Healthy male volunteers (19 to 45 years old with a body mass index (BMI) of 17.5 to 30.5 kg/m²) who had no congenital abnormalities and no chronic disease within the past 3 years were recruited. These volunteers were informed about the details of the study (including the purpose, benefits, and risks) and provided signed informed consent before participating in the study. Physical examinations, measurements of vital signs, 12-lead electrocardiograms (ECGs), and clinical laboratory assessments (i.e., hematology, biochemistry, urinalysis and urine drug screening) were performed to confirm the health of the individuals within 3 weeks before the first administration of the investigational products. Individuals who had no clinically significant findings in these screening tests were eligible to become study subjects. The exclusion criteria consisted of any evidence or history of the following: hemorrhagic, renal, endocrine, pulmonary, gastrointestinal, urinary, cardiovascular, hepatic, psychiatric, neurological, or allergic disease; hypersensitivity reaction to the components of the investigational products; use of any drug known as a significant inducer or inhibitor of drug-metabolizing enzymes within 30 days before the beginning of the study; or a genetic problem such as galactose intolerance, Lapp lactase deficiency and glucose-galactose malabsorption. Subjects were asked to avoid smoking and the consumption of caffeinated food and beverages during the hospitalization periods of the study. Subject compliance with these restrictions was determined by self-reporting and by taking the subject's history.

According to previous pharmacokinetic studies, the intrasubject coefficient of variation (CV_intra) of the area under the curve up to the last sampling time (AUC_last) and the maximum plasma concentration (C_max) of solifenacin were 11.8% and 11.4%, respectively.[4] Considering the increase in the coefficient of variation due to different salt formulation, the CV_intra was set to approximately 20% in this study. When the true ratio (test/reference) of the mean is 1.05 and the CV_intra is 0.2, a sample size of 24 achieves 90% power at a 5% significance level. The total sample size was set to 36, considering a 30% dropout rate.

This study was a clinical trial conducted at the Chonbuk National University Hospital clinical trial center. The study was designed as a randomized, open-label, single-dose, two-way crossover study. Subjects were hospitalized in the clinical trial center on the evening before drug administration and were allocated to two groups in a 1:1 ratio according to a predesigned randomization table. During the first treatment period, subjects received one of the two treatments (either the test formulation or the reference formulation). The test formulation was solifenacin tartrate 10.66 mg (Besigum Tab, Hanmi Pharmaceuticals Co. Ltd., Seoul, Republic of Korea),[9] and the reference formulation was solifenacin succinate 10 mg (VESIcare, ASTELLAS PHARMR KOREA Co., Ltd., Seoul, Korea).[6] After the scheduled procedures in the first treatment period were finished (day 2), the subjects were discharged. After a 21-day washout period (more than 5 times the half-life of solifenacin), each subject received the other formulation.

After 10 hours of overnight fasting, the investigational products were administered to each subject with 150 mL of water. An oral check was immediately performed after administration to ensure compliance. All subjects received standardized lunch and dinner at 4 hours and 10 hours post-dose, respectively. Water was not permitted for 1 hour before and 1 hour after drug administration. In addition, subjects were not allowed to consume grapefruit or grapefruit-containing products from 7 days before the first drug administration until the collection of the final blood sample for pharmacokinetic assessments.

For pharmacokinetic analysis, blood samples containing 6 mL of blood each were collected to measure solifenacin concentrations during each treatment period at the following time points: pre-dose and 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 48 and 72 hours post-dose. The blood samples were immediately centrifuged at 3,000 rpm for 10 minutes at 4℃, and the plasma was transferred to polypropylene tubes and then stored in a freezer at −70℃ until further analysis.

Plasma concentrations of solifenacin were determined by a validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS, Waters ACQUITY UPLC™ System and Waters Xevo™ TQ MS; Waters, MA, USA) method using solifenacin-d₅ as the internal standard. Chromatographic separation of the compounds was accomplished using a Waters ACQUITY UPLC®BEH C18 column (2.1 mm ID × 50 mm L, 1.7 µm; Waters, MA, USA). A gradient elution procedure was employed using 0.1% (v/v) formic acid in distilled water and 0.1% (v/v) formic acid in acetonitrile at a flow rate of 0.3 mL/min. Briefly, 10 µL of the internal standard (0.1 µg/mL) and 300 µL of acetonitrile were added to 100 µL of plasma sample. This sample was stirred for 3 min at 1,500 rpm using a MixMate (Eppendorf, Hamburg, Germany), followed by centrifugation for 1 min at 4,000 rpm. A 150-µL aliquot of supernatant was placed in a 1.1-mL deep-well plate (Axygen, NY, USA), and 300 µL of 0.1% (v/v) formic acid in distilled water was added. After stirring for 3 min at 1,500 rpm and centrifuging for 1 min at 4,000 rpm, 10 µL of diluted sample was injected into the UPLC-MS/MS system. Electrospray ionization in positive ion mode was used for detection and quantification. The multiple reaction monitoring (MRM) transitions were m/z 363.21→110.05 for solifenacin and 368.19→110.05 for the internal standard. A calibration curve covering the range of 0.200 to 100 ng/mL was constructed. For solifenacin, the intraday accuracy was 97.2% to 100.0% (with a precision of 1.0%–1.5%), and the interday accuracy was 96.3% to 100.5% (with a precision of 1.6%–6.0%). These results indicated that the bioanalytical method for the drug assay was adequate (r²≥0.9950).

