Bioequivalence Study of Zidovudine 100 Mg Capsule with Two- Way Crossover Design

Introduction

Zidovudine is a synthetic nucleoside analogue, specifically a nucleoside reverse transcriptase inhibitor (NRTI), that inhibits the replication of retroviruses, including HIV. Intracellularly, Zidovudine is phosphorylated into its active metabolite, 5′-triphosphate and zidovudine triphosphate (ZDV-TP). The level of phosphorylation varies depending on the cell type. ZDV- TP inhibits the activity of HIV by inhibiting the reverse transcriptase (RT) enzyme through the terminal DNA chain after separation with a nucleoside analogue. The lack of a 3’-OH group in the incorporated nucleoside analogue prevents the formation of the 5’ to 3’ phosphodiester linkage that is important for DNA chain elongation, and thus the growth of the DNA virus is halted. ZDP-TP is a weak inhibitor of α-DNA polymerase and mitochondrial γ-polymerase [1, 2, 3].

• Absorption After oral administration, Zidovudine is rapidly absorbed from the GI tract. Absorption is delayed by food. Bioavailability: Approximately 64%. Peak plasma concentration is reached within 0.5-1.5 hours.
• Distribution Zidovudine is distributed to CSF, crosses the blood-brain barrier and placenta, enters breast milk, and is found in semen. Its volume of distribution is 1-2.2 L/kg. Its plasma protein binding is 34-38%.
• Metabolism Zidovudine is metabolized intracellularly into active metabolite 5´-triphosphate and undergoes metabolism in the liver, mainly into inactive glucuronide.
• Excretion Zidovudine is excreted in the urine as both an unchanged drug and antimetabolites. The plasma half- life is around 0.5-3 hours [4, 5, 6].
• Adverse Event The side effects of Zidovudine include headache, malaise, anorexia, nausea, vomiting [4].
• This study objective to determine the bioequivalence of Zidovudine 100 mg capsules produced by PT Kimia Farma Tbk compared to Retrovir 100 mg capsules produced by GlaxoSmithKline Pharmaceuticals S.A., in healthy subjects.

Health Screening

Health screening is conducted prior to the study to evaluate the subject’s health condition based on inclusion and exclusion criteria. Subjects were through medical examination within 7 days prior to their first study drug administration day. These include assessment of physical examination, vital signs (i.e., blood pressure, pulse rate, and body temperature), and ECG was conducted by Responsible Physician in Equitrust Lab. Laboratory values of routine haematology, liver function, kidney function, blood glucose, and urinalysis were tested by Clinical Laboratory. Immunology test for HBsAg, HCV, HIV, and Rapid Test Antigen Covid-19 was conducted in Equitrust Lab. During the screening and immunology test (HBsAg, HCV, and HIV) approximately 10 mL of blood samples were drawn from each subject.

Drug Product Administration

This study consists of two periods during 10 hours for each period. At the specified time, the subjects will be asked to come to the Equitrust Lab on a day before drug administration for quarantine. The subjects are requested to report to the investigator regarding any health disturbances experienced and the medication or food supplements taken since their last arrival.

The subjects must fast for 8 hours before taking the investigational drug or reference drug. About 1 hour before taking the drug, the condition of the subjects is checked by a doctor to assess their health (blood pressure, pulse rate, body temperature, oxygen saturation (SpO2) examination, and respiratory rate). The results of the examination are recorded in the Case Report Form (CRF).

Starting at 7a.m, on the first sampling day, the subjects are given the investigational drug (1 capsule of 100 mg Zidovudine) or the reference drug (1 capsule of 100 mg Retrovir) with 220 mL of water while sitting according to randomization. The subjects are asked to maintain an upright position, either standing or sitting, for 1 hour after drug administration.

After 2 hours, water was provided. No food was allowed until 4 hours after drug administration. Standard meals were reserved for 4 hours (lunch), 8 hours (snack), and 12 hours (dinner) after study drug administration. Subjects were remaining in a sitting position until 4 hours period after drug administration. Subjects were not allowed to exit the clinical facility.

Blood Samples Collection

Blood samples were taken at specific time points to represent the drug absorption, distribution, and elimination phases. Most drugs require 12-18 blood samples, including 1 sample before dosing, 2-3 samples before Cmax, 4-6 samples around Cmax, and 5-8 samples after Cmax [6]. In the Zidovudine test, blood samples were taken for 10 hours. A pre-dose blood sample was taken at 6.30 a.m within 30-minutes prior to drug administration and a 5 mL of blood was collected through venipuncture using a syringe at the following time points: 0 hours (before drug administration); at 0.16, 0.33, 0.50, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, and 10 hours after drug administration (15 points). Every deviation exceeding the predetermined time is recorded in the Case Report Form (CRF) as a protocol deviation.

The blood samples are collected via venipuncture using a syringe and transferred into CPDA blood collection tubes. Plasma is centrifuged at 3000 rpm for 10 minutes and immediately transferred plasma into clean microtubes 2.0 mL. Plasma separation was carried out in preparation room at room temperature. Then, the plasma is stored in the freezer at a maximum temperature of -20 ºC until Zidovudine analysis is performed. The total amount of blood collected from each subject was 165 mL (including 15 mL for initial health screening).

