Ethics
The trial was performed abiding by the Declaration of Helsinki [7], Good clinical practice (GCP) [8] and the guidelines of China National Medical Products Administration (NMPA). Relevant documents, including protocol, informed consent and drug inspection report were all approved independently by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University (No.: QYFYEC 2018-065-01). All protocol violations have been reported to the Medical Ethics Committee.
Subjects
The inclusion criteria for the volunteers included as follows: 1) Healthy male and female aged 18 and above. 2) The body mass index is in the range of 18.6~28.5 kg/m2 (including the boundary value). The weight of male is not less than 50.0 kg, and that of female is not less than 45.0 kg. 3) The following inspection indexs are normal and abnormal without clinical significance. The inspection including: vital signs, physical examination, blood routine, blood biochemistry, urinalysis, serological tests for hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), and syphilis virus, 12-lead electrocardiogram (ECG), breath test for alcohol, drug abuse test, pregnancy test for female. 4) The subjects have no family planning within 3 months and could select contraceptive method. 5) Before the study, all subjects have been informed of the study’s purpose, protocal, benefits and risks, and signed the informed consent voluntarily.
The exclusion criteria included as follows: Being allergy to the study medications, smoking, alcohol abuse, and participation in another clinical trial within 3 months.
Study design
The single-dose randomized, open-label, two-period crossover study was executed in the Phase I Clinical Research Center of the Affiliated Hospital of Qingdao University. According to the random table generated by SAS 9.4, the subjects were divided into four groups (Table 1). The qualified volunteers were hospitalized in the Phase I Clinical Research Center, and fasted for 10 hours overnight until administration. Levamlodipine at a single dose of 5 mg or amlodipine at a single dose of 10 mg was swallowed with 240 ml water at room temperature. 4 mL blood samples were taken before administration and at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 24, 36, 48, 72, 96, 120, 144, 168 hours after administration. The samples were centrifuged at 1,800 g for 10 min at 4 °C to separate the plasma. The plasma samples were divided into two aliquots and stored at -80 °C until bioanalysis. Since the half-life of levamlodipine is about 30~50 hours, washout period, the interval between two administration, was set at 21 days. The operation of the two periods was consistent. Moreover, in the fed group, the high-fat breakfast was arranged within half an hour before taking the medicine.
Safety assessment
The safety was assessed by monitoring vital signs and laboratory tests. Vital signs, such as body temperature, blood pressure, and heart rate, were measured before administration and at 3, 8, 24, 36, 48, 72, 96, 120, 144, 168 hours after administration. Before removal from this study, the subjects were evaluated with blood routine, blood biochemistry, urinalysis, pregnancy test for female, and 12-lead ECG. For the adverse events (AEs), clinical symptoms, severity, occurrence and ending time, duration, treatment measures and the correlation with the drugs were recorded. All of the AEs that occur within the 7 half lives of the drug were recorded and followed up, unless the subjects returned to normal or stable, or failed to visit.
Bioanalysis
The analysts were blinded to the randomization. Plasma samples were determined by the liquid chromatography-tandem mass spectrometry (LC-MS/MS), which was tested by Suzhou Shenglin Pharmaceutical Technology Co., Ltd. An ACQUITY ultra-high-performance liquid chromatography unit (SHIMADZU, Nexera UHPLC LC-30A, Japan) and a mass spectrometer (Applied Biosystems, MDS Sciex, Triple Quad 6500 plus, Concord, Canada) were used in the study. Under multiple reaction monitoring, LC-MS/MS system adopts positive ionization mode. Data collection and analysis was employed with Analyst 1.6.3 software (Applied Biosystems, Foster City, CA, U.S.A.). The solid-phase extraction (SPE) experiments [9] were performed by HLB 96-well Plate (Waters Oasis, WAT058951). The plate contains a reversed phase functionalized polymeric sorbent (30 mg/well), in which particle size is 30 μm.
Figure 1 summarized the process of the cleaning and extraction. The cartridges were activated by 800 μL of methanol and cleaned by 800 μL of deionized water. The column was loaded with 150 μL of plasma sample or calibrator, 50 μL of working fluid (5 ng/mL) and 100 μL of deionized water. Clean the column twice with 500 μL deionized water. The column was depolarized with 80% methanol and washed twice with 500 μL pure acetonitrile to elute levamlodipine. Dry the solution with the pure nitrogen stream. 150 μL of pure acetonitrile was add into each sample, and mixed at room temperature for 10 minutes. Finally, 20 μL of the sample was injected into the LC-MS / MS system.
The method is verified fully by selectivity, accuracy, precision, calibration curve and stability. The drug concentration was linear within the range of 0.05~10.0 μg × L–1. The lower limit of quantification was 0.05 μg × L–1, and the equation was Y = 0.55667X - 0.0030182 (r2 = 0.9949). The intra- and interday maximum precision was 5.4% and 4.8%, respectively. The intra- and interday accuracy was -6.7~3.9% and -3.3~3.3%, respectively. The extraction recovery of levamlodipine was 94.7±3.9%. There was no significant interference in selectivity and stability.
Pharmacokinetic analysis
All subjects completed the study and the data were included in the pharmacokinetic analysis. The pharmacokinetic parameters were calculated according to non-compartment model with Phoenix™ WinNonlin® 8.0 software (Pharsight, St. Louis, MO, USA). The value below the lower limit of quantification that occur before the first measurable concentration were set as zero. Subsequent values below the lower limit of quantification were excluded. The primary PK parameters were the maximum plasma concentration (Cmax), the area under the plasma concentration-time curve from 0 to the last measured time point (AUC0-t), and the area under the plasma concentration-time curve from 0 to infinity (AUC0-∞). The secondary PK parameters were the observed time to Cmax (Tmax) and the apparent terminal half-life (T1/2).
Statistical analysis
Analysis of variance (ANOVA) was performed on the logarithmically transformed Cmax, AUC0-t, and AUC0-∞ to assess the effects from subject, treatment, period, and preparation. Statistical data were presented as mean ± standard deviation (SD). The probability value less than 0.05 is considered statistically significant. The GMRs of the primary PK parameters and their 90% confidence intervals (CIs) were calculated. If it is within the equivalent range (80 ~ 125%), it is judged as bioequivalence, and the results of double unilateral t-test are listed. Tmax was analyzed by non-parametric statistical test. Statistical analyses were performed by SAS 9.4 (SAS Institute Inc. Cary, NC, USA).