Pharmacokinetic assessment
For pharmacokinetic evaluation, serial blood samples were collected at 0 (pre-dose), 1, 2, 3, 4, 5, 6, 8, 12 and 24 hours after last dose for evogliptin and its metabolites (M7, M8), and at 0 (pre-dose), 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12 and 24 hours after last dose for pioglitazone and its metabolites (M3, M4). Urine samples of evogliptin, pioglitazone and their metabolites were collected up to 24 hours after last dose.
Individual steady-state pharmacokinetic parameters of each period were calculated by non-compartmental methods using Phoenix WinNonlin® software version 8.0 (Certara, Princeton, NJ, USA) software. Maximum plasma concentration of each analyte at steady-state (Cmax,ss) and the time to reach Cmax,ss (Tmax,ss) was directly derived from observed data. Area under the plasma concentration-time curve during a dosing interval at steady-state (AUCτ,ss) was calculated by the linear trapezoidal method when concentrations are increasing in the interval, and by the log trapezoidal method when concentrations are decreasing in the interval. Apparent clearance at steady-state (CLss/F) was calculated as administered dose / AUCτ,ss. Renal clearance at steady-state (CLR,ss) was calculated as amount of unchanged drug excreted into the urine during a dosing interval at steady state (Aeτ,ss) / AUCτ,ss. Metabolic ratio at steady state was calculated as AUCτ,ss of metabolite / AUCτ,ss of parent drug.
Plasma and urine concentrations of evogliptin, pioglitazone and their metabolites (M7, M8 of evogliptin and M3, M4 of pioglitazone) were analyzed with a validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method (LC: Shimadzu UFLC, Shimadzu, Japan. MS: TQ5500(3)/5500QTRAP, SCIEX, USA). Internal standards (ISs) for evogliptin, M7 and M8 were evogliptin-d9, M8-d9 and M8-d9, respectively, and ISs for pioglitazone, M3 and M4 were pioglitazone-d4, M3-d4 and M4-d5, respectively.
For plasma sample analysis, mobile phase consists of 5 mM ammonium formate with 0.1 % formic acid and acetonitrile for evogliptin; deionized water with 0.1% formic acid and methanol for M7 and M8. Mobile phase consists of 10 mM ammonium formate with 0.1 % formic acid and acetonitrile for pioglitazone, M3 and M4. All plasma analytes and their ISs were separated in C18 column (evogliptin: 100 × 2.1 mm, 1.7 μm, M7/M8: 100 × 2.1 mm, 5 μm, pioglitazone/M3/M4: 50 × 2.1 mm, 3 μm). Positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) was used to detect transition (m/z ) of evogliptin (402.2 → 346.2), evogliptin-d9 (411.2 → 347.2), M7 (418.2 → 362.2), M8 (418.2 → 362.2), M8-d9(427.2 → 363.2), pioglitazone (357.2 → 134.1), pioglitzone-d4 (361.2 → 138.2), M3 (371.2 → 148.1), M3-d4 (375.3 → 152.3), M4 (373.2 → 150.2) and M4-d5 (378.3 → 154.4). Calibration curves of plasma analytes were linear within the range of 0.1 – 60 ng/mL for evogliptin, 10 – 10,000 pg/mL for M7 and M8, 10 – 10,000 ng/mL for pioglitazone and 10 – 5,000 ng/mL for M3 and M4 (r ≥ 0.9950). Accuracy and precision of intra-batch quality control (QC) is 98.0 – 106.3 % and < 8.3 % for evogliptin, 96.0 – 107.8 % and < 6.7 % for M7, 93.9 – 105.9 % and < 6.5 % for M8, 89.4 – 108.2 % and < 6.7 % for pioglitazone, 95.6 – 106.9 % and < 7.3 % for M3, and 92.6 – 111.2 % and < 6.2 % for M4.
For urine sample analysis, mobile phase consists of 5 mM ammonium formate with 0.1% formic acid and acetonitrile for evogliptin; 5 mM ammonium formate with 0.1% formic acid and methanol for M7 and M8. Mobile phase consists of 10 mM ammonium formate with formic acid and acetonitrile for pioglitazone, M3 and M4. Other LC and MS/MS condition was same as in plasma sample analysis. Calibration curves of plasma analytes were linear within the range of 5 – 5,000 ng/mL for evogliptin, 0.5 – 500 ng/mL for M7 and M8, 30 – 10,000 ng/mL for pioglitazone and 30 – 10,000 ng/mL for M3 and M4 (r ≥ 0.9950). Accuracy and precision of intra-batch quality control (QC) is 100.4 – 107.1 % and < 3.6 % for evogliptin, 95.3 – 99.4 % and < 5.2 % for M7, 97.4 – 110.8 % and < 6.2 % for M8, 92.4 – 105.4 % and < 9.9 % for pioglitazone, 103.3 – 109.1 % and < 4.3 % for M3, and 99.2 – 107.8 % and < 8.2 % for M4.