The study was conducted ethically in accordance with the principles of the ICMR guidelines (2017) [18] . New Drugs & Clinical Trials Rules 2019, India [19] , and adhered to the ethical principles of the Declaration of Helsinki [20] and the International Conference on Harmonization Good Clinical Practice Guidelines [21] . The study protocol was approved by an Independent Ethical Committee (ECR/141/indt/KA/2013/RR-19), Application No. EC/RENEW/IND2019/6255, and certified by CDSCO/DGHS to ICBio Clinical Research Pvt, Ltd., Study numbers: ICBio/010/0224 and ICBio/013/0224.

The test formulation was an empagliflozin/linagliptin 25 mg/5 mg coated tablet provided by Laboratorios Leti, S.A.V., República Bolivariana de Venezuela, batch number: EP 0623553-6R; expiry date: February 2026. The reference formulation was Jardianz® DPP coated tablets from Boehringer Ingelheim Pharma GmbH & Co. (empagliflozin/linagliptin) 25 mg/5 mg coated tablets, batch number: 303018; expiry date: April 2026. Each test or reference formulation was administered during each treatment period.

Healthy adult human subjects aged over 18 years (21 - 44), with body mass index (BMI) between 19.96 and 29.76 kg/m². A complete clinical history valid for the 6 months prior to the start of the study; normal laboratory values as determined by medical history and physical examination at the time of screening; normal vital signs (blood pressure, pulse rate, and axillary temperature) and physical examination; creatinine clearance (CrCl) value of more than 50 mL/min; negative tests for hepatic transaminases, hepatitis B and C, HIV, and VDRL; normal 12-lead EKG values, normal chest radiography, and negative result in urine drug tests. Subjects included non-smokers as well as smokers who had not smoked for at least 10 hours before the start of the study. All the subjects were informed about the possible risks and the benefits of their participation, such as blood lab tests, electrocardiograms, and travel and food expenses. They all signed the informed consent.

The exclusion criteria were as follows, a history of hypersensitivity to the study medication or to any other medication belonging to the study group, or any cardiovascular, renal, hepatic, metabolic, gastrointestinal, neurological, endocrine, hematopoietic, psychiatric, or other organic abnormalities; taking medications that interfere with the quantification and/or kinetics of the study medication, or potentially toxic medications within 30 days prior to the start of the study; having taken any medication, been hospitalized for any reason, or been seriously ill within the 90 days prior to the study; donated or lost 300 mL or more of blood within 90 days prior to the start of the study; recent history of drug abuse, including alcohol; consumed products such as cola drinks containing caffeine, theobromine, or theophylline in the 48 h prior to the study; consumption of grapefruit juice within 72 hours prior to the study.

This study comprised two independent clinical trials (fasting and fed study), each of which was a randomized, open-label, two-formulation, single-dose, two-sequence, two-period crossover bioequivalence study performed at the Phase Ι, screening, clinical study center, and bioanalytical facility by ICBio Clinical Research Private Limited, India.

All subjects in each trial were randomly assigned to either the T-R or R-T group (T was the test product and R was the reference product) at a 1:1 ratio according to a random number table generated by SAS statistical software (version 9.1.3). Group T-R subjects, who received the test product in the first treatment period, received the reference product in the second treatment period, whereas Group R-T received the opposite administration sequence. A 42-day washout period was used between the two treatment periods for both fed and fasting studies.

Under fasting conditions, each subject received a single dose of the test or reference tablet, administered orally with 240 mL of a 20% glucose solution in water, followed by 60 mL of the glucose solution administered every 15 minutes for up to 4 hours after dosing in a sitting position 2 h after at least a 10-hour overnight fast [22] in a sitting position, whereas the subjects under fed conditions received a standard high-fat (1394 kcal) breakfast (approximately 32.8 g of protein, 266 g of carbohydrate, and 22.5 g of fat) 30 min before each drug administration and followed the same scheme. Water intake was prohibited for 1 h before and after administration; outside of this restriction, it was provided ad libitum. Standardized lunches, snacks, and dinners were provided at 4, 8, and 12 hours after drug administration in each period.

A total of 16 × 6 mL blood samples of 6 mL each were collected through a cannula from each subject during each period at pre-dose and at 0.50, 1.00, 1.25, 1.50, 1.75, 2.00, 2.50, 3.00, 4.00, 6.00, 8.00, 12.00, 24.00, 48.00, and 72.00 hours after dosing. Samples at 48.00 and 72.00 hours were collected by direct venipuncture in both fasting and fed studies.

