Drugs and preparations Test preparation (T): Azilsartan tablets, strength: 20mg per tablet, batch number: 20180108, produced by Zhaoke Pharmaceutical (Guangzhou) Co., Ltd.; reference preparation (R): Azilsartan tablets (trade name: Azilva®), strength: 20mg per tablet, batch number: AH2101, produced by Takeda Pharmaceutical Company Limited. Azilsartan standard, analyte: Azilsartan, purity: 97%, batch number: 5-SYQ-120-1; internal standard: Azilsartan-d5, purity: 98%, batch number: 5-XDD-19-2, all purchased from Toronto Research Chemicals, Japan.
Instruments LC-30AD high performance liquid chromatograph, Shimadzu, Japan; Triple Quad 5500 mass spectrometer, AB Sciex, USA.
2.2 Subject selection
The trial protocol was approved by the Clinical Medical Research Ethics Committee of the First Affiliated Hospital of Bengbu Medical College (ethics approval number: 2018112, the clinical trial registration platform registry number: CTR20191688, clinical trial registration platform website: www.chinadrugtrials.org.cn). All the subjects signed the informed consent. A total of 30 subjects were enrolled for the fasting test, including 19 males and 11 females aged (26.50±6.87) years old with body weight (62.20±8.44) kg and body mass index (BMI) (22.10±2.22) kg·m-2. A total of 30 subjects were enrolled for the postprandial test, including 20 males and 10 females aged (30.20±7.83) years old with body weight (64.88±7.56) kg and BMI (22.50±2.05) kg·m-2.
Inclusion criteria (1) Healthy Chinese aged 18-45 years old, male or female; (2) Male body weight ≥50.0kg, female body weight ≥45kg, BMI 19.0~26.0kg·m-2 (inclusive); (3) No history of abnormalities in heart, liver, kidney, digestive tract, nervous system and metabolic system, no history of postural hypotension, no history of syncope; (4) Non-lactating women and male subjects who are willing to adopt appropriate contraceptive methods throughout the study period.
Exclusion criteria (1) Abnormality with clinical significance in the vital signs, physical examination, clinical laboratory testing (blood routine, urine routine, blood biochemistry, four indicators for blood transfusion, coagulation routine, female pregnancy testing, drug screening), 12-lead ECG; (2) Subjects with diseases of the nervous system, cardiovascular system, blood and lymphatic system, immune system, digestive system, respiratory system, metabolism and skeletal system, or with any other diseases or physiological conditions that can interfere with the trial results; (3) Subjects (female) are pregnant or breastfeeding; (4) Subjects with a history of allergy to angiotensin receptor antagonists or other antihypertensive drugs or antihypertensive biological agents; (5) Usage of any drug or food with mutual effect with Azilsartan and that can affect its absorption, distribution, metabolism and excretion within one month before screening; (6) Subjects who have donated blood, lost blood (greater than 200ml) or participated in another clinical trial within 3 months prior to screening; (7) History of smoking addiction, alcoholism and drug abuse.
2.3 Grouping, administration and blood sample collection
The study was designed as a single-center, randomized, open-label, single-dose, double-cycle, double-cross clinical trial.
30 subjects were enrolled in both fasting and postprandial tests and randomized to 2 administration sequence groups (T-R or R-T) by 1:1. Each subject underwent 2 cycles of test according to the determined administration sequence with a 7-day wash out period between 2 cycles. The subjects were admitted to the ward one day before administration, and were given test or reference Azilsartan tablet 20mg (1 tablet) with 240ml warm water without food or after high-fat meal in the first cycle (day 1 of the trial) and the second cycle (day 8 of the trial), respectively. Subjects were asked to take the drug after fasting overnight for at least 10 hours for the fasting test, while the recommended high-fat meal was provided 30 minutes before drug administration and the meal would be finished within 30 minutes for the postprandial test. Subjects in both groups were fasted for 1 hour before and after drug administration, and fasted for 4 hours after drug administration. The lunch and supper were provided after 4 hours and 10 hours, respectively.
