Evaluation of Ornidazole Tablets Bioequivalence in Chinese Healthy Participants Under Fasted and Fed Conditions Using Pharmacokinetic Parameters

For bioequivalence evaluation, the test formulation was 0.25 g per tablet (lot no.: 020211001; expiration date: 7 October 2023) ornidazole manufactured by Zhejiang Aisheng Pharmaceutical Co., LTD and supplied by OrPha Swiss GmbH, whereas the reference formulation (marketed as Tiberal^®) was 0.5 g per tablet (0.5 g per tablet; lot no.: 3026; expiration date: January 2025) ornidazole produced by Zhejiang Aisheng Pharmaceutical Co., LTD and supplied by Orpha Swiss GmbH.

Healthy Chinese females (weighing ≥ 45 kg) and males (weighing ≥ 50 kg) aged ≥ 18 years who had a body mass index (BMI) between 19.0 and 26.0 kg/m² (including the cut-off value) were eligible for participation in our study. The volunteers underwent a comprehensive medical examination involving medical history review, routine physical examinations, vital signs measurements [armpit temperature, pulse, and heart rate, as well as sitting blood pressure (BP)], laboratory investigations [blood routine, blood biochemistry, hepatitis, coagulation function, human immunodeficiency virus testing, urinalysis, syphilis specificity antibody screening, human chorionic gonadotropin (HCG) test, alcohol breathalyzer test, and drug screening], chest X-ray, and 12-lead electrocardiography (ECG). Individuals who exhibited acute or chronic illnesses, clinically significant diseases, allergic reactions to any component of ornidazole tablets, allergic constitutions, needle stick or blood draw reactions, smoking or alcohol abuse, drug abuse within the past 2 years, medication intake within 14 days before the initial dosing, participation in clinical studies within the past 3 months, blood loss or donation of 200 mL or more, and female participants who were pregnant, breastfeeding, or may be likely to become pregnant were excluded from the study.

Before participating in the study, all participants were informed by the clinical investigators of the research’s objectives, methodologies, and risk–benefit evaluations. Written informed consent was obtained from each participant before their participation in the study. Subjects were allowed to withdraw at any given point in time.

The present investigation was subjected to approval by the ethics committee of the Cangzhou Central Hospital (approval number 2021-061-01) and was conducted at Phase I Clinical Research Center of the Cangzhou Central Hospital from December 2021 to April 2022 following the International Conference on Harmonisation Good Clinical Practice Guideline [13] and the Guideline for Good Clinical Practice suggested by the National Medical Products Administration (NMPA) [14]. Before participating in this investigation, every participant provided written informed consent.

The primary objective of this single-center, randomized, open-label, two-period, two-sequence crossover, and single-dose bioavailability study for fasting or fed participants was to determine bioequivalence. There was a 7-day washout period between each administration. The participants in the study were randomly classified into either the test/reference (T/R) or reference/test (R/T) group in a 1:1 ratio for both the fed and fasted trials. Each participant was administered a single oral dose of either test or reference tablets, consisting of test tablets (two tablets × 0.25 g) or reference tablets (one tablet × 0.5 g), followed by 240 mL of warm water. The recruited participants in the fasting trial were subjected to a minimum of 10 h of overnight fasting. Additionally, the enlisted participants in the fed trial were administered a standard high-fat, high-calorie breakfast (800–1000 kcal) consisting of 52.1% fat, 17.7% protein, and 30.2% carbohydrate, 30 min before the ingestion of the medications. Participants were not allowed to drink additional water for 2 h after drug administration. In addition, 4 and 10 h following drug administration, each participant received a standardized meal during the study, participants were not allowed to consume alcoholic drinks, engage in vigorous exercise, or smoke.

Blood samples were collected to the EDTA-K₂ vacuum tube by inserting a cannula into the forearm vein prior to administration. In the fasted trial, pre-dose 4 mL blood samples were at baseline (0 h), and 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 24, 36, 48, and 72 h after the dose. Similarly, in the fed trial, pre-dose 4 mL blood samples were taken at baseline (0 h), and 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 24, 36, 48, and 72 h after the dose. Blood samples were centrifuged at 1700g at 4 °C for 10 min within 1 h of collection and kept at − 70 ± 10 °C within 2 h of collection until they were utilized for subsequent analysis purposes.

The plasma concentrations of ornidazole were determined by a validated liquid chromatography tandem mass spectrometry (LC–MS/MS) method. The method involved protein precipitation and the quantitation of the target compounds, which was performed using a positive ion mode and multiple reaction monitoring (MRM). The lower limit of quantification (LLOQ) for the plasma assay was 0.100 μg/mL and the linear calibration range was 0.100–15.0 μg/mL. The overall precision of the ornidazole assay ≤ 2.9% coefficient of variation (CV).

