Pharmacokinetics and metabolite identification of a novel VEGFR-2 and Src dual inhibitor 6-chloro-2-methoxy-N-(2-methoxybenzyl) acridin-9-amine in rats by liquid chromatography tandem mass spectrometry
Highlights
► A robust LC-MS/MS method was developed for MBAA analysis in rat body fluid samples. ► Main pharmacokinetics parameters of MBAA were measured. ► Metabolic pathways of MBAA were presumed in the liver of rats. ► MBAA was suggested to metabolism and a strong first-pass clearance in the liver.
Introduction
Acridine derivatives possess numerous biological activities, such as anti-inflammatory [1], [2], anti-bacterial [3], [4], anti-parasite [5], and anti-cancer [6]. Some of acridines, such as amsacrine [7], [8] and pyrazoloacridine [9], [10], have been clinically used as anticancer agents which act by intercalating with DNA and thus inhibiting DNA related enzymes, such as topoisomerase II [11] and telomerase [12], [13]. Recent study indicates that the nature and position of substituents on the acridine scaffolds play a critical role in their anticancer properties. Based on a molecular docking and SVM virtual screening method [14], we have synthesized a series of 9-aminoacridine derivatives as VEGFR-2 [15], [16] and Src [17] inhibitors (such as tyrosine inhibitor) to obtain acridines with better anticancer activity. Among which 6-Chloro-2-methoxy-N-(2-methoxybenzyl) acridin-9-amine (MBAA) [18] showed promising activity against K562 and HepG-2 cancer cells by inhibiting VEGFR-2 and Src activity, but not topoisomerase. MBAA at 10 μM significantly reduced the level of activated ERK 1/2 in a time dependant manner. Such selectivity and specificity of MBAA is beneficial to reducing host toxicity. However, the pharmacokinetic and metabolism of MBAA remain unknown.
As a novel anticancer lead compound and tyrosine kinase inhibitor with an acridine scaffold, pharmacokinetic characteristics and metabolites of MBAA in vivo are important parameters for the evaluation of its potential as an anticancer agent. Multi-analyses of the pharmacokinetics and metabolic processes in vivo may comprehend the mechanism of action and provide appreciable information for further structure modifications to develop more effective antitumor analogues of acridines. On the other hand, to know the process (absorption, metabolism, and elimination) of the new compound candidates in order to select the route of administration and the dose. Several methods have been reported for the quantitation of acridine derivatives, such as liquid chromatography (LC) with ultraviolet (UV) [19], [20], electrochemical [21] and fluorescence detection [22]. Up to date, HPLC with different detection modes is well established, but is unable to distinguish the same kinds of mixture. Liquid chromatography tandem mass spectrometry (LC-MS/MS) with APCI source can provide the information of distinct quality [22], but uncertainty of derivatization procedures and the LOD value of APCI-MS tests limit its use as a quick biopharmaceutical analysis. Besides, in order to satisfy the requirement of sensitivity, a large volume of body fluid samples were usually necessary. To accurately know the pharmacokinetics and metabolism behavior of acridine derivatives in vivo, it is necessary to establish a suitable method for measuring lower concentrations of acridine derivatives in bio-samples and reducing the time and requirements for sample preparation.
In this study, an accurate, sensitive and rapid LC-ESI-MS/MS method is developed for MBAA quantification in rat plasma, urine and bile. Pharmacokinetics of MBAA was studied in rats when administered by oral or intravenous injection. Based on previous pharmacokinetic indicators of acridine derivatives [23], [24], the compounds responsible for the first-pass effect in the body were supposed. The phase I/II metabolites of this novel 9-aminoacridine derivative and the fragmentation rules of metabolic products were defined.
Section snippets
Chemicals and reagents
6-Chloro-2-methoxy-N-(2-methoxybenzyl) acridin-9-amine (MBAA) was synthesized and purified as previously described [18]. Internal standard (IS) fluoxetine and formic acid (LC-MS grade) were purchased from Sigma-Aldrich (St Louis, MO, USA). Methanol and acetonitrile (HPLC grade) were obtained from Fisher Co., Ltd. (Emerson, IA, USA). Methylcellulose, Tween-80, DMSO and other reagents of analytical grade were purchased from Fuchen Chemical Factory (Tianjin, China). A Milli-Q water system
LC-MS/MS
For the quantitation of MBAA, the mobile phase was optimized. Gradient elution was used for narrowing the peaks of MBAA and IS and shortening running time of the chromatography. The running time for each sample was just for 2.5 min. ESI was chosen as the interface based on the structure of compounds which made an intense signal. Sensitivity ratio of the MRM scan mode was significantly superior to that of the selected ion recording (SIR). Cone voltage and collision energy were optimized for each
Conclusions
This work developed and validated a specific LC-MS/MS method for the quantitation of 6-Chloro-2-methoxy-N-(2-methoxybenzyl) acridin-9-amine and identification of its metabolites in the rats’ body fluid samples. This method showed excellent sensitivity, linearity, precision and accuracy. It was successfully applied to evaluation of pharmacokinetics of MBAA in rats. A LC-MS/MS based systematic approach for rapid metabolite identification of compounds such as MBAA that undergo multiple metabolic
Acknowledgements
This work was supported by grants from the International S&T Cooperation Program of China (2011DFA30620), the Ministry of Science and Technology of China (2009ZX09501-004) and the Chinese National Natural Science Foundation (21172129, 20872077, 90813013).
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