The blood samples were collected in pre-labeled K₂ and ethylenediaminetetraacetic acid (EDTA) vacutainers and were centrifuged at 4000 rpm for 10 min at 2˚C - 8˚C. Plasma was separated, labeled, and stored at −70˚C ± 5˚C before analysis. Empagliflozin and linagliptin concentrations in K₂EDTA, Human plasma samples collected from study ICBio/010/0224, were determined using the Method SOP N˚ MV-020-00. Sample analysis was conducted from January 27, 2025 to February 09, 2025 for analytes by liquid-liquid extraction method using a validated high-performance liquid chromatographic method with mass spectrometric detection (LC-MS/MS). All processes are inspected by the Quality Assurance Unit. The method demonstrated high accuracy and precision in measuring calibration standards for Empagliflozin (Analyte 10.030 to 1016.135 ng/ml, mean accuracy 100.06% and mean precision 3.15%) and Linagliptin (Analyte 0.213 to 13.585 ng/ml, mean accuracy 100.86% and mean precision 3.92%).

Empagliflozin: Subsequently, the plasma samples were processed. The calibration curve of internal standards (IS), Empagliflozin D4 (Vivian Life Sciences, Bangalore, India), and quality control (QC) samples were thawed and vortexed for preparation and analysis. Empagliflozin was selectively isolated from 300 μL plasma by a liquid-liquid extraction method. Aliquots of 0.300 μL were mixed with 0.300 μL of extraction buffer (50 mM sodium carbonate in water) and vortexed. After adding 2.500 mL of TBME (methyl tert-butyl ether) and shaking for 10 min, the samples were centrifuged at 4000 rpm for 5 min at 4˚C. The supernatant layer (2.000 mL) was transferred and dried in a nitrogen evaporator at 40˚C ± 2˚C. The samples were reconstituted with 0.50 mL of reconstitution solution and spiked with IS over the concentration range of 10.030 - 1016.135 ng/mL. Analytes, IS, and QC samples were transferred to pre-labeled vials in the autosampler at 5˚C ± 3˚C and injected into a liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) instrument (ICBio II/BA/LCMS/0003, Bangalore, India). The chromatographic separation was performed by a BDS Hypersil C18, 4.6 × 50 mm, 5 μm column (Exion LC, Bangalore, India). The mass spectrometer was operated in positive electrospray mode. Identifications were based on multiple reactions monitoring transitions; m/z 355.200 - 468.300 for empagliflozin and m/z 359.100-472.100 for IS. The inter-batch calibration standard accuracy was 96.70% - 102.68% with precision values of 2.34% - 5.50% and a linearity range of 10.030 - 1016.135 ng/mL.

Linagliptin: The plasma samples were processed. The calibration curve of internal standards (IS) Linagliptin D4 (Vivian Life Sciences Pvt. Ltd., Bangalore, India) and quality control (QC) samples were thawed and vortexed for preparation and analysis. Linagliptin was selectively isolated from 300 μL of plasma by a liquid-liquid extraction method using a validated high-performance liquid chromatographic method with mass spectrometric detection. Aliquots of 0.300 mL were mixed with 50 μL IS dilution, 300 μL of extraction buffer (10% ammonia solution in water), and 2.5 mL of ethyl acetate, then vortexed. They were then centrifuged at 4500 rpm for 5 min at 4˚C. Was transferred 2.0 mL of the supernatant layer and dried in a nitrogen evaporator at 40˚C ± 2˚C. The samples were reconstituted with 0.400 mL of reconstitution solution and spiked with IS over a concentration range of 0.213 ng/mL to 13.584 ng/mL. Analytes, IS, and QC samples were transferred to pre-labeled vials in the autosampler at 5˚C ± 3˚C and injected into a liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) instrument (Vivian Life Sciences, VL/S-LG-052/c, Bangalore, India). The chromatographic separation was performed by a BDS Hypersil C18, 4.6 × 50 mm, 5 μm column (Exion LC, Bangalore, India). Identifications were based on multiple reaction monitoring transitions; m/z 420.000 - 473.300 for linagliptin and m/z 424.000 - 477.200 for IS. The inter-batch calibration standard accuracy was 97.53 - 104.10%, with precision values of 1.11% - 4.63%, and a linearity range of 0.213 - 13.584 ng/mL.

PK parameters were evaluated and adhered to FDA regulations [22] . The PK parameters calculated were maximum peak concentration (C_max), area under the curve (AUC) from time 0 h to the last measurable concentration (AUC_0-72). Other secondary PK parameters evaluated were: time to reach C_max (T_max), T_1/2, and Kel in plasma concentration for empagliflozin and linagliptin. PK analysis was performed using the non-compartmental model of SAS® Statistical Analysis System (software version 9.1.3, SAS Institute Inc., CARY, USA).

