Determination of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid and its acyl glucuronide in Caco-2 monolayers by liquid chromatography with fluorescence detection: application to transport studies

Abstract

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a potent cytokine inducer, with a bioavailability of >70% in the mouse. The aim of this study was to develop and validate HPLC methods for the determination of DMXAA and DMXAA acyl glucuronide (DMXAA-G) in the human intestinal cell line Caco-2 monolayers. The developed HPLC methods were sensitive and reliable, with acceptable accuracy (85−115% of true values) and precision (intra- and inter-assay CV < 15%). The total running time was within 6.8 min, with acceptable separation of the compounds of interest. The limit of quantitation (LOQ) values for DMXAA and DMXAA-G were 14.2 and 24 ng/ml, respectively. The validated HPLC methods were applied to examine the epithelial transport of DMXAA and DMXAA-G by Caco-2 monolayers. The permeability coefficient ( P app) values (overall mean ± S.D., n = 3–9) of DMXAA over 10–500 μM were independent of concentration for both apical (AP) to basolateral (BL) (4.0 ± 0.4 × 10 −5 cm/s) and BL-AP (4.3 ± 0.5 × 10 −5 cm/s) transport, and of similar magnitude in either direction, with net efflux ratio ( R net) values of 1–1.3. However, the P app values for the BL to AP transport of DMXAA-G were significantly greater than those for the AP to BL transport, with R net values of 17.6, 6.7 and 4.5 at 50, 100 and 200 μM, respectively. Further studies showed that the transport of DMXAA-G was Na +- and energy-dependent, and inhibited by MK-571 [a multidrug resistance associated protein (MRP) 1/2 inhibitor], but not by verapamil and probenecid. These data indicate that the HPLC methods for the determination of DMXAA and DMXAA-G in the transport buffer were simple and reliable, and the methods have been applied to the transport study of both compounds by Caco-2 monolayers. DMXAA across Caco-2 monolayers was through a passive transcellular process, whereas the transport of DMXAA-G was mediated by MRP1/2.