At a total of 18 timepoints at 0h (within -1 h) before administration and 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 16, 24, 36 and 48 hours after administration each cycle, 4mL of venous blood samples were collected each time from subjects into the blood collection tube with heparin sodium as anticoagulant. After mixing by gentle inversion, the samples were centrifuged by 2000g, 2-8°C for 10 min. After centrifugation, the plasma was evenly divided into two aliquots, one for assay and one for backup. The exact collection time for each blood sample were recorded in detail. If an venous indwelling needle was used for blood collection, approximately 0.5ml of blood was discarded before each blood collection, and an appropriate amount of normal saline was used to seal the indwelling needle after each blood collection. The blood samples to be tested and the backup blood samples were temporarily stored in a low-temperature refrigerator at -20°C in the Phase I ward, and transferred to an ultra-low temperature refrigerator at -60~-90°C at a later time for testing and analysis.
During the trial, the research physicians and nurses asked the subjects about their subjective feelings and observed the subjects for the occurrence of adverse events. Vital signs were measured for the subjects during the screening period, before admission, 1h before administration, 2h, 6h, 12h, 24h and 48h after administration, and the permissible time window for vital signs monitoring was ±30min. In case of serious adverse events, corresponding emergency measures and treatment would be taken. After the completion of the two-cycle trial, the subjects underwent physical examination and laboratory testing, and those without abnormalities left the ward and the study was completed.
2.4 Assay and sample handling
Chromatographic conditions Chromatographic column: Phenomenex Synergi 4 μm Fusion-RP 80A (50 × 2 mm); mobile phase A: 0.5% formic acid and 10 mmol/L ammonium acetate in water, mobile phase B: 100% acetonitrile; column temperature: 40°C; flow rate: 0.4mL·min-1; injection volume: 5.00 μL. Gradient elution procedure: 0.01~1.3min, 45% B; 1.3-1.5 min, 45%-85% B; 1.5~2.3min, 85% B; 2.3~2.4min, 85%-45% B; 2.4~3.2min, 45% B.
Mass spectrometry conditions Electrospray ion source: positive ion detection; scanning mode: multiple reaction monitoring (MRM); ion source voltage: 5500V; curtain gas: 310.26kpa; collision gas (CAD): 68.95kpa; nebulizer gas (GS1) pressure: 275.79kpa; auxiliary gas (GS2) pressure: 344.74kpa; ion source temperature: 550°C; the ionic reactions used for quantitative analysis were Azilsartan m/z 457.1→233.0 and Azilsartan-d5 m/z 462.2→233.0.
Plasma sample handling After the sample was thawed at room temperature and fully vortexed, 50ul sample was added into a 0.6mL centrifuge tube, 200ul internal standard working solution (100ng/mL of Azilsartan-d5 in acetonitrile) was added to the sample, and centrifuged at 12000g and 4°C for 10min after shaking for 3min. 30ul supernatant was transferred to a 96-well plate containing 270ul of 45% methanol aqueous solution. After shaking the 96-well plate for mixing, the samples were centrifuged at 3000g and 4°C for 10min before placing the 96-well plate into an automatic sampler for injection.
2.5 Methodology investigation and evaluation
Specificity 6 different sources (batches) of human blank plasma were used for assay of blank samples without internal standard and for the addition experiment spiking Azilsartan standard solution and internal standard, respectively, according to the “plasma sample handling” section. The results showed that all the 6 batches of matrix passed the test with the retention times of 1.05min and 1.04min for Azilsartan and the internal standard Azilsartan-d5, respectively. The results indicated that the endogenous substance in blank plasma did not interfere with Azilsartan and internal standard, and there was no mutual interference between Azilsartan and internal standard, showing good specificity of this method. The chromatograms are shown in Figure 1.