The sample size was based on the intra-subject coefficient of variation (CV%) for empagliflozin and linagliptin. With an expected coefficient of variation for both C_max and AUC not exceeding 20% and 26% respectively and assuming the true ratio of the test and reference formulations would fall between 92% -108%, the study required at least 32 evaluable subjects to demonstrate bioequivalence with a power greater than 90% at a 5% level of significance [5] [6] [23] [24] .

The Ln-transformed pharmacokinetic parameters C_max and AUC_0-72 of empagliflozin and linagliptin, under fasting and fed conditions, will be subjected to analysis of variance (ANOVA) for bioequivalence assessment. The ANOVA model will include the main effects of period and treatment as fixed effects and subject nested within sequence as a random effect. A separate ANOVA model will be used to analyze each of the parameters. The period, treatment, and sequence effects will be considered statistically significant if the probability values of the respective effects (p-values) are ≤0.05. The significance of the sequence effect will be tested using the mean square of subjects nested within sequences as the error term and reported.

Bioequivalence assessment was performed on the basis of the 90% confidence intervals of the differences of the least squares treatment means for Ln-transformed pharmacokinetic parameters C_max and AUC_0-72 of empagliflozin and linagliptin obtained after single-dose administration under fed and fasting conditions.

Safety evaluation was performed before, during, and at the end of the study. Vital signs, blood pressure, pulse rate, respiratory rate, and ancillary temperature were measured and recorded at check-in, before dosing (within 2 hours), 3.00 hours, 7.00 hours, and 11.00 hours post-dosing, during check-out, and at ambulatory visits of each period. A twelve-lead electrocardiogram and glycated hemoglobin (HbA1c) were recorded during the screening. Blood glucose level monitoring was done pre-dose (within 2 hours of dosing) at 1.0 hours, 3.0 hours, and 9.0 hours post-dose of each period for fed and fasting condition studies. Subjects were monitored for adverse events or serious adverse events throughout the study.

The demographic profile of the subjects completing the bioequivalence study under fasting and fed conditions is summarized in Table 1 .

A total of 32 subjects were enrolled in the fasting BE study in period I, and 28 subjects in period II. 4 participants were considered dropouts because they did not return to period II. In the fed condition study, 28 subjects completed the study and were included in the PK and statistical evaluation. The demographic characteristics of the individual sequence groups did not differ significantly. The washout period of 42 days was maintained between IP administrations in each treatment period in both studies, in fasting and fed conditions.

A non-compartmental PK analysis was applied to estimate the plasma concentration vs. time of empagliflozin and linagliptin. The oral dosing of empagliflozin/linagliptin for 72 h post-dose is represented on linear and logarithmic scale plots for the mean ± SD plasma concentration vs time curves for both test and reference products, Figures 1-4 for fasting conditions and Figures 5-8 for fed conditions. Descriptive statistics for all PK parameters are presented in Tables 2-7 for test and reference products, in fasting and fed conditions.

As shown in Figure 1 and Figure 2 for the empagliflozin test (T) and reference (R)

products and Figure 3 and Figure 4 for linagliptin, T and R products were comparable across treatments. Mean values for empagliflozin PK parameters were: C_max 391.03 ng/mL and T_max 2.43 h for T product, and C_max 398.68 ng/mL and T_max 2.30 h for R product, respectively.

The AUC_0-72 was 3277.27 ng·h/mL for T and 3452.26 ng·h/mL for R product. ( Table 2 )

Mean peak linagliptin plasma concentrations were comparable across treatments and PK parameters: C_max 6.35 ng/mL and T_max 4.17 h for the T product and C_max 7.04 ng/mL and T_max 3.39 h for the R product. The AUC_0-72 was 243.85 ng·h/mL for T and 261.94 ng∙h/mL for the R product. ( Figure 3 and Figure 4 )

The PK parameters C_max, AUC_0-t, T_max, Kel (h⁻¹), and T_1/2 of empagliflozin and linagliptin plasma concentrations are presented in Table 3 .

Data presented as a mean ± SE. C_max, maximum concentration, AUC_0-t, area under the plasma concentration-time curve from time 0 to the last measurable concentration; AUC_0-72, area under the plasma concentration-time curve from time 0 to 72 hours, T_max time to reach C_max, Kel (1/h) is the fraction of drug that is eliminated per unit of time, T_1/2 time required for plasma concentration to decrease by 50%.

