Determination of NVP-BEZ235, a dual PI3K and mTOR inhibitor, in human and mouse plasma and in mouse tissue homogenates by reversed-phase high-performance liquid chromatography with fluorescence detection.
NVP-BEZ235 is a novel dual inhibitor of PI3K/mTOR and currently undergoing phase I/II clinical trials for advanced solid tumors. We developed a sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) assay with fluorometric detection for quantification of NVP-BEZ235 in biological matrices. Liquid-liquid extraction with tert-butyl methyl ether was used for sample pre-treatment, yielding a recovery of >84%. Chromatographic separation of NVP-BEZ235 and the internal standard (IS) NVP-BBD130 was achieved on a GraceSmart C-18 column by isocratic elution with a mobile phase which consisted of acetonitrile, methanol, and milliQ water adjusted with acetic acid to pH 3.7 (20:36:44, v/v/v). Fluorescence detection using excitation and emission wavelengths of 270 and 425 nm, respectively, provided a selectivity and sensitivity allowing quantification down to 1 ng/ml in human plasma and linear calibration curves within a range of 1-1000 ng/ml. The assay was validated for human plasma, mouse plasma and a range of tissues. The accuracy, within-day and between-day precision for all matrices, was within the generally accepted 15% range. NVP-BEZ235 was stable for 72 h in pretreated samples in reconstitution mixture (acetonitrile-water (30:70, v/v)), but unstable in mouse tissue homogenates upon repeated freeze-thaw cycles or long term storage (≥24 h) at room temperature. A pilot pharmacokinetic study in mice demonstrated the applicability of this method for pharmacokinetic purposes. Overall, this assay is suitable for the pharmacokinetic studies of NVP-BEZ235 in mice and in human plasma.
Department of Clinical Chemistry/Preclinical Pharmacology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Fan
F
Lin
Gayathri
G
Chandrasekaran
Mark C
MC
de Gooijer
Jos H
JH
Beijnen
Olaf
O
van Tellingen
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D007093:Imidazoles / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics; D016014:Linear Models; D059625:Liquid-Liquid Extraction; D051379:Mice; D011804:Quinolines / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014018:Tissue Distribution
D007093:Imidazoles; D011804:Quinolines; C531198:dactolisib