The study was conducted in accordance with the principles outlined in the ICMR Guidelines [15], the New Drugs and Clinical Trials Rules 2019 of India [16], the Declaration of Helsinki [17], the International Council for Harmonisation (ICH) Good Clinical Practice (GCP) guidelines [18], and the FDA Product-Specific Guidance for Bioequivalence Studies [19].

The protocol (ICBio/021/0823) was reviewed and approved by the ACE Independent Ethics Committee on 14 May 2024 (Version 00, dated 03 August 2023), and certified by the CDSCO/DGHS for ICBio Clinical Research Pvt. Ltd.

This was an open-label, randomized, two-treatment, two-period, two-sequence, single-dose, crossover bioequivalence (BE) study under fed conditions comparing two 300 mg ursodeoxycholic acid (UDCA) formulations.

Test formulation (T): Ursodeoxycholic acid 300 mg tablets, Laboratorios Leti S.A.V., Venezuela; Batch EP-0416445-5; expiry 06/2026, and Reference formulation (R): Ursobilane® 300 mg tablets, Laboratorios ESTEDI S.L., Spain; Batch 0178T028; expiry 05/2025.

A total of 24 healthy male subjects were randomized to one of the two sequences (T-R or R-T). The randomization schedule was generated using SAS® 9.1.3 (SAS Institute Inc., Cary, NC, USA). Each subject received a single oral dose in each period. Subjects who received T in Period I received R in Period II, and vice versa.

The pre-screening period lasted 28 days. Participants were housed in the ICBio Clinical Research facility from 36 h pre-dose until 24 h post-dose in each period. The entire study was conducted from July 19 to August 18, 2024, including a 30-day washout, compliant with at least five elimination half-lives of UDCA [3][4].

Twenty-four healthy male volunteers who met the inclusion and exclusion criteria were enrolled. The mean age was 36.63 years, the mean weight 70.79 kg, the mean height 1.71 m, and the mean BMI 24.08 kg/m² (Table 1).

Table 1.Mean demographic characteristics of enrolled subjects (N = 24).

Screening procedures included a complete medical history, physical examination, vital signs, liver transaminases, tests for hepatitis B and C, HIV, VDRL, 12-lead ECG, chest radiography, and urine toxicology including alcohol. Urine tests for drugs of abuse and alcohol were repeated on each check-in day. Eligible subjects were non-smokers or smokers who abstained from smoking for at least 10 hours before dosing. All subjects were capable of understanding and signing the informed consent form.

Exclusion criteria included any condition affecting the gastrointestinal tract or hematopoietic system, history of gastric or duodenal ulcer, gastrointestinal bleeding, or blood in stools, recent infection, major or minor surgery, hypersensitivity to UDCA or related drugs cardiovascular, renal, hepatic, metabolic, neurological, endocrine, psychiatric, or hematopoietic disorders, and use of medications interfering with UDCA metabolism or quantification.

Screening was completed within 28 days before check-in. Subjects were housed from 36 h pre-dose to 24 h post-dose in each period. After a 10-hour overnight fast, a high-fat, high-calorie breakfast (1100 kcal) was served exactly 30 minutes prior to dosing, consistent with FDA fed-state requirements. Standardized meals totaling 2500 kcal/day were provided. Water was allowed ad libitum except 1 hour before and after dosing.

All subjects received a single oral dose of 600 mg UDCA (two 300 mg tablets) of either T or R according to the SAS-generated randomization schedule. Study personnel responsible for investigational product (IP) dispensing ensured compliance with randomization.

Blood Sampling

A total of 22 samples were obtained per period in baseline samples at −24.00, −18.00, −12.00, −06.00, and 0.00 h and post-dose samples at 0.334, 0.667, 1.00, 1.334, 1.667, 2.00, 2.334, 2.667, 3.00, 3.35, 4.00, 6.00, 8.00, 12.00, 24.00, 48.00, and 72.00 h.

The last three samples were collected by direct venipuncture. Pre-dose samples were 7 mL each; post-dose samples were 5 mL each.

Venous blood samples were collected in pre-labeled K₂EDTA (ethylenediaminetetraacetic acid) vacutainers and were centrifuged at 4000 rpm for 10 min at 2˚C - 8˚C within 45 minutes of sample collection. Plasma was separated, labeled, and stored at −70˚C ± 15˚C in an appropriate container.