The safety evaluation approach involved tracking alterations in vital signs, physical examination results, and adverse events (AEs) incidence. Physical examination, 12-lead ECG, routine blood test, liver function, blood glucose, renal function, urinalysis, and female HCG test were performed in the laboratory at baseline and 72 h after the second dose administration. The study recorded the vital signs, including temperature, pulse rate, and blood pressure, of the participants at several timepoints. These timepoints included 1 day and 1 h before administration, and 2, 24, 48, and 72 h after each administration. Medical observations and spontaneous reports from individual participants were used to collect AEs that occurred during the study. According to the Medical Dictionary for Regulatory Activities, v20.0, all AEs were preferred terms and systemic organ classes.

The PK analysis set included participants who completed all clinical trials without any substantial protocol deviations. The area under the curve from t = 0 to infinity (AUC_0–∞); AUC from t = 0 to the final measurable concentration (AUC_0–t); and maximum plasma concentration (C_max) were the primary assessment indices. Time to C_max (T_max), terminal elimination rate constant (z), and half-life (t_1/2) were the secondary assessment indices.

Analyst 1.6.3 workstation (Sciex, USA) was used to collect data and chromatogram integration, and regression analysis was performed to calculate the original concentration. Blood concentration analysis was conducted using Phoenix WinNonlin Software, v8.3, and the non-compartmental model using PKCS. C_max and T_max were calculated using the data obtained. AUC_0–t was calculated using the linear trapezoidal rule. The terminal log-linear phase slope (K_e) was divided by the final measurable concentration (C_t) to determine the extrapolated area, which was added to the AUC_0–t to get the AUC_0–∞. The terminal log-linear phase slope was used to compute the t_1/2 value, which was 0.693/K_e. The tested formulation’s relative bioavailability (F) was calculated based on the equation below: F = AUC_0–t (test)/AUC_0–t (reference) × 100%. To ascertain the effects of therapy, sequence, time, and individuals nested in sequence, the logarithm (ln)-transformed PK parameters (AUC_0–t, C_max, and AUC_0–∞) were evaluated using a one-way analysis of variance following the NMPA regulatory standards. T_max values were compared across groups using the Wilcoxon signed-rank test. The general linear model (GLM) approach was used to conduct the statistical analysis using SAS, V9.4 (SAS Institute Inc, Cary, North Carolina, license number 000063317058). The geometric mean ratio (GMR) AUC_0–∞, AUC_0–t, and C_max of the two formulations were regarded to be bioequivalent when the disparities between the measured parameters were found to be negligible (P > 0.05) and the 90% confidence interval (CI) for these measurements ranged between 80.00 and 125.00%.

Parametric 90% CIs for the GMR between the two formulations (test–reference) were determined using the Schuirmann method.

In total, 148 healthy Chinese participants were evaluated (Fig. 1). Both the fed and fasted trials involved 24 patients (17 men and 7 women and 16 men and 8 women, respectively). No participant included was excluded thereafter. Table 1 displays the demographic information for all the included participants.

In the fed and fasted trials, all 24 participants completed both phases and were analyzed for pharmacokinetics.

In the fed and fasted phases after an oral single dose of the reference and test ornidazole formulations, respectively, Figs. 2 and 3 depict ornidazole’s mean plasma concentration–time profiles. The PK characteristics for ornidazole are presented in Table 2.

For the bioequivalence assessment in the fast trial, the prime PK parameters comparison between the reference and test formulation revealed that the GMR (90% CI) values for AUC_0–t, C_max, and AUC _0–∞ were 100.97% (99.12–102.85%), 99.88% (90.63–110.08%), and 101.12% (99.17–103.11%), respectively. The defined equivalency margin of 80.00–125.00% included all the estimated 90% CIs. For the bioequivalence assessment in the fed trial, the key PK parameters comparison between the reference and test formulations revealed that the GMR (90% CI) values for AUC_0–t, C_max, and AUC_0–∞ were 103.00% (100.94–105.11%), 101.90% (99.63–104.22%), and 102.99% (100.87–105.16%), respectively. The fed trial’s 90% CIs, similar to those of the fast trial, fell within the anticipated range of 80.00 to 125.00%. Tables 3, 4 and 5 provide the average bioequivalence assessments for ornidazole plasma PK parameters under fasting or fed settings, as well as the variance analyses for AUC_0–∞, AUC_0–t, and C_max after logarithmic transformation.

The incidence rate of AEs during the fasting trial was 33.33%, with nine AEs recorded in eight participants. Laboratory testing detected 100% of these AEs, which were mild and did not need treatment. No SAEs occurred and no participants withdrew from the trial due to adverse events. Among them, the relationship between two AEs in two participants and the experimental drug was determined to be “possibly related” and recorded as adverse reactions. Table 6 summarizes the AE incidence during the fasted trial.

The AE incidence during the fed trial was 58.33%, with 23 AEs recorded in 14 patients. Laboratory examinations revealed that all these AEs were mild. Except for two cases of AEs, subjects were instructed to drink more warm water and keep warm, and no measures were taken for the other AEs. Among all the AEs, the relationship between five AEs in four subjects and the experimental drug was determined to be “possibly related” and recorded as adverse reactions. Table 7 summarizes the AE incidence in the fed trial.