2 METHODS
2.1 Subjects
The fasting and feeding study protocols were approved by the ethics committee of the General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China and the written informed consents were obtained from the volunteers. The ethics committee’s appproval identification number is 2015–133. Forty-four healthy Chinese volunteers were recruited for this study. Subjects were all male, weighted 60-80 kg, aged 18-28 years, and had a normal body mass index range (19-24 kg/m2) [14]. Drugs, alcohol and caffeine-containing beverages, cigarettes, and nutritional supplements were refrained a week before commencement and throughout the study[15].
2.2 Study protocol[14]
The clinical protocols of fasting and feeding study were designed in a randomized two-process, two-phase, two-sequence, and crossover manner with a 2-week washout period[16,17]. In fasting study without breakfasting on day 1, subjects orally received 150 mg BUP (a tablet of 150 mg BUP SR; Disha, Shandong, China) or 150 mg BUP (a tablet of 150 mg BUP SR; Jingxin, Zhejiang, China) with 200 mL of water at 8:00 am. Then they drank 200 mL of water at 10:00 am, and ingested the meals at 12:00 pm and 18:00 pm. Subjects had no other foods except standard meals applied during the study. On day 15, subjects received another tablet at the same condition. Feeding study was designed as the same as fasting study except the high-fat breakfasting, containing one fried egg, one portion of hash brown potatoes, 220 mL of pure milk, and one drumstick 30 min prior to administration. Serial blood samples (5 ml) were collected using a forearm in-dwelling venous catheter 1 h prior to dosing and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 10, 12, 24, 48, 72 and 96 hour after BUP oral administration[14]. These blood samples were stored in EDTA-K2 tubes, and then centrifuged at 3000 rpm for 30 min. The separated plasma samples and blood cells were instantly stored at –80° until analysis.
2.3 Concentration assay[14]
The concentrations of BUP and HBUP in plasma were determined[18,19] with a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method[20,21](LC-30ATM, Shimadzu, Kyoto, Japan; API 4000TM, Applied Biosystems, Framingham, MA, USA). A Shimpack XR-ODSIII column (1.6 µm, 50×2.0 mm, Japan) and a mobile phase (acetonitrile:10 mM ammonium formate/ B:A) : 0 min, 5% B; 2.5 min, 30% B; 3.0 min, 30% B; 3.5 min, 5% B; 4.0 min, stop (v/v) at a flow rate of 0.3 mL/min were applied. Venlafaxine was used as the internal standard.
2.4 Calculation of pharmacokinetic parameters
AUC(0→96), Cmax, CL, Vd, tmax and t1/2 of BUP and HBUP of 44 subjects were calculated by non-compartmental method used DAS3.0 software package (Bojia Corp., Shanghai, China). Concentration-time curves and Tables were designed.
2.5 Genotyping of CYP2B6
The blood cells were extracted by Blood DNA Kit (50) (e.z.N.A.TM, OMEGA, Norcross, GA, USA) for genomic DNA. CYP2B6*4 (A785G ),CYP2B6*6 (A785G , G516T ) andCYP2B6*9 (G516T ) genotypes were ascertained after amplified by polymerase chain reaction (PCR) (Eppendorf AG, Germany) and restriction fragment length polymorphism (RFLP) [14]. The PCR conditions consisted of initial denaturation at 94° for 5 min, followed by 34 cycles of denaturation at 94° for 30 s, annealing at 60° for 40 s for A785G and 58° for G516T , and extension at 72° for 1 min. The final volume of the PCR was 50 uL, including 200 ng of DNA, 10 uM of each primer pair, 2.5 uM of dNTPs, 19 uL of dd H2O and 25 uL of Taq DNA polymerase (Takara, Dalian, China). Genotype of A785G was confirmed by StyI(thermo scientific, EU) at 60 ℃ overnight, and G516T was ascertained by BsrI (New England Biolabs, America) at 65° for 15 min[22].
2.6 Groups among high-fat diet, genotypes and pharmacokinetic parameters
Numbers of fasting and feeding groups were counted. Subjects were grouped into 4 groups by wild type and mutants under fasting and feeding condition. Pharmacokinetic parameters of 44 subjects were grouped based on CYP2B6 genotypes and feeding condition.CYP2B6*1/*1 and CYP2B6mutants were both contained in fasting and feeding groups. Concentration-time curves were drawn and Tables were tabulated according to categories.
2.7 Statistical analysis
Independent-Sample T and Mann-Whitney U or Kruskal-Wallis tests were used to evaluate AUC(0→96), Cmax, CL, Vd, tmax and t1/2between different groups of BUP and HBUP with 95% confidence intervals (CIs). The results were expressed as the mean ± standard deviation (Mean ± SD) in the Tables and Figures. Statistical results were performed with SPSS (version 22.0, Chicago, IL, USA) for windows. P<0.05 was considered statistically significant.