Standard curve and lower limit of quantitation(LLOQ) Standard curve samples were taken for 8 mass concentration levels: 3.00, 6.00, 20.0, 90.0, 300, 1000, 2500 and 3000 ng/mL, with 2 replicates for each mass concentration level. The samples were processed according to the “plasma sample handling” section to record the chromatogram for LC/MS-MS analysis. The linear regression was performed by the least square method with the plasma concentration of Azilsartan as the abscissa (x) and the ratio of Azilsartan peak area to Azilsartan-d5 peak area as the ordinate (y), and the weight factor of 1/x2. With the fitting equation of y=ax+b, the linear equation of the standard curve was y=2.49×10-3x+3.74×10-4 (r=0.9969). The linearity range of Azilsartan plasma concentration was 3.00 ~ 3000 ng·mL-1, showing good linear relationship with the lower limit of quantification of 3.00 ng·mL-1.
Precision and recovery Control samples were prepared at 5 levels, i.e. LLOQ, low, geometric medium, moderate and high concentration(3.00, 9.00, 100.00, 1500.00, 2400.00 ng·mL-1). According to the “plasma sample handling” section, 6 replicates were prepared for each mass concentration, and 3 batches were independently analyzed to calculate the accuracy, precision and absolute recovery within and between batches. The results showed that the relative standard deviations (RSDs) of both within and between batches were < 10%, and the relative error (RE) for accuracy was -2.02%~5.28%, which met the acceptance criteria of biological samples. The relative recovery was the ratio of the concentration of QC sample, calculated from the standard curve of the day, to the amount spiked. The mean recovery rates of Azilsartan were 90.11%, 88.40% and 87.17% respectively in QC samples with low, medium and high concentrations, and the coefficient of variation was no greater than 15%, meeting the requirements for recovery. See Table 1 for details.
Matrix effect The normal blank matrix of 6 different individuals was investigated at the QC levels of low concentration (9.00 ng/mL) and high concentration (2400 ng/mL) for each individual matrix. The matrix factor (MF) for analyte and internal standard was calculated by the ratio of peak area in the presence of matrix (pure solution of normal blank matrix after extraction with the analyte and internal standard added) to the peak area in the absence of matrix (pure solution of ultrapure water, instead of normal blank plasma, after extraction with the analyte and internal standard added). The normalized MF of the internal standard was calculated by dividing the MF of analyte by the MF of internal standard. The mean values of the internal standard normalized matrix factors of the low and high QC concentrations of the normal blank matrix of 6 different individuals were 1.01 and 1.00, respectively, and the coefficient of variation (%CV) was 1.83% and 0.40%, respectively. The total coefficient of variation of the internal standard normalized MF of the low and high concentrations was 1.30%, meeting the acceptance criteria.
Stability The short-term and long-term stability of analyte stock solutions and working solutions, plasma stability, freeze-thaw cycle stability (5 cycles of -75°C freezing/room temperature thawing), sample pretreatment stability (room temperature, 25.5h), sample stability after preparation (8°C, 180h), long-term stability of analyte in biological matrix (-20°C 40.5d, -75°C 40.5d), analyte stability before sample centrifugation (room temperature, 2h) were investigated. The results showed that both Azilsartan and internal standard could maintain stable under the above conditions.
2.6 Statistical analysis
WinNonlin8.2 software was used to analyze the pharmacokinetic parameters with the noncompartmental model for the plasma concentration data of Azilsartan. SAS 9.4 statistical software was used for the descriptive and inferential statistical analysis. After logarithmic transformation of major pharmacokinetic parameters (Cmax, AUC0-t, AUC0-∞), the mixed effect model was applied for multi-factor analysis of variance and two one-sided t test. The 90% confidence interval of the geometric mean ratio of the main pharmacokinetic parameters of the two drugs was calculated, non-parametric test was conducted for Tmax, and bioequivalence evaluation was conducted with the equivalence interval set to 80.00% ~ 125.00%.