| An integrated approach on basis of rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS) was developed for in vivo drug metabolites screening and identification. This approach was applied to analyzing metabolites of a potential anticancer agent,3,6,7-trimethoxyphenanthroindolizidine (CAT) in rats'urine. And the metabolic differences between healthy and Walker256tumor-bearing rats were compared to evaluate the influence of Walker256tumor. Based on the research above, pharmaco-metabonomic study of CAT was accomplished to obtain the alternations of endogenous metabolites resulting from the intervention of drugs under different pathological states. In the metabolite study,21metabolites were detected in rat urine after oral administration of CAT and1metabolite was found to have significant difference (P<0.05) between healthy and Walker256tumor-bearing rats. In the pharmaco-metabonomic study, the integration of sign changes helped to find the potential biomarkers that were closely associated to the toxicity and the efficacy of drug. As a result, two endogenous metabolites related to the drug toxicity with healthy rats and three endogenous metabolites related to drug efficacy with Walker256tumor-bearing rats were found. The identification of CAT and its metabolites as well as the endogenous metabolites that were closely related to the toxicity or the efficacy of drug will provide important in vivo metabolic transformation information for the successfully development of potential anticancer agent CAT and offer a better understanding of its mechanism of action.1. Integrated RRLC-MS/MS approach for screening and identification of metabolitesAn integrated approach combining data acquisition using MSE and multi-period product ion scan (mpMS/MS), with high-resolution characteristic extracted ion chromatograms (hcXIC) as a data mining method, was developed for in vivo drug metabolites screening and identification. Untargeted full-scan MSE enabled the high-throughput acquisition of potential metabolites, and targeted mpMS/MS contributed to the sensitivity and specificity of the acquisition of molecules of interest. This approach efficiently combined the superiority of MSE and mpMS/MS. The data processing method hcXIC, based on the structure of CAT, was shown to be highly effective for the metabolite discovery. Through the double-filtering effect of the characteristic ion and accurate mass, conventional extracted ion chromatograms that contained a substantial number of false-positive peaks were simplified into chromatograms essentially free of endogenous interferences.2. Metabolic study of CAT in healthy rats and Walker256tumor-bearing ratsUsing established integrated RRLC-MS/MS approach, the urine samples after oral administration of CAT were analyzed and21metabolites were detected, including9phase â… and12phase â…¡ metabolites. Based on the characteristic fragmentation patterns of the phenanthroindolizidine alkaloid, the structures of9metabolites were identified. Then semi-quantitative analysis of detected metabolites was carried out by average comparison and t-test to find metabolic differences bwteen healthy and Walker256tumor-bearing rats. As a result, metabolite M10was at a relative higher level in Walker256tumor-bearing group (p<0.05). In order to further explore the reason for the metabolic difference, the enzyme activity of UDP-glucuronosyltransferase (UGT) was analyzed. The results showed that there were no significant difference in UGT enzyme activity between the two groups, which indicated that biological environment difference leads to the higher level of M10in Walker256tumor-bearing rats. The research is helpful for understanding the transformation and possible mechanism of CAT in rats.3. Metabonomic study on Walker256tumor modelBased on the established metabonomic approach, the metabonomic research on Walker256tumor-bearing rats was performed using peak area normalization and volumes of24h urine as the normalization method for metabonomic analysis of urine sample, combined with time-course analysis of discriminating metabolites. As a result,29low-molecular-weight discriminating metabolites were detected to have significant differences between healthy and Walker256tumor-bearing rats, whose abnormal changes might be highly associated with the perturbations in metabolism, which were believed as potential biomarkers. Then high resolution MS and MS/MS spectra analysis were performed for the identification of the metabolites of interest,7of which were eventually confirmed with further validation by metabolite database searching as well as standard samples comparison, including L-carnitine, acetyl-carnitine, cytidine,2'-deoxycytidine, hypoxanthine, urocanic acid and creatine.4. Pharmaco-metabonomics study of CATIn this study, the sign changes were integrated into pharmaco-metabonomic study to find the potential biomarkers that were closely associated to the toxicity or the efficacy of drug. First of all, according to the physical signs such as body weights and survival time, the toxicity and efficacy of CAT were analyzed. It was found that CAT showed dose-dependent toxicity in healthy rats, while double effects (toxicity and efficacy) in Walker256tumor-bearing rats administrated with high, middle and low dosage CAT. For high dosage, toxicity was greater than efficacy. For middle and low dosage, efficacy was greater than toxicity. In addition, metabolic changes resulting from administration of CAT and sign changes were complemented to acquire discriminating metabolites between CAT administration groups and corresponding control groups. As a result, two endogenous metabolites related to the toxicity with healthy rats and three endogenous metabolites related to efficacy with Walker256tumor-bearing rats were found using this approach, which provided a novel way for pharmaceutical research. The results will provide important in vivo information for a better understanding of the efficacy and toxicity of potential anticancer agent CAT.
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