Investigation of in vitro drug-drug interactions and analysis of candesartan cilexetil by various analytical techniques

Candesartan cilexetil (KSSL) is a prodrug of candesartan (KS) which is used for hypertension therapy. Capillary electrophoretic (CE) and high performance liquid chromatographic (HPLC) methods are described for the investigation of the in vitro pharmacokinetic interaction between KS and diclofenac sodium (DIC) which is a standard CYP 2C9 substrate using rat liver microsomal enzymes, in this study.In the first step, a CE method was developed for the simultaneous determination of KS, KSSL and DIC by using metoprolol succinate (MTP) and lansoprazole (LNS) as internal standards (IS). The signals were recorded at 214 nm by UV detector. The optimum conditions for the best separation were 15 mM borate buffer (pH 8.5) containing 10 % (v/v) methanol as run buffer, 25 kV for separation potential and 1 s for injection time. The migration times were 3.3 min for MTP, 4.7 min for LNS, 5.1 min KSSL and 7.5 min for KS. The method was validated in the concentration range of 1.88 x 10-6 ? 9.41 x 10-5 M for KSSL and 1.91 x 10-6 ? 9.58 x 10-5 M for KS for linearity, precision, accuracy, ruggedness, robustness and selectivity. The limit of detection (LOD) of the method for KSSL and KS were 3.07 x 10-7 M and 4.79 x 10-7 M respectively and limit of quantitation (LOQ) for KSSL and KS were 9.15 x 10-7 M and 1.45 x 10-6 M respectively. The method was also applied to the commercial tablet for the determination of KSSL and the results obtained were in the limits of USP XXIV.In the second step the analysis of DIC, MTP and KS by HPLC was performed with fluorescence detector at 290 nm as excitation and 360 nm as emission wavelengths, and a pH 3.5, 57 % methanol (v/v) solution containing 0.01 % (w/v) 1-heptane sulphonic acid was used as mobile phase. Separation was achieved using C18 column (3.0 x 150 mm, 5 µm) at 1 mL/min flow rate with isocratic system. Retention times were 2.5 min for MTP, 4.6 min for KS and 16.4 min for DIC. The method validation was also investigated in the concentration range 1.14 x 10-8 ? 1.46 x 10 -6 M for KS and 3.67 x 10-7 ? 3.91 x 10-5 M for DIC to show the linearity, accuracy, precision, ruggedness, robustness and selectivity. LOD was 2.46 x 10-7 M and LOQ was 7.47 x 10-7 M for DIC, and LOD was 1.72 x 10-8 M and LOQ was 5.22 x 10-8 M for KS.The in vitro pharmacokinetic interaction between KS and DIC has been investigated using rat liver microsomal enzymes in the last part of the study. The effects of the microsomal protein and substrate (DIC and KS) concentration on the drug metabolization were investigated to find out optimum parameters. The samples obtained from in vitro KS and DIC interaction studies were analyzed by using the developed methods and the results were compared.Key Words: candesartan cilexetil, capillary electrophoresis, cytochrome P450 2C9, diclofenac sodium, high performance liquid chromatography