, sensitivity, precision, accuracy, extraction and stability.(2) Twelve Sprague Dawley (SD) male rats were randomly assigned into two groups, namely, control group:Atorvastatin, and test group:Wuzhi capsule+Atorvastatin. Each group consisted of 6 male or female rats. Rats underwent overnight fasting but were allowed to drink water freely. Blood samples were collected at 13 time points for 24 hours and atorvastatin concentration in plasma was measured.(3) Six SD female rats were treated with Wuzhi capsule (150 mg/kg, i.g.) daily for 6 days, and on the seventh day they underwent overnight fasting and were allowed to drink water freely before atorvastatin administration. Blood samples were collected for at 13 time points 24 hours and atorvastatin concentration in plasma was measured.(4) Twelve healthy SD female rats were randomly assigned into two groups:A:low dose of SchisantherinA (0.86 mg/kg)+Atorvastatin; B:high dose of SchisantherinA (12.69 mg/kg)+Atorvastatin. Rats underwent overnight fasting but were allowed to drink water freely. Blood samples were collected for 24 consecutive hours and atorvastatin concentration in plasma was measured.(5) Female rat liver microsomes were used in vitro incubation to study Wuzhi capsule and its main components (SchisandrolB, SchisantherinA, SchizandrinA) on the inhibition of atorvastatin.Results(1) Quantitative analysis method of atorvastatin was developed in rat plasma, which met the current FDA criteria for bio-analytical method validation and was successfully applied to the determination of atorvastatin concentrations in in vivo and in vitro. The calibration curve of atorvastatin showed good linearity in the range of 50-2000 ng/mL, The limit of quantification was 50 ng/mL. The RSD (%) of intra-and inter-precision was less than 15%(n= 5) and the extraction recoveries was higher than 80%(n= 5). Atorvastatin was stable under room temperature for 24 hours or -70℃ for one month.(2) The in vivo results showed that WZC increased the mean plasma concentration of atorvastatin in female rats but not in male rats. Compared to the control group of atorvastatin alone (Cmax:293.83±188 ng/mL and AUC0-24h:2227.64±670 ng·h/mL), the pharmacokinetic parameters of atorvastatin (Cmax:979.87±529.59 ng/mL and AUCoâ†'24h:6817.81±2045 ng-h/mL) was increased by about 3-fold in female rats with a single intragastric administration of WZC. Pretreatment of female rats with WZC for 6 days could also significantly increase the systemic exposure of atorvastatin (Cmax: 621.87±199.2 ng/mL and AUC0â†'24h:3768.83±659.63 ng·h/mL), but to less extent than did the single intake of WZC. However, Cmax (468.30±102.01 ng/mL) and AUC0-24h (3108.87±949.25 ng·h/mL) of atorvastatin in the control group for male rats were not significantly different from those in the group with the concomitant use of WZC (Cmax:378.78±123.02 ng/mL and AUC0â†'24h:2611.66±629.37 ng-h/mL). Similar to WZC, the high dose of SchisantherinA (12.86 mg/kg) could significantly elevate the Cmax and AUCo-24h of atorvastatin in female rats by 2.3 and 2.1-fold, respectively, but low dose of SchisantherinA had no influence.(3) The final protein concentration of female rat liver microsome was 1 mg/mL and the atorvastatin final concentration was 1μM in the incubation system. The mixture (100μL) was incubated in a 37℃ shaking water bath for 40 min. The IC50 for SchisantherinA, SchisandrolB, SchisandrinA was 0.68μM,9.13μM,27.68μM, respectively.ConclusionThe HPLC method developed is convenient, accurate, selective and sensitive, which was successfully applied to the determination of atorvastatin concentrations in in vivo and in vitro samples. We also built a stable and reliable incubation system in vitro for the study of atorvastatin and its interactions. WZC and one of its major components (SchisantherinA) could markedly increase the plasma exposure of atorvastatin in female rats, but not in male rats. The inhibition of CYP3A might explain the drug-drug interaction between atorvastatin and WZC. Long term administration of WZC exhibited less effect than short term administration of WZC, suggesting possible induction effects on CYP3A by WZC. |
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