Research Design
The study of the Lafsefik DS bioequivalence test containing cefixime trihydrate 100 mg/5 ml quantitatively examined the in vitro comparative dissolution test and in vivo bioequivalence test with a randomized, cross-sectional study two-way, double-blind, cross-sectional study design with a single dose. The study compared the results of the Comparative Dissolution Test and Bioequivalence Test of the Lafsefik DS test drug product containing cefixime trihydrate 100 mg/5 ml produced by the Air Force Pharmaceutical Institute with the drug product Suprax oral suspension containing cefixime trihydrate 100 mg/5 ml produced by Odan Laboratories, Canada, with healthy subjects.
Subject
The total sample size (actual size) was 21 individuals, consisting of 8 men and 13 women. Subjects met the inclusion and exclusion criteria by being aged between 21 and 28 years, weighing between 45 and 74 kg, having a height between 148 and 179 cm, having a BMI between 18 and 25 kg/m2, and being willing to participate in the study from the beginning to the final study. Before commencement of the study, the patient was declared healthy by the general doctor during a standard medical check-up. Then, all the subjects received information regarding the study objective, and they signed informed consent forms. Prior to research, the patient’s information sheet, the protocol, and informed consent were reviewed and approved by the Ethics Committee of the Faculty of Medicine, The University of Indonesia (Protocol number: 092/BE/FL-IU/VII/2019). This study was carried out by the Declaration of Helsinki [14], Good Clinical Practice [15], Good Laboratory Practice [16], and ISO/IEC 17025:2017 [17].
Study Product
The test drug preparation Lafsefik DS (containing Cefixime 100 mg/5 ml, no. batch: A0110819) was manufactured by Air Force Pharmaceutical Industry (Bandung, Indonesia). The reference preparation Suprax OS is produced by Odan Laboratories Canada (containing Cefixime 100 mg/5 mL/, no. batch: E202762) as an innovator/comparator product.
The test product was subjected to a few comparative dissolution tests before the bioequivalence investigation, with the innovator product serving as the comparator. The dissolving test was carried out in three different pH media (pH 1.2, pH 4.5, and pH 6.8), reflecting the pH throughout the gastrointestinal tract where the study drug may be absorbed. In each medium, the dissolution profiles of the test and the comparison products had similarity factors of more than 50 (the value of similarity factors was 62.99% at pH 4.5 and 69.90% at pH 6.8, respectively, but pH 1.2 was 28.816% (<50%), which means there is no similarity with comparator drug).
Blood Sampling
The night before medication administration, all subjects attended PT. Farmalab Indoutama (Jakarta, Indonesia), where they were instructed to avoid all food and beverages besides mineral water beginning at 9 p.m. A predose pharmacokinetic blood sample was collected at approximately 6 a.m. on the dosage day (day 1), following an overnight fast. The study medication (one tablet of the test medicine or the reference drug) was administered with 200 mL of water at 7:00 a.m.
Blood samples (5 mL) were taken 17 times using a syringe at the following times: before drug administration (0 hours), 1 hour; 1.5; 2; 2.5; 3; 3.5; 4; 4.5; 5; 6; 8; 10; 12; 16; 24; and 32 after administration of the drug with a time deviation of 10%, within a maximum of 30 minutes from taking. Blood samples were centrifuged at 3000 rpm (1399 rcf and a rotor diameter of 27.8 cm) for 10-15 minutes, and the plasma obtained was transferred to labeled tubes.
After a week (i.e., the washout period), the same procedure was performed with the alternate drug. Using a 22 G drawing needle, blood samples from each subject were taken in vacuum polypropylene tubes containing citrate phosphate dextrose adenine (CPDA). Samples were processed at room temperature, and plasma was stored at -20°C to -45°C until cefixime analysis was performed.
The dates and timings of each sample collection were noted on the case report forms.
Analysis Method
The plasma concentrations of cefixime were measured using an ultra-pressure, high-performance liquid chromatography technique with a tandem mass spectrometer detector that was well-validated (UPLC Acquity H Class and Waters Xevo TQD equipped with an autosampler and MassLynx software). The analytical method of the comparative dissolution test for the test drug product Lafsefik DS and the comparison drug product/innovator Suprax Oral Suspension were carried out in triplicate with three solutions, namely, an HCl solution pH 1.2 (HCl 0.1 N), citrate buffer solution pH 4.5, and phosphate buffer solution pH 6.8 with a dissolution tester. Samples were taken at 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes. Then, the cefixime levels were examined with a spectrophotometer with a wavelength of 288 nm. The data were obtained in the form of percentage levels of cefixime of the Lafsefik DS test drug product and the percentage of cefixime levels of the comparison drug product Suprax OS.
Methods for the bioequivalence test were carried out in two stages randomly, crossed, two-way, and double-blind, with a single dose where everyone controlled himself for the test product and a comparison product they received. After the first stage, individuals did not receive drug administration for two weeks for residual drug clearance. Following this, they enter the second stage using the same method as the Lafsefik DS test drug product with the Suprax OS comparison drug product. All research subjects were given 15 mL of blood before being given the drug at T0 (0 hours), then given the drug according to the group, followed by taking blood at the following times at 1 hour; 1.5; 2; 2.5; 3; 3.5; 4; 4.5; 5; 6; 8; 10; 12; 16; 24; and 32 after 8 hours of fasting. Furthermore, the blood is separated from the plasma, and cefixime is added as an internal standard, followed by extraction with a precipitation process. The clear solution/supernatant is injected automatically into the UPLC–MS/MS system. Data on cefixime levels at stage one and stage two from each subject were collected.
Data Analysis
Data from the comparative dissolution test obtained from the examination of levels in buffer solutions of pH 1.2, pH 4.5, and pH 6.8 were compared for the Lafsefik DS test drug product and the comparison drug product Suprax OS. Calculation of the F2 value with the formula was conducted to see the similarity between the test drug product and the comparison/innovator drug product, according to the standard F2 value >50%, having similarities:
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)
where F2 is the similarity factor, Rt is the cumulative percentage of drug that dissolves at each sampling time of the drug product comparator, and Tt is the cumulative percentage of drug dissolved at each sampling time of the test drug product.