Bioequivalence study of two Esomeprazole40 mg MUPS tablets in healthy adult Bangladeshi human subjects

Background : The primary objective of this study is to investigate the bioequivalence of two formulations (test& reference) of Esomeprazole 40 mg MUPS Tablet in healthy, adult human subjects. Method : In this study, a single dose, two-sequence, two-way crossover randomized design was used to investigate the bioequivalence under fasting conditions. 14 healthy volunteers received the two formulations over the course of two treatment days, each followed by a seven-day washout period. Following the administration of a single dose of 40 mg of each formulation, blood samples were taken at predetermined time points and subjected to a validated LC-MS/MS method for Esomeprazole concentration analysis. The plasma concentration-time profiles of both formulations were used to determine the pharmacokinetic parameters C max , AUC 0-t , and AUC 0-∞ . Result : Pharmacokinetic parameters C max , AUC 0-t , and AUC 0-∞ did not show any statistically significant differences. The 90 % confidence intervals of C max , AUC 0-t , and AUC 0-∞ for Esomeprazole was found 89.35% (82.27-97.03%), 107.61% (99.78-116.05%), and 107.72 (99.87-116.20%) respectively which is within predetermined bioequivalence acceptance limits of 80 - 125 %. Conclusion : In terms of absorption rate and extent, the test product of Beximcopharmaceuticals has met the regulatory requirements for bioequivalence with the reference product Nexium MUPS.


Introduction
One of the most widespread illnesses, gastroesophageal reflux disease (GERD), is characterized by symptoms like regurgitation, heartburn, and tissue damage brought on by the reflux of gastric contents into the esophagus [1].For the treatment of gastroesophageal reflux disease (GERD), proton-pump inhibitors (PPIs) is extensively used [2].The most recent drug in the proton pump inhibitors family is Esomeprazole (PPIs) which was developed as the S-isomer of omeprazole to selectively inhibit the gastric H+/K+-ATPase enzyme, which is liable for secreting acid in the parietal cells of the stomach [3,4].
The purpose of this study was to determine the pharmacokinetic profiles of the two formulations of Esomeprazole in healthy adult male Bangladeshi subjects while they were fasting.

Subjects
The volunteers were chosen randomly from a database of registered volunteers and asked to take part in a pre-study screening process.On the basis of pre-study medical history, physical examination, clinical laboratory tests, ECG, X-ray, and inclusion-exclusion criteria volunteers were determined to be healthy.14healthy male volunteers (age 19-31 years, Body Mass Index (BMI) of 19.43-27.17kg/m 2 ) were enrolled in the study.Volunteers with pertinent clinical abnormalities were kept out of the trial.
Before participating in the study, each individual provided informed consent in writing and orally.
Post-study clinical examination was also carried out.

Study design
The bioequivalence study was carried out as an open-label, randomized, laboratory blind, single dose, two period, two treatments, two sequence, crossover study in which subjects received two formulations of Esomeprazole (either test or reference) with 240 ml water under fasting conditions [7].To ensure the fasting state, individuals were housed in the facility for one night prior to the trial (10 hours before study drug administration) [7].Study participants were confined till 24 hours post-dose.A standardized meal was given at 4 hours following dosing [7].To reduce the impact of food on the study's results, meal consumption was closely regulated and the same food was provided to all volunteers during both periods.Two periods were separated by a washout period of 7 days.Study medications were administered in two different sequences (Test-Reference) (TR); (Reference-Test) (RT) as per the randomization scheme generated by Statistical Analysis Software (SAS).All the subjects were divided into equal sizes so that the allotment of sequences was balanced over the period and sequence.
To separate plasma, blood sample vacutainers were centrifuged at 3500 Revolutions Per Minute (RPM) for 10 minutes at 5℃±3℃

Bioanalytical Method
Esomeprazole in plasma was determined by protein precipitation method using a validated Liquid Chromatography Mass Spectrometry(LC-MS/MS) method.Chromatographic separations were performed on a Hypersil Gold column (4.6 X 50 mm, 5.0µ particle size) at a flow rate of 0.600 mL/min.The method included the following: 100 μl of plasma sample, 100 μl of internal standard (rabeprazole, 0.8 μg/ml), and 1000 μl of a mixture of acetonitrile: water

Tolerability
Every subject was constantly monitored for the occurrence of any adverse events.Safety data were evaluated by monitoring vital signs at pre dose, 1.00, 3.00, 5.00, 7.00, 13.00, and 24.00 hours, check-in, and check-out in each period.Pre-study & post-study laboratory results were also evaluated for monitoring of adverse events.
In accordance with US Food and Drug Administration (FDA) requirement, the test product was considered to be bioequivalent to the reference product, if 90% confidence intervals for ratio (test/reference) of geometric least square means based on log-transformed primary PK parameters -Cmax, AUC0-t and AUC0-∞ fall within 80% to 125% [9].Following strict inclusion-exclusion criteria, fourteen healthy subjects were enrolled.Table 2represents demographic summary data for all evaluable subjects in the study.

Bioanalytical method validation
Bioanalytical method validation was carried out as per GLP & USFDA guidance on Bioanalytical Method Validation [10].
The method was sensitive & selective.The obtained chromatograms showed no interferences.Lower limits of quantification were 0.02μg/ml & the method's range was 0.02 to 4.00 μg/ml.With a coefficient of determination of 0.9969, the method was determined to be linear over this concentration range.

Tolerability
No incidence of serious adverse effects was reported in this study.Thus, in this study, the test product and reference product were safe and well tolerated by the subjects under fasting conditions.The 90% Confidence Intervals (CI) of the ratios (Test/Reference) between the two formulations regarding Cmax, AUC0-t, and AUC0-∞ were found 89.35% (82.27-97.03%),107.61% (99.78-116.05%),and 107.72 (99.87-116.20%)respectively which included into the range of bioequivalence acceptance limit of 80.00-125.00%(Table 4).

Figure 1
Figure 1 Structural formula of Esomeprazole

Figure 2 Figure 3
Figure 2 Linear Plot of Mean Plasma Concentrations versus Time profile of Esomeprazole

Table 1
Investigational Medicinal Product

Table 2
Summary of subject demographics

Table 3
Summary of Pharmacokinetic Parameters (N=14) : maximum plasma concentration of drug, AUC0t: area under the plasma concentration-time curve from time zero to the time of the last measurable concentration, AUC0-∞: area under the plasma concentration-time curve from time zero to infinity, Tmax: time to reach maximum plasma Concentration, Kel : elimination rate constant, T1/2: half-life of drug, Summary of statistical analysis is presented in Cmax

Table 4 Table 4
Summary of Statistical Analysis (N=14)