Individual pharmacokinetic parameters were analyzed using Phoenix® WinNonlin® version 6.3 software (Pharsight Corporation, CA, USA) with the noncompartmental method. Of the pharmacokinetic parameters, AUC_last and C_max were evaluated as primary parameters. The area under the plasma concentration-time curve up to infinity (AUC_inf), the time to C_max (T_max) and the plasma elimination half-life (t_1/2) were also estimated as secondary parameters. AUC_last was calculated using the linear trapezoidal method. AUC_inf was calculated with the following equation: AUC_last + C_last/λ_z, where C_last is the last measured concentration and λ_z is the elimination rate constant estimated from the log-linear terminal phase of the concentration-time curve as the slope of the natural logarithm of concentration against time. C_max and T_max were determined by observing the data of the plasma concentration-time profile, and t_1/2 was calculated as ln 2/λ_z. In pharmacokinetic analyses, actual sample times were used.

Safety was assessed by monitoring adverse events (AEs), physical examinations, laboratory tests (hematology, biochemistry and urinalysis), vital sign measurements (blood pressure, pulse rate and body temperature), and ECG. AEs were identified by asking the subjects about their condition during the study period. AE data were recorded from the pre-dose until the post-study visit. AEs were summarized by treatment groups in terms of severity (mild, moderate, or severe) and relationship. Physical examinations, laboratory tests, and vital sign measurements were conducted at screening, pre-dose, 72 hours post-dose, and at the post-study visit. ECGs were recorded at screening and the post-study visit.

Subjects who completed the pharmacokinetic blood sampling as scheduled were included in the pharmacokinetic analysis. Statistical analyses were performed using SAS software, version 9.3 (SAS institute, Cary, NC, USA). To summarize the pharmacokinetic data from the two treatments, descriptive statistics that included arithmetic means, standard deviations (SDs), and median values for continuous data were used. The log-transformed pharmacokinetic parameters of AUC_last and C_max were analyzed using a mixed-effects analysis of variance (ANOVA) model with fixed effects of sequence, period, and formulation and a random effect of subjects within the sequence to compare the pharmacokinetic parameters of the two formulations. The 90% confidence intervals (CIs) of the geometric least-square mean ratios of the test to reference formulations for AUC_last and C_max of solifenacin were calculated to assess bioequivalence.

Of the 45 volunteers who were screened, 36 subjects were enrolled in the study and randomized into the two treatment groups. The demographics of the subjects (mean ± SD) included a mean age of 22.1 ± 2.3 years, height of 174.7 ± 6.1 cm, weight of 69.6 ± 9.4 kg, and BMI of 22.8 ± 2.6 kg/m² (Table 1). There were no significant differences in demographic characteristics between the sequence groups.

A total of 36 subjects were randomized in this study. Three subjects withdrew consent after the first dose and were dropped from the study. After the second period, one subject withdrew consent and did not participate in the post-study visit; however, this subject completed the scheduled pharmacokinetic sampling schedule and was included in the pharmacokinetic analysis. The pharmacokinetic assessment was conducted based on 33 subjects who completed the entire pharmacokinetic blood sampling schedule without significant violations.

The geometric mean plasma concentration-time curves of solifenacin for the test or reference formulation in healthy male volunteers are shown in Figure 1. The pharmacokinetic parameters of solifenacin in plasma by formulation are summarized in Table 2.

After oral administration of the test or reference formulation, the mean values of AUC_last of solifenacin were 486.98 ± 138.47 h·ng/mL and 469.07 ± 128.29 h·ng/mL, respectively. The mean C_max values of solifenacin were 14.66 ± 3.85 ng/mL and 14.10 ± 3.37 ng/mL, with median(min-max) T_max values of 5.00(3.00–6.00) hours and 5.00(3.00–6.00) hours, respectively. The mean t_1/2 of solifenacin was 33.38 ± 8.06 hours and 32.40 ± 6.29 hours, respectively. The apparent total body clearance and the apparent volume of distribution of solifenacin were very similar for the two formulations.

The point estimates and 90% CIs for the geometric mean ratio (test/reference) of the AUC_last and C_max of solifenacin were 1.0376 (0.9702 – 1.1097) and 1.0369 (0.9779 – 1.0993), respectively (Table 3). The 90% CIs for these pharmacokinetic parameters of solifenacin met the acceptance range of 0.80 – 1.25 for bioequivalence.

No serious adverse events occurred during the entire study period. A total of 15 AEs occurred in 11 of the 36 subjects after administration of the investigational products. Among these AEs, 14 cases were evaluated as mild, and 1 case was evaluated as a moderate AE in intensity. There were 5 cases of adverse drug reactions (ADRs) in 2 subjects who were treated with the test formulation. The reported ADRs were epistaxis, dry mouth and oropharyngeal pain and all of these ADRs were of mild intensity. There were no significant differences between the test formulation and reference formulation when evaluating laboratory tests, vital signs, physical examinations and ECG results.