ABSTRACT
JJH201501, a deuterated modification for multi-target antidepressant vortioxetine, is currently in phase I clinical trial. This study aimed to establish a sensitive and rapid UPLC-MS/MS method that was capable of simultaneously detecting JJH201501 and its major metabolite JJH201501-01 quantitatively in human plasma. The pretreatment was achieved by protein precipitation using 4-fold(v:v) acetonitrile with 5 ng/mL fluoxetine as internal standard precipitant. For method validation, the method was investigated in terms of the selectivity, inter- and intra-run precision and accuracy, carryover, matrix effect, extraction recovery and stability. The total running time was 3 min, and the retention time of JJH201501 and JJH201501-01 was 1.17 min and 1.05 min, respectively. The linear concentration range for JJH201501 and JJH201501-01 was 0.2 to 50 ng/mL and 0.4 to 100 ng/mL, respectively. The results showed that this method was in line with the guidelines for bioanalytical method proposed by FDA. In addition, the method was successfully applied to a plasma pharmacokinetic study of JJH201501 tablets in healthy volunteers which was part of the phase I trial.
Subject(s)
Chromatography, High Pressure Liquid/methods , Tandem Mass Spectrometry/methods , Vortioxetine/blood , Vortioxetine/pharmacokinetics , Adolescent , Adult , Deuterium , Female , Humans , Limit of Detection , Linear Models , Male , Middle Aged , Reproducibility of Results , Vortioxetine/analogs & derivatives , Vortioxetine/chemistry , Young AdultABSTRACT
Human cytochrome P450 1B1 (CYP1B1)-mediated formation of 4-hydroxyestradiol (4-OHE2) from 17ß-estradiol plays an important role in the progression of human breast cancer, while the biotransformation of 17ß-estradiol to 2-hydroxyestradiol mediated by cytochrome P450 1A1 (CYP1A1) is considered as a less harmful pathway. In this study, inhibitory effects of flavonoids baicalein and oroxylin A, a metabolite of baicalein in human body, on CYP1A1 and 1B1 activities were investigated in vitro. The inhibition intensities of baicalein and oroxylin A towards CYP1B1 were greater than towards CYP1A1 with a mixed mechanism. In addition, oroxylin A showed a stronger inhibitory effect than baicalein towards the CYP1B1-mediated 17ß-estradiol 4-hydroxylation, with the IC50 values of 0.0146 and 2.27 µM, respectively. Docking studies elucidated that oroxylin A had a stronger binding affinity than baicalein for CYP1B1. In MCF-7 cells, compared with baicalein-treated groups, oroxylin A with lower doses decreased and increased the formation of 4-OHE2 and 2-hydroxyestradiol, respectively, with a preferential induction of mRNA of CYP1A1 over CYP1B1. In conclusion, this study demonstrated that oroxylin A showed a stronger inhibitory effect than baicalein on CYP1B1-mediated 4-OHE2 formation in MCF-7 cells, providing crucial implications for their possibly preventive/therapeutic potential against breast cancer via inhibition of CYP1B1, particularly of oroxylin A.
Subject(s)
Carcinogenesis/drug effects , Carcinogenesis/genetics , Cytochrome P-450 CYP1B1/genetics , Estradiol/analogs & derivatives , Estrogens, Catechol/metabolism , Estrogens, Catechol/toxicity , Flavanones/pharmacology , Flavonoids/pharmacology , Breast Neoplasms/chemically induced , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carcinogenesis/chemically induced , Carcinogens/metabolism , Carcinogens/toxicity , Down-Regulation/drug effects , Down-Regulation/genetics , Estradiol/metabolism , Female , Flavanones/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 CellsABSTRACT
Targeting therapy of anti-cancer drugs has been gaining increasing attention. Cell pharmacokinetics have been used for in vitro disposition evaluation, as well as drug-drug interaction for anti-cancer drugs, revealing their fate after entering tumor. Flavonoid compound oroxylin A (OA) possesses strong anti-cancer effects especially on the liver and breast cancer. However, despite the low bioavailability, the disposition of OA and its active metabolite in the target cancer cells remained unclear. In current study, a highly sensitive and selective solid phase extraction (SPE)-UPLC-MS/MS method was developed and validated to simultaneously quantify the concentrations of OA and its major active metabolite oroxylin A 7-O-d-glucuronide (OG) in HepG2 cell lysate and multiple subcellular organelle fractions. The proposed method appeared to be suitable for the analysis with desirable linearity(R2â¯>â¯0.99). The relative standard deviations (RSDs) of intra- and inter-assay precision and accuracy were less than 9.9% and -7.7%, 8.4% and 11% for OA and OG in cell lysate respectively. The intra- precision and accuracy was less than 9.5% and -11.3%, 9.4% and 12.3% for OA and OG in subcellular organelles respectively. The range of absolute recovery of this method in the cell lysate was from 73.1%⯱â¯1.4% to 87.9%⯱â¯6.7%. The RSDs of matrix effects of the quality control (QC) samples were below 15%. The uptake and distribution experiments demonstrated a time-dependent transport characteristic in HepG2 cell lines. Furthermore, both OA and OG were mainly distributed into nuclei after taken up by the tumor cells. In addition, OG was also distributed into mitochondria, which indicates another potential target of OG. The present study, for the first time, reports the in vitro cell pharmacokinetics profiles of OA and OG in tumor cell lines in vitro.
