Bioequivalence of levamlodipine besylate tablets in healthy Chinese subjects: a single-dose and two-period crossover randomized study

Levamlodipine, a calcium channel blocker, has been show act as a cardiovascular drug. To compare the pharmacokinetic parameters between levamlodipine (test formulation) at a single dose of 5 mg and amlodipine (reference formulation) at a single dose of 10 mg, the bioequivalence study was carried out. A single-dose randomized, open-label, two-period crossover study was designed in healthy Chinese subjects. 48 subjects were divided into fasted and fed groups equally. The subjects randomly received the test or reference formulations at the rate of 1:1. Following a 21-day washout period, the alternative formulations were received. The blood samples were collected at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 24, 36, 48, 72, 96, 120, 144, 168 h later. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to determine the plasma concentrations of levamlodipine. Adverse events were recorded. The 90% confidence intervals (CIs) of the ratio of geometric means (GMRs) of Cmax, AUC0-t, and AUC0-∞ under both fasted and fed conditions were within the prespecified bioequivalence limits between 80 ~ 125%. Under fasted conditions, 24 subjects were enrolled and completed the study. The mean Cmax was (2.70 ± 0.49) ng/mL, AUC0-t was (141.32 ± 36.24) ng × h/mL and AUC0-∞ was (157.14 ± 45.65) ng × h/mL after a single dose of 5 mg levamlodipine. The mean Cmax was (2.83 ± 0.52) ng/mL, AUC0-t was (153.62 ± 33.96) ng × h/mL and AUC0-∞ was (173.05 ± 41.78) ng × h/mL after a single dose of 10 mg amlodipine. Under fed conditions, 24 subjects were enrolled and completed the study. The mean Cmax was (2.73 ± 0.55) ng/mL, AUC0-t was (166.93 ± 49.96) ng × h/mL and AUC0-∞ was (190.99 ± 70.89) ng × h/mL after a single dose of 5 mg levamlodipine. The mean Cmax was (2.87 ± 0.81) ng/mL AUC0-t was (165.46 ± 43.58) ng × h/mL and AUC0-∞ was (189.51 ± 64.70) ng × h/mL after a single dose of 10 mg amlodipine. Serious adverse event was not observed. The trial confirmed that levamlodipine at a single dose of 5 mg and amlodipine at a single dose of 10 mg were bioequivalent under both fasted condition and fed condition. Cinicaltrials, NCT04411875. Registered 3 June 2020 - Retrospectively registered


Background
Hypertension is one of the most common risk factors of cardiovascular disease and stroke, which can lead to serious complications [1]. Amlodipine, a dihydropyridine calcium antagonist, has therapeutic effect on hypertension and angina pectoris. Amlodipine acted by inhibiting the influx of calcium through L-type calcium channels into vascular smooth muscle cells, preventing vasoconstriction while simultaneously improving blood flow. Usually, amlodipine is prescribed at a daily dose of 5 and 10 mg, may reach bioavailability of 60~90% [2]..Amlodipine is an racemic mixture, including (R)-amlodipine and (S)-amlodipine, but only the latter has therapeutic activity [3]. (S)-amlodipine, known as levamlodipine, similar to amlodipine in pharmacology, also play a role in vasodilation and decreasing blood pressure [4,5].
Pharmacokinetics of levamlodipine besylate 2.5-mg tablet in healthy male subjects was studied previously [6]. However, pharmacokinetic studies for other doses of levamlodipine have not been fully carried out yet. Furthermore, the bioequivalence between levamlodipine and amlodipine has never been verified. The bioequivalence study was designed to compare the pharmacokinetic parameters between levamlodipine at a single dose of 5 mg and amlodipine at a single dose of 10 mg.

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.   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 h 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 h after administration. The samples were centrifuged at 1800 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 h, 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 h 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 chromatographytandem 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 96well 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 min. 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 (r 2 = 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 noncompartment 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 (C max ), the area under the plasma concentration-time curve from 0 to the last measured time point (AUC 0-t ), and the area under the plasma concentration-time curve from 0 to infinity (AUC 0-∞ ). The secondary PK parameters were the observed time to C max (T max ) and the apparent terminal half-life (T 1/2 ).

Statistical analysis
Analysis of variance (ANOVA) was performed on the logarithmically transformed C max , AUC 0-t , and AUC 0-∞ 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. T max was analyzed by non-parametric statistical test. Statistical analyses were performed by SAS 9.4 (SAS Institute Inc. Cary, NC, USA).

Characteristics of the subjects
All subjects completed the study. A total of 24 subjects including 6 women and 18 men enrolled in the fasted group. Among them, 23 were Han and 1 was Manchu nationality. Parameter, mean ± SD (

Pharmacokinetics
Following a single dose of test formulations or reference formulations, the mean plasma concentration-time curves were shown in Fig. 2 (the fasted group) and Fig. 3 (the fed group). Individual plasma concentration-time data of levamlodipine was show supplementary file.   Mean pharmacokinetic parameters from the fasted group (Table 2) and the fed group (Table 3) are summarized. In the fasted group (Table 4), ANOVA for C max , AUC 0-t , and AUC 0-∞ indicated a lack of effects on treatment. ANOVA for C max indicated a significant difference in subjects (p ≤ 0.05). ANOVA for AUC 0-t (p ≤ 0.05) and AUC 0-∞ (p ≤ 0.05) indicated a significant difference in formulations and preparations. In the fed group (Table 5), ANOVA for C max , AUC 0-t , and AUC 0-∞ indicated a lack of effects in treatments and preparations. ANOVA for C max (p ≤ 0.05), AUC 0-t (p ≤ 0.05), and AUC 0-∞ (p ≤ 0.05) indicated a significant difference in subjects. ANOVA for AUC 0-t indicated a significant difference in periods (p ≤ 0.05).
The 90% CIs for the GMRs of C max , AUC 0-t , AUC 0-∞ and the power were presented in Table 6 (the fasted group) and Table 7 (the fed group). These ratios were within the predefined equivalence limit of 80~125%.

