Title

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

Document Type

Article

Publication Date

9-2004

Keywords

5, 6-dimethylxanthenone-4-acetic acid, Caco-2 cells

Digital Object Identifier (DOI)

https://doi.org/10.1016/j.jchromb.2004.06.009

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 (Papp) 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 (Rnet) values of 1–1.3. However, the Papp values for the BL to AP transport of DMXAA-G were significantly greater than those for the AP to BL transport, with Rnet 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.

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No

Citation / Publisher Attribution

Journal of Chromatography B, v. 809, issue 1, p. 87-97

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