Evaluation of the metabolism of PEP06,an endostatin-RGDRGD 30-amino-acid polypeptide and a promising novel drug for targeting tumor cells / 药物分析学报

PEP06 is a novel endostatin-Arg-Gly-Asp-Arg-Gly-Asp(RGDRGD)30-amino-acid polypeptide featuring a terminally fused RGDRGD hexapeptide at the N terminus.The active endostatin fragment of PEPO6 directly targets tumor cells and exerts an antitumoral effect.However,little is known about the kinetics and degradation products of PEP06 in vitro or in vivo.In this study,we investigated the in vitro metabolic stability of PEP06 after it was incubated with living cells obtained from animals of different species;we further identified the degradation characteristics of its cleavage products.PEP06 underwent rapid enzymatic degradation in multiple types of living cells,and the liver,kidney,and blood play important roles in the metabolism and clearance of the peptides resulting from the molecular degradation of PEP06.We identified metabolites of PEP06 using full-scan mass spectrometry(MS)and tandem MS(MS2),wherein 43 metabolites were characterized and identified as the degradation metabolites from the parent peptide,formed by successive losses of amino acids.The metabolites were C and N terminal truncated products of PEP06.The structures of 11 metabolites(M6,M7,M16,M17,M21,M25,M33,M34,M39,M40,and M42)were further confirmed by comparing the retention times of similar full MS spectrum and MS2 spectrum information with reference standards for the synthesized metabolites.We have demonstrated the metabolic stability of PEP06 in vitro and identified a series of potentially bioactive downstream metabolites of PEP06,which can support further drug research.

Rapid characterization of chemical constituents and rat metabolites of Fufang Gancao Tablets by UHPLC-LTQ-Orbitrap mass spectrometer / 中国中药杂志

Ultra-high-performance liquid chromatography coupled with tandem high-resolution mass spectrometry (UHPLC-HR-MSn ) method was used to analyze the constituents of Fufang Gancao Tablets and its main metabolites in rat plasma. Rat plasma was collected both before and after oral administration of Fufang Gancao Tablets. After solid phase extraction, ACQUITY UHPLC BEH C₁₈ (2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid-acetonitrile solution as the mobile phase for gradient elution. The chemical components in Fufang Gancao Tablets and their prototypes and metabolites in plasma samples were analyzed by LTQ-Orbitrap equipped with an ESI ion source in a positive ion mode. Based on the accurate mass measurements, the retention time and mass fragmentation patterns, a total of 55 compounds were tentatively identified from Fufang Gancao Tablets, including 42 flavonoids, 9 triterpenes and 4 alkaloids. Furthermore, metabolites in rat plasma after oral administration of Fufang Gancao Tablets were also analyzed. A total of 26 compounds were identified, including 20 prototypes and 6 metabolites mainly through metabolic pathways of hydroxylation, glucuronide conjugation, and sulfate conjugation, etc. Our results showed that the ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap) could comprehensively elucidate the chemical constituents of Fufang Gancao Tablets and their migrating components in rat plasma, providing scientific basis for further studying the metabolism process and pharmacodynamic substance of Fufang Gancao Tablets.

Rapid characterization of chemical constituents and rats metabolites of Kudiezi injection by UHPLC-LTQ-Orbitrap / 中国中药杂志

A rapid and sensitive UHPLC-HR-MSn method was used for the identification of Kudiezi injection and its main metabolites in rat plasma. After the tail intravenous injection of Kudiezi, ACQUITY UHPLC BEH C₁₈ (2.1 mm×100 mm, 1.7 μm) was used, with 0.1% formic acid-acetonitrile solution as the mobile phase for gradient elution. Kudiezi injection and plasma were detected by ESI-LTQ-Orbitrap equipped with an ESI ion source in a negative mode. Based on the accurate mass measurements, the retention time and the mass fragmentation patterns, a total of 53 compounds were tentatively identified and characterized. Furthermore, metabolites in rat plasma after the intravenous administration of Kudiezi injection were also analyzed. A total of 38 compounds were identified, including 27 prototypes and 11 metabolites through metabolic pathways of methylation, glucuronide conjugation, sulfate conjugation and hydrolysis. As a result, UHPLC-LTQ-Orbitrap technique was applied to comprehensively expound Kudiezi injection's chemical components and constituents migrating to rat plasma, and provide scientific basis for further studies on Kudiezi injection's in vivo metabolic process and effective material base.