Pharmacokinetics Study Of Domestic Pitavastatin Calcium Tablet

IntroductionToday 3 -hydroxy-3 -methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, more commonly known as the statins, are the most often prescribed drugs among the therapeutics for hypercholesterolemia. Statins inhibit the synthesis of mevalonate, a ratelimiting step in cholesterol biosynthesis, leading to a reduction in the plasma low density lipoprotein (LDL)-cholesterol level. High plasma LDL-cholesterol is a risk factor of cardiovascular diseases and, therefore, cholesterol-lowering drugs are used to prevent them. Some randomized controlled trials have shown that statins have potent cholesterol-lowering effects and reduce the risk of cardiovascular diseases in everyday medical practice. Pitavastatin (Kowa Company Ltd, Tokyo, Japan) is a novel statin used in the treatment of hyperlipemia. It is a new synthetic HMG-CoA reductase inhibitor, which is developed, and has been available in Japan since 17 July 2003. It is becoming available in Europe.In studies, pitavastatin at doses of 1, 2 and 4 mg has been well tolerated with a safety profile that is comparable to the other statins. Pitavastatin at 1, 2 and 4 mg doses appears to be as efficacious as 10, 20 and 40 mg atorvastatin. At 12 weeks, pitavastatin reduced total and LDL cholesterol by 28 and 38%, respectively, compared to 14 and 18%, respectively, in the pravastatin arm. The effect on HDL was similar in both groups. Both drugs were equally well tolerated and adverse events were similar. The first domestic tablet of pitavastatin is designed by domestic pharmaceutical company. In the following study, we have set up the method of determination of pitavastatin in human plasma by HPLC-MS/MS in this experiment. Then we investigate the pharmacokinetics of pitavastatin for clinical application of the drug after oral administration of different dosages of pitavastatin calcium tablets in Chinese healthy volunteers.ObjectiveTo establish a HPLC-MS/MS method for determination of pitavastatin in human plasma and application in pharmacokinetic study after oral administration of different dosages of pitavastatin calcium tablets in Chinese healthy volunteers.Methods1. Establishment the method for determination of pitavastatin in plasma with HPLC-MS/MS.Pitavastatin was determinated by HPLC-MS/MS with hydrochloride paroxetine as internal standard. Plasma sample pretreatment involved simple protein precipitation by addition cetonitrile to 200μl of plasma sample volume. Separation was achieved on an Agilent Zorbax SB-C18 (4.6×150 mm, 1.8μm) column at a temperature of 25℃using an isocratic elution method with methanol-0.1% formic acid in water (85:15, v/v)) at a flow rate of 0.4 mL·min^-1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 422.0→290.1 for pitavastatin, and m/z 330.1→192.1 for paroxetine. The method was corroborated according to acceptance criteria of industrial guidance for the bioanalytical method validation, including examination of method specificity, standard curve preparation, base effect investigation, absolute rate of recovery, relative rate of recovery as well as stability investigation.2. Investigation of pharmacokinetics for pitavastatin in human plasma. Before study, this project was approved by ethics committee of the First Affiliated Hospital, College of Medicine, Zhejiang University. Twelve healthy volunteers were recruited. According to 3×3 crossover trial design, 12 healthy adult volunteers were given three oral dosage levels (1 mg, 2 mg, 4 mg) of pitavastatin calcium tablets before meal, respectively. 4ml of venous blood sample were collected, then centrifuged for 15min at 4000 rpm and the plasma was preserved at -70℃in PP tube. The time for collection of each dose were set at 10min,20min,30min,45 min,1 h,1.5 h,2h,3 h,4h,6h,8h,12 h,16 h,24 h,48 h,72 h after administration. The concentration of pitavastatin in plasma was quantitated by HPLC-MS/MS. Main pharmacokinetics parameters such as Cma_x,t_max,Ke,t_1/2,AUC0-t and so on were calculated. The AUC0-t was calculated with trapezia method. AUC0-iμf = AUC0-t + Ct /Ke. Tmax was statistical managed by non-parametric method. The main parameters of pitavastatin was analysed by DAS 2.0 software.The results demonstrated that:(1) Calibration curves were linear in the concentration range of 0.15-150 ng·mL^-1, with limit of detection (LOD) of 0.06 ng·mL^-1 (S/N≥10). Method recoveries were between 95.6% - 102.8%. Intra-day precision (RSD) and inter-day precision was less than 8.78%. A high recover efficency, degree of precision and degree of accuracy was obtained. The HPLC-MS/MS method had a high sensitivity (having low quantitation limits as 0.06 ng·mL^-1), specificity, linear correlation, consistent to analysis requirement.(2) Healthy subjects were administrated pitavastatin, the blood samples were determined by HPLC-MS/MS and the pharmacokinetics data was calculated. Two-compartment Model was fitted better than others. The main pharmacokinetic parameters of pitavastatin after single oral doses (1, 2 and 4mg) were as follows respectively: t_1/2β: (11.39±7.66)h, (10.00±7.30)h, (11.30±7.95)h; T_max: (0.833±0.289)h, (0.729±0.198)h, (0.854±0.458)h; C_max: (30.48±11.66)n·mL^-1, (60.80±22.97)ng·mL^-1, (120.98±35.51) ng·mL^-1; K₁₀: (0.299±0.144 )h^-1, ( 0.217±0.0454 ) h^-1, ( 0.318±0.120 ) h^-1; AUC_{0→t}: ( 93.19±26.61 ) ng·mL^-1·2 h, (179.5±52.85 ) ng·mL^-1·h, ( 364.4±94.74 ) ng·mL^-1·h;AUC_{0→?}: ( 96.70±27.42 ) ng·mL^-1·h, (183.3±53.62 ) ng·mL^-1·h, ( 372.9±95.84 ) ng·mL^-1·h; MRT_{0→t}: (10.82±1.24) h, (10.05±1.13) h, (10.05±1.76) h;MRT_{0→∞}: (13.98±2.08) h, (11.90±1.87) h, (12.09±2.05 ) h; CUF: (0.0151±0.0064) L·h^-1, (0.0152±0.0036) L·h^-1 (0.0149±0.0050) L·h-1; V/F: (0.2154±0.128) L, (0.2195±0.1699) L, (0.2293±0.1709) L.(3) Safety evaluation: The whole trial was under continuous medical supervision by physicians. Eleven of the twelve volunteers had good tolerance to pitavastatin and was not found obvious adverse reactions. There was no significant difference in the results of hematology, blood biochemistry, urine and electrocardiogram analysis before and after the trial. Creatine kinase (CK) out of normal range was observed in only one of the twelve volunteers. After several days of surveillance and observation, without any medical interfere , the indicator fell to normal.ConclusionThis HPLC-MS/MS technique was found to improve the sensitivity of pitavastatin in plasma and well successfully used in pharmacokinetic studies; the main pharmacokinetic parameters of Chinese are similar to foreign who were different ethnics.