Statistical Analysis

Pharmacokinetic parameters, including Cmax, AUC0-t, AUC0-inf, tmax, and t1/2, were calculated for each subject and each period using the Ms. Excel program. Bioequivalence

between the test and reference products was determined based on the average ratios of Cmax and AUC0-t with a 90% Confidence Interval (90% CI) of the log or ln-transformed data. The log or ln-transformed values of Cmax and AUC0-t for the two products are analysed using a two-way Analysis of Variance (ANOVA) and R program. The compared factors were the drug products (Test and Reference), drug administration period (I and II), subject, and sequence (TR and RT). The mean differences in Cmax and AUC0-t between the test and reference products were considered bioequivalent if the ratio of the geometric mean (AUC)T/ (AUC)R= 1.00 with 90% CI= 80.00-125.00% (α: 0.05) and (Cmax)T/(Cmax)R= 1.00 with 90% CI= 80.00-125.00% (α: 0.05). The study had a power of 80% with a significance level (alpha) of 5% (two-tailed) [7, 8, 11].

The 90% Confidence Interval (90% CI) is calculated using the following equation: (90% CI) diff = Difference ± t0.1(n-2) × SE diff Difference : the mean ln T - the mean ln R n : the number of subjects α: 0.05

1 1 1 SEdiff=[( Mresidual×( )) ] 2 nRT 2 The 90% CI for the ratio is calculated as follows: (90% CI) ratio = antilog (90% CI) diff × 100%

Assay Methodology and Validation

Prior to the assay of Zidovudine in the sample, bioanalytical method validation was evaluated for selectivity; carry-over effect; calibration curve and Lower Limit of Quantification (LLOQ); precision and accuracy; matrix effect; dilution integrity; and stabilities (i.e., freeze- thawed stability, short-term stability at room temperature, post preparative stability / autosampler batch integrity, stock solution and internal standard stability, and long-term stability). An assay of Zidovudine concentration in plasma was carried out by a fully validated LCMS/MS with LLOQ 10.00 ng/mL. The anticoagulant used during the validation and bioanalytical phase was CPDA (Citrate Phosphate Dextrose Adenine).

Data Quality Assurance

During the bioanalytical phase of the plasma samples, the analysis was monitored by the quality control process include system suitability test, linearity of calibration curve, and quality control samples (Low QC, Medium QC, and High QC) referred to requirement described in European Medicinal Agency (EMA) guideline 2011 [12].

The calibration curve will be conducted with eight concentrations at each batch. The acceptance criteria are %LLOQ deviation + 20.00% and %other concentration deviation + 15.00% with a minimum of 75% of the calibration curve with at least 6 concentration levels must meet these criteria.

QC Samples will be conducted with three concentrations at each batch. The acceptance criteria are %deviation value and RSD of QC samples must be <15% with a minimum of 67% of the total QC samples and a minimum of 50% at each concentration level must meet these criteria.

The investigator has conducted the study in accordance with the protocol requirements, Good Clinical Practice (GCP), and Good Laboratory Practice (GLP) set by the National Agency of Drug and Food Control. The Quality Assurance (QA) department is responsible for conducting periodic audits of the BA/BE study to ensure the integrity of the study, both internally and externally. The QA department keeps written records and signs audit notes, findings, and issues related to the study. Any research issues that arise will be evaluated or audited to ensure the integrity of the study data that will be conveyed to the investigator, and the evaluation results can be used for handling according to the problems faced. A representative from the sponsor’s QA will ensure that there are no deviations from the approved protocol or standard operating procedures made in the study.

Result and Discussion

The total number of subjects who participated the study was 32 subjects (21 males and 11 females), where S18 dropped out of the study in the 2nd period because of personal reasons. The demographic data of the subject are tabulated in Table 1.

There was one adverse event during this bioequivalence study, i.e. dyspepsia. All of these adverse events were recorded in the CRF and the deviation during the study is reported in protocol deviation point at full study report.

Conclusion

This study aims to determine the bioequivalence of Zidovudine 100 mg capsules produced by PT Kimia Farma Tbk compared to Retrovir 100 mg capsules produced by GlaxoSmithKline Pharmaceuticals S.A, in healthy subjects.

The study was conducted in a randomized, single- dose, open-label, two-way crossover design (2 treatments, 2 periods, and 2 sequences) under fasting state with 7 (seven) days washed-out period between each period. The number of subjects who participated and completed the study were 31 of 32 adult male and female. One subject dropped out from 2nd period because of personal reason. The subjects received an explanation of the study and signed informed consent. The primary parameters to assess the bioequivalence between the test and reference drug of Zidovudine are AUC0-t and Cmax. Based on the bioequivalence criteria of a 90% confidence interval, the ratio of the geometric mean values (AUC)T/(AUC)R=1.00 with 90% CI=(80-125)% (α: 0.05) and (Cmax)T/(Cmax)R=1.00 with 90% CI=(80-125)% (α: 0.05) were used.

The geometric mean ratios (90% confidence intervals) of the test drug/ comparator drug for Zidovudine were 101.65% (96.77- 106.83%) for AUC0-t and 95.63% (84.09- 108.76%) for Cmax. Based on the AUC0- t and Cmax values, Zidovudine 100 mg capsules produced by PT Kimia Farma Tbk is bioequivalent to Retrovir 100 mg capsules produced by GlaxoSmithKline Pharmaceuticals S.A. In this study, the intra-subject coefficient of variation (%CV) obtained from ANOVA was 11.49% for AUC0-t and 30.47% for Cmax.