Safety
During the whole study period, both test preparation and reference preparation showed good tolerance. The AEs found in physical examination, 12-lead ECG and laboratory examination were listed in Table 8 (the fasted group) and Table 9 (the fed group). None of them were judged as serious adverse events (SAEs).

Discussion
On March 5, 2016, the general office of the State Council of the people's Republic of China issued opinions on the quality and efficacy consistency evaluation of generic drugs. Accordingly, the present study was performed to compare the pharmacokinetics of a newly-developed levamlodipine besylate tablet at a single dose of 5 mg (test formulation, anglikang Pharmaceutical Company Co., Ltd., Zhejiang, China) with that of a marketed amlodipine besylate tablet at a single dose of 10 mg (reference formulation, Pfizer Pharmaceuticals Limited, USA) for assessment of bioequivalence in healthy Chinese volunteers under fasted and fed conditions. The two medicinal products are bioequivalent when their 90% CI of the AUC 0-t , AUC 0-∞ and C max of the reference preparation over the test preparation fall between the predetermined limits of 80~125%. The two medicinal formulations were well tolerated. No subject withdrew from the study due to any AEs, and no SAEs occurred.
Amlodipine besylate tablets were developed by Pfizer Pharmaceutical Co., Ltd., in which the ratio of (R)-amlodipine to (S)-amlodipine is 1:1. The active component of amlodipine is (S)-amlodipine, also known as levamlodipine. In vivo, there is no mutual transformation between (R)-amlodipine and (S)-amlodipine. Before this study (November 13, 2019), there is no approved levamlodipine besylate tablet in the European Union, the United States, Japan, and China. Moreover, the pharmaceutical research of the the new developed test formulation is carried out according to the amlodipine besylate tablets developed by Pfizer. Combined with Chinese drug consistency evaluation catalogue, amlodipine besylate tablets (Norvasc®) produced by Pfizer was selected as the reference formulation in this study. On December 19, 2019, levamlodipine maleate tablets (Conjupri®) produced by CSPC Ouyi pharmaceutical Co., Ltd. was  approved by FDA. However, the acid radical of this product is different from the test formulation in this study. Therefore, NMPA approved that amlodipine besylate tablets developed by Pfizer was selected as the reference preparation in this study.
In this study, the parameters such as recoveries, matrix effects, linear range, lower limit of quantification, stability by specificity, precision, and accuracy specifications, were investigated to confirm the method of LC-MS/MS. Concentration of levamlodipine in human plasma showed good linear relationship within 0.05~10 μg × L − 1 . The relative standard deviation (RSD) values of intra-and interday precision were both less than 10%. The matrix effect induced by endogenous interfering substances in biological samples did not affect the ionization efficiency and signal intensity of levamlodipine [9]. According to FDA bioequivalence guidance, the final elimination half-life of amlodipine is about 30~50 h. Thus, washout period is set to 21 days, which is a length of time greater than seven half lives. And the blood samples taken for 168 h was enough.
In view of the inhibitory effect of amlodipine on CYP3A4 [10], subjects with behaviors of drinking, smoking and drug intake through CYP3A4 metabolism were excluded. During the trial, the administration of drugs metabolized by CYP3A4 did not occur. As an open-label study, AEs assessment may not be objective enough. When the test formulation passes the evaluation and enters the market, the efficacy and side effects need to be further explored.

Conclusions
The trial confirmed that levamlodipine at a single dose of 5 mg and amlodipine at a single dose of 10 mg were bioequivalent under both fasted condition and fed condition. If the test formulation levamlodipine can be approved by NMPA, it can be used in the treatment of hypertension in clinic.

Acknowledgments
We are grateful to anglikang Pharmaceutical Company Co., Ltd. Suzhou and Shenglin Pharmaceutical Technology Co., Ltd.

CONSORT guidelines
The study adheres to CONSORT guidelines and include a completed CONSORT checklist as an additional file.

Authors' contributions
The study was designed by YC. XL wrote the paper, participated in data statistics and performance. CJW, T L, YPL, SQL, YT, YPM and XMG performed research. The author(s) read and approved the final manuscript.

Funding
Test execution and data collection in this work were supported by grants from the National Major Scientific and Technological Special Project for "Significant New Drugs Development" (2020ZX09201-018). Layout fee of publication was supported by the China Postdoctoral Science Foundation (2016 M602100).

Availability of data and materials
We have shared the raw data by providing it in a supplementary file.

Ethics approval and consent to participate
The study passed the review of Medical Ethics Committee of the Affiliated Hospital of Qingdao University on August 30, 2018, and obtained the approval (No.: QYFYEC 2018-065-01). The written informed consents was obtained from all participants.

Competing interests
The authors report no conflicts of interest.