Formulations
The test product of Lisinopril/amlodipine tablets (batch no.:180101; expiration date: December 2019) was produced by Sichuan MEIDAKANG Pharmaceutical Co. Ltd (Sichuan Province,China), and developed by Sichuan Sunrise Biopharm Co. Ltd (Sichuan Province, China).
The reference product of Lisonorm® (batch no.:T79030A; expiration date: September 2019) was produced by Gedeon Richter Ltd (Hungary).
Subjects
Healthy volunteers that meet the inclusion criteria and not the exclusion criteria were enrolled in the study after the clinical and laboratory examinations. The inclusion criteria included as follows: 1) healthy male and female aged over 18 years; 2) the Body Mass Index is in the range of 19.0 to 26.0 kg/m2 (both inclusive), and males with minimum of 50 kg weight, females with minimum of 45 kg weight; 3) subjects have no clinically significant abnormalities, including vital signs (BP should not be less than 90/60 mmHg and the heart rate ranges from 50–100 beats per minute), physical examinations, laboratory tests, and ECG as determined by clinical examination; 4) agree to follow approved birth control methods.
Subjects were excluded if any of the following conditions were present: 1) allergic diathesis or hypersensitivity to investigational products; 2) history or presence of significant cardiovascular, urogenital, pulmonary, hepatic, renal, gastrointestinal, endocrine, immunological, dermatological, neurological or psychiatric disease or disorder, or other medical history affecting drug absorption; 3) use of any drugs or herbal medicine within 14 days; 4) smoking more than five cigarettes a day, abuse of alcohol or drugs, drinking too much tea, coffee or caffeinated drinks (more than 8 cups a day, 250 ml/cup); 5) donation or loss of blood or plasma > 400 mL in the past 3 months; 6) consumption of any beverages or food containing caffeine or products rich in grapefruit, such as coffee, tea and chocolate, etc., within 48 h prior to receiving study drug.
Ethic
The bioequivalence study has been registered on ClinicalTrials.gov (No.: NCT04885660, retrospectively registered in 13/05/2021) and been approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University on August 01, 2018 (No.: QYFYEC 2018–055–01). The study was performed at Phase I Clinical Research Center of the Affiliated Hospital of Qingdao University and was conducted in accordance with the Declaration of Helsinki, Good Clinical Practice (GCP) and applicable laws and regulations of China National Medical Products Administration (NMPA).Written informed consent was obtained from all subjects before their participation in the study.
Study design
This was a single center, randomized, open-label, single-dose, two-period crossover bioequivalence study in both fasting and fed conditions. According to pre-test and previous studies, the intra-subject coefficient of variation (CV), which was calculated by the standard deviation (SD) according to the formula: CV = [exp (SD2)-1]1/2, of lisinopril ranged from 20%-28% [8,9,10], while amlodipine ranged from 10–18% [13, 14]. Under the condition that α = 0.05, statistical test efficiency 1-β = 0.9, intra-subject CV was 0.28, equivalent lower limit was 0.80, upper limit was1.25 and actual ratio was 1.05, 35 samples were needed. Considering the possibility of shedding, 40 subjects were planned to be selected in each fasting and fed bioequivalence study. If the number of finally assessable subjects is less than 35, the substitute subjects can replace the subjects falling off. All eligible subjects were assigned according to the random table generated by SAS 9.4 and took the test (T) and reference(R) drugs respectively with 240 ml water after an overnight fast of at least 10 h (fasting study) or after the high-fat breakfast within half an hour before dosing (fed study). The high-fat breakfast contained about 900 kcal calories, and consisted of three pork buns with cabbage, spinach mixed with Yuba, and millet gruel. Subjects were forbidden to drink water within 1 h before and after taking the drug, and the lunch and dinner were provided at 4 h and 8 h respectively post-drug administration. No other food and beverage intake was permitted except the provided diets. A washout of 14 days was set between the two administrations, according to the half-life recorded in original drug instructions. 4 ml venous blood samples were collected before drug administration and at 1,2,3,4,5,6,7,8,9,10,11,12,13,24,36,48,72,96,144,168 h after administration. The samples were centrifuged at 1700gear per minute for 10 min at 4℃ to separate the plasma, which was divided into two aliquots (drug monitoring at least 800ul and backup) and stored at - 80℃until analysis. The study flow chart is presented in Fig. 1.