Data presented as a mean ± SE. C_max, maximum concentration, AUC_0-t, area under the plasma concentration-time curve from time 0 to the last measurable concentration; AUC_0-72, area under the plasma concentration-time curve from time 0 to 72 hours, T_max time to reach C_max, Kel (1/h) is the fraction of drug that is eliminated per unit of time, T_1/2 time required for plasma concentration to decrease by 50%.

The geometric mean ratios (GMR), 90% CI, and power of variation of both products for Ln-transformed pharmacokinetic parameters C_max and AUC_0-72 for empagliflozin and linagliptin are presented in Table 4 .

Geometric Least Square Mean (GLSM) for C_max and AUC_0-72, and their respective 90% CI ratios, fell within the accepted bioequivalence range.

For empagliflozin, the GLSM was: C_max 95.39% (CI: 88.56% - 102.75%) and AUC_0-72: 92.87% (88.46% - 97.50%), and for linagliptin, the GLSM was: C_max 89.23% (81.88% - 97.23%) and AUC_0-72 94.99% (86.68% - 104.09%) under fasting conditions.

As shown in Figure 5 and Figure 6 for empagliflozin and Figure 7 and Figure 8 for linagliptin, across test and reference treatments, plasma concentration-time profiles were similar.

Mean values for empagliflozin PK parameters were comparable: C_max 188.59 ng/mL with a T_max of 3.36 h for the T product and C_max 202.78 ng/mL with a T_max of 3.5 h for the R product, respectively. The AUC_0-72 was 175.35 ng·h/mL for the T and 179.10 ng·h/mL for the R product ( Table 5 ).

Mean linagliptin 5 mg PK parameters were comparable across treatments: C_max 4.18 ng/mL with a T_max of 4.55 h for the T product and C_max 4.21 ng/mL with a T_max of 5.78 h for the R product.

The AUC_0-72 was 243.85 ng·h/mL for the T and 261.94 ng·h/mL for the R product ( Table 6 ).

The PK parameters C_max, AUC_0-t, T_max, Kel (h⁻¹), and T_1/2 of linagliptin plasma concentrations are presented in Table 6 .

Geometric least square mean (GLSM) and the ratios of the GLSM for C_max and AUC_0-72 are summarized in Table 7 .

Data presented as a mean ± SE. C_max, maximum concentration, AUC_0-t, area under the plasma concentration-time curve from time 0 to the last measurable concentration; AUC_0-72, area under the plasma concentration-time curve from time 0 to 72 hours, T_max time to reach C_max, Kel (1/h) is the fraction of drug that is eliminated per unit of time, T_1/2 time required for plasma concentration to decrease by 50%.

Data presented as a mean ± SE. C_max, maximum concentration, AUC_0-t, area under the plasma concentration-time curve from time 0 to the last measurable concentration; AUC_0-72, area under the plasma concentration-time curve from time 0 to 72 hours, T_max time to reach C_max, Kel (1/h) is the fraction of drug that is eliminated per unit of time, T_1/2 time required for plasma concentration to decrease by 50%.%. Note: Kel and T_1/2 were calculated only for the 27 subjects in these 2 samples.

For empagliflozin and linagliptin, their respective 90% CIs for C_max and AUC_0-72 (87.17% - 100.94%) and (80.78% - 92.02%) for empagliflozin, and (90.83% - 105.94%) and (86.12% - 109.51%) for linagliptin, respectively, fell within the acceptable bioequivalence range (80% - 125%).

The ANOVA results ( Table 8 ) for log-transformed C_max data of empagliflozin showed no statistically significant variation for period, treatment, or sequence values between the test and reference formulations in fasting and fed conditions. However, the log-transformed AUC_0-72 data showed significant variations for treatment values in fed conditions only, with no significant variation for period or sequence in the fasting and fed condition study. Linagliptin log-transformed Cmax data showed statistically significant variation for treatment in fasting condition study only, and the other parameters in both conditions; the ANOVA results for C_max and AUC_0-72 indicated no significant variation for treatment, period, or sequence values.

All subjects were monitored for adverse events throughout the study. At the end of the study, a post-study safety evaluation was done, which included hematology and clinical biochemistry tests. Post-study safety evaluation was performed on all the subjects (except subjects S11, S14, S17, and S21) who came for period II of the clinical study.

Twenty-eight subjects’ post-study safety samples were collected, and upon evaluation, all post-study safety laboratory parameters were found to be normal.

Post-study safety evaluation was performed on all the subjects (except subjects S05, S10, S26, and S32) who came for period II of the clinical study. Post-study safety samples were collected from 28 subjects, and upon evaluation, all post-study safety laboratory parameters were found to be normal.

In these studies (fasting and fed), no adverse events or serious adverse events were reported.