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: applicati

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• 作者：Zhou ; Shufeng ; Feng ; Xia ; Kestell ; Philip ; Baguley ; Bruce C. ; Paxton ; James W.
• 关键词：AP ; apical ; BL ; basolateral ; CYP ; cytochrome P450 ; DMSO ; dimethyl sulphoxide ; DMXAA ; 5 ; 6-dimethylxanthenone-4-acetic acid ; DMXAA-G ; DMXAA acyl glucuronide ; HBSS ; Hank’ ; s balanced salt solution ; HEPES ; N-[2-hydroxyethyl]piperazine-N&prime ; -[4-butanesul
• 刊名：Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2004
• 出版时间：September 25, 2004
• 年：2004
• 卷：809
• 期：1
• 页码：87-97
• 全文大小：167 K

文摘

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 6.8min, with acceptable separation of the compounds of interest. The limit of quantitation (LOQ) values for DMXAA and DMXAA-G were 14.2 and 24ng/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 10–500μM were independent of concentration for both apical (AP) to basolateral (BL) (4.0 ± 0.4 × 10⁻⁵cm/s) and BL-AP (4.3 ± 0.5 × 10⁻⁵cm/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 200μM, 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.