Bioanalytical assay
Plasma concentrations of lisinopril and amlodipine were measured by an established and validated liquid chromatography-tandem mass spectrometry (LC–MS/MS) method at Suzhou Haike Pharmaceutical Technology Co., Ltd (Suzhou, Jiangsu Province, China). For the analysis of lisinopril, plasma samples were pretreated by liquid liquid extraction with isopropanol: ethyl acetate(1:2,V/V); LSN-d5 was used as internal standard; 5 mM ammonium acetate aqueous solution, 0.01% formic acid and methanol were used as mobile phase; chromatographic separation was performed on Atlantis-dC18 column(Waters, Massachusetts, USA) and the analytes were detected using Triple Quad TM 5500 tandem mass spectrometer(Sciex, Canada) in positive ion mode, with ion spray in multiple reaction monitoring mode. The lower limit of quantification (LLOQ) was 0.500 ng/mL and the assay dynamic range was 0.500-100 ng/mL. The intra- and inter-day accuracy ranges were 93.8–108.5% and 98.7–104.6%, while 93.8–99.9% and 96.5% for LLOQ, respectively. The intra- and inter-day precision coefficient of variation (CV) % were < 4.3% and < 5.6%, while 5.5% and 5.2% for LLOQ, respectively. The analytes in matrix were stable when stored at -20℃ for 26 days, at -80℃ for 169 days and after four freeze–thaw cycles.
Amlodipine plasma concentrations were determined using a liquid chromatography unit (Shimadzu, LC-30AD, Japan) and a mass spectrometer (Sciex, Triple Quad TM 6500 plus, Canada). Under multiple reaction monitoring, LC–MS/MS system adopts positive ionization mode. For the analysis of amlodipine, the LLOQ was 0.050 ng/mL and the assay dynamic range was 0.050–10.0 ng/mL. The intra- and inter-day accuracy ranges were 99.4–105.5% and 102.0–103.8%, while 104.7–107.6% and 105.8% for LLOQ, respectively. The intra- and inter-day precision coefficient of variation (CV) % were < 6.0% and < 5.3%, while 8.1% and 5.2% for LLOQ, respectively. The analytes in matrix were stable when stored at -20℃ for 91 days, at -80℃ for 207 days and after four freeze–thaw cycles.
Data collection and analysis was performed with Analyst 1.6.3 software (Sciex, Canada) and Watson LIMS (Thermo, USA). Calibration curves were constructed using linear regression equation obtained by the weighted (W = 1/X2) least square method fitting for both analytes. Quantitation of quality control and clinical samples were also performed by the Analyst software using the same mathematical algorithm as that used in the calibration of standard curves.
Pharmacokinetic analysis
PK parameters for lisinopril and amlodipine in plasma were estimated by a non-compartmental model (NCA) using Phoenix WinNonlin version 7.0 software (Pharsight Corporation, St Louis, MO, USA). For the purpose of bioequivalence analysis, the maximum observed concentration (Cmax), the area under the plasma concentration–time curve from time 0 to the last measured time point (AUC0-t), and the area under the plasma concentration–time curve from time 0 to infinity (AUC0-∞) were considered as primary PK parameters. The secondary PK parameters were the observed time to Cmax (Tmax) and the apparent terminal half-life (T1/2). Cmax and Tmax were the factually measured data and AUC0-t was calculated using the linear and logarithmic trapezoidal methods. AUC0-∞ was calculated according to the following formula: AUC0-∞ = AUC0-t + Clast/λz (Clast is the last measurable concentration and is the first order rate constant of terminal elimination determined from a linear regression line after logarithmic transformation at the end of concentration time curve. λz is the slope calculated by linear regression after logarithmic conversion at the end of the concentration–time curve). T1/2 was calculated to be ln2/λ.
Safety assessment
The safety was evaluated by monitoring vital signs, physical examination, laboratory tests, electrocardiogram (ECG) and adverse events (AEs) collected after dosing throughout the study. Vital signs, including body temperature, blood pressure (BP) and heart rate, were measured at screening, before drug administration and at 2, 4, 6, 8, 12, 24, 36, 48, 72, 96, 144, 168 h after administration. Routine laboratory tests (hematology, urinalysis, serum chemistry and pregnancy test for females) and 12-lead ECG were conducted at screening and before removal from the study. The AEs, including all subjective symptoms reported by subjects and objective signs observed by clinical investigators, were recorded and assessed for their severity and the correlation with research drugs.
Statistical analysis
Statistical analysis was performed by SAS 9.4. Demographic characteristics, safety parameters and pharmacokinetic data were summarized using descriptive statistics, the results were presented as the mean ± SD and the differences between groups were determined by two one-sided tests. The probability value (P) less than 0.05 was considered statistically significant. AUC and Cmax were logarithmically transformed and analyzed by linear mixed effect model. Sequence, period and formulation were fixed effects, and subject within sequence was included as a random effect. Analysis of variance (ANOVA) of cross-over design was performed on the log-transformed variables. The geometric mean ratios (GMRs) of the primary pharmacokinetic parameters and their 90% confidence intervals (CIs) were calculated, and the test formulation was judged as bioequivalence if it fell within the equivalent range (80–125%). Bioequivalence was assessed separately in both the fasting and fed groups.