A sensitive and selective LCMS/MS method to quantify ursodeoxycholic acid was developed and validated using ursodeoxycholic acid D4 as internal standards (Vivian Life Sciences). The calibration curve (CC) of the internal standard (ISTD) showed a linearity range for UDCA of 0.010 to 10.034 µg/ml, as per method SOP N˚MVR-087-02-23 at the bioanalytical laboratory of ICBio Research Pvt. Ltd., Bangalore. The method with a linearity range of 0.010 - 20.000 µg/ml was validated to determine the UDCA endogenous levels.

Liquid-liquid extraction was carried out by adding 50 µL of ISTD dilution to all the samples. Ursodeoxycholic acid was selectively isolated from plasma by the solid phase extraction method. Estimation was done by the mass spectrometric method and high performance liquid chromatography (HPLC) using a BDS Hypersil C18, 4.6 × 100 mm, 5 µm column (Shimadzu). The mass spectrometer was operated in positive electrospray mode. Identifications were based on multiple reaction monitoring transitions; m/z 391.30 - 391.30 for UDCA drug and 395.30 - 395.30 for UDCA D4 IS, m/z 448.40 - 448.40. The inter-batch calibration standard for UDCA showed a precision range of 5.26% to 7.37%, with accuracy from 102.57% to 106.68%, and UDCA D4 IS 5.77% to 10.94% precision range with accuracy 96.67% to 116.67%. Inter-batch precision and accuracy for UDCA met acceptance criteria [20][21].

Sample size was based on intra-subject CV% from published data [22][23]. The highest reported intra-subject CV% was ~24.08% for C_max. Assuming CV% ≤26% for C_max and AUC, and a true ratio between 98% and 102%, 21 evaluable subjects provided 80% power at α = 0.05. To allow for dropouts, 24 subjects were enrolled.

Pharmacokinetic analysis was performed on subjects completing both periods using SAS® 9.1.3. Primary PK Parameters (per FDA guidance [19]): C_max and AUC_0-72.

Secondary PK Parameters T_max, t_½ and K_el and N points of terminal phase C_max and AUC_0-72 were ln-transformed and analyzed using a generalized linear model (GLM) ANOVA with fixed effects (treatment, period) and random effect (subject nested in sequence).

Bioequivalence was concluded if the 90% confidence intervals for T/R geometric mean ratios of C_max and AUC_0-72 fell within 80% - 125% [24].

For UDCA (unconjugated) and total UDCA (conjugated + unconjugated), descriptive statistics included the mean, SD, CV%, geometric mean, median, and range.

Least-square means (LSMs) of ln-transformed parameters were compared (T-R). Anti-logged LSM differences produced geometric mean ratios (T/R). Schuirmann’s two one-sided t-tests generated 90% CIs for BE determination.

Safety was evaluated through adverse event monitoring, vital signs, screening/end-of-study laboratory tests, ECG at screening, and urine drug testing before check-in for each period.

Pharmacokinetic (PK) analyses were completed for 22 of the 24 randomized subjects. Subjects 17 and 23 were classified as dropouts because they did not attend Period II. PK parameters are presented in Table 2. A non-compartmental analysis was performed to estimate the peak plasma concentration (C_max), the area under the plasma concentration-time curve from zero to 72 hours (AUC_0-72), time to reach peak concentration (T_max), elimination rate constant (λ_z/K_el), and half-life (T_1/2). These parameters were calculated for baseline-corrected and uncorrected unconjugated UDCA (Table 2) and for total UDCA (conjugated + unconjugated) (Table 3) for both test and reference formulations.

Table 2.UDCA unconjugated, uncorrected, and corrected (N = 22).

Note: For mean along with minimum and maximum values represented. Data are presented as mean ± standard deviation. C_max = Maximum concentration, AUC_0-t = Area under the plasma concentration-time curve from time 0 to the last measurable concentration, K_el = Elimination rate constant, T_max = Time to reach C_max, T_1/2 = time required for plasma concentration to decrease by 50%, PK: Pharmacokinetic.

Table 3.Total UDCA (conjugated and unconjugated) (N = 22).

Note: For the mean along with the minimum and maximum values represented. Data are presented as mean ± standard deviation. C_max = Maximum concentration, AUC_0-t = Area under the plasma concentration-time curve from time 0 to the last measurable concentration, K_el = Elimination rate constant, T_max = Time to reach C_max, T_1/2 = time required for plasma concentration to decrease by 50%. PK: Pharmacokinetic.

The ANOVA results for log-transformed C_max and AUC_0-72 confirmed that, for both unconjugated and total UDCA, there were no statistically significant effects of treatment, sequence, or period between the test and reference formulations (Table 4 and Table5).

The geometric mean ratios (Test/Reference) and their corresponding 90% confidence intervals for log-transformed PK parameters demonstrated that bioequivalence was achieved. For unconjugated UDCA, the 90% CI for C_max (103.42%; 90.34% - 118.40%) and AUC_0-72 (105.09%; 94.95% - 116.30%) were within the acceptance limits of 80% - 125%. For total UDCA (conjugated + unconjugated), the 90% CI for C_max (100.27%; 89.50 - 112.33%) and AUC_0-72 (96.96%; 80.49% - 116.80%) also fell within the predefined bioequivalence interval (Table 6).

Table 4.ANOVA unconjugated UDCA (Baseline corrected, P value).

Table 5.ANOVA total UDCA, conjugated and non-conjugated (Baseline corrected, P value).

Table 6.UDCA unconjugated and total (conjugated and unconjugated) in Plasma.

There were no significant differences between the PK parameters of both UDCA formulations (p > 0.05). These findings confirm that the test product meets the regulatory bioequivalence criteria [13].

Mean UDCA plasma concentrations following oral administration of 600 mg (2 × 300 mg tablets) for up to 72 hours post-dose are shown in Table 7 and Table8 and are graphically represented in Figures 2-5, using both arithmetic and logarithmic scales.

Table 7. Plasma concentration UDCA unconjugated corrected.

Ln = neperian logarithm; SE = standard error.

Table 8.Plasma concentration of UDCA total (conjugated and unconjugated) corrected.

Ln = neperian logarithm; SE = standard error.

Figure 2.Ursodeoxycholic acid 600 mg dose (2 tablets of 300 mg), mean plasma concentration vs time (h) profile for each formulation is presented on a linear scale, following a single oral dose in the fed condition. The blue line indicates UDCA 600 mg, test product of Laboratorios Leti S.A.V, and the red line indicates Ursobilane® 600 mg, reference product of Laboratorio Estedi S.L., Spain.

Figure 3. The ursodeoxycholic acid 600 mg dose (2 tablets of 300 mg), mean plasma concentration vs time (h) profile for each formulation is presented on a logarithmic scale, following a single oral dose in the fed condition. The blue line indicates UDCA 600 mg, Test product of Laboratorios Leti S.A.V., and the red line indicates Ursobilane® 600 mg, Reference product of Laboratorio Estedi S.L., Spain.

Figure 4.Ursodeoxycholic acid 600 mg dose (2 tablets of 300 mg) total (conjugated and unconjugated) mean plasma concentration vs time (h) profile for each formulation is presented on a linear scale, following a single oral dose in fed condition. The blue line indicates UDCA 600 mg, Test product of Laboratorios Leti S.A.V., and the red line indicates Ursobilane® 600 mg, Reference product of Laboratorio Estedi S.L., Spain.

Figure 5. Ursodeoxycholic acid 600 mg dose (2 tablets of 300 mg) total, (conjugated and unconjugated) mean plasma concentration vs time (h) profile for each formulation is presented on a logarithmic scale, following a single oral dose in the fed condition. The blue line indicates UDCA 600 mg, Test product of Laboratorios Leti S.A.V, and the red line indicates Ursobilane® 600 mg, Reference product of Laboratorio Estedi S.L., Spain.

All subjects were monitored for clinical well-being and vital signs during the study. Measurements were recorded at check-in (−36 h), at pre-dose (−24 h, −18 h, −12 h, and within 2 h prior to dosing), at 3, 7, and 11 hours post-dose, at check-out for each period, and during all scheduled ambulatory visits.

No adverse events (AEs) or serious adverse events (SAEs) were reported during the study. Post-study safety evaluations were completed for all subjects who attended Period II, except for subjects 17 and 23, who were withdrawn as dropouts. All clinical laboratory parameters were within normal limits.