Background As a prodrug of 5-fluorouracil (5-FU), orally administrated capecitabine (CAP) undergoes preliminary conversion into active metabolites in the liver and then releases 5-FU in the gut to exert the anti-tumor activity. Since metabolic changes of CAP play a key role in its activation, a single kind of intestinal or hepatic cell can never be used in vitro to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) nature. Hence, we aimed to establish a novel in vitro system to effectively assess the PK and PD of these kinds of prodrugs. Methods Co-culture cellular models were established by simultaneously using colorectal cancer (CRC) and hepatocarcinoma cell lines in one system. Cell Counting Kit-8 (CCK-8) and flow cytometric analysis were used to evaluate cell viability and apoptosis, respectively. Apoptosis-related protein expression levels were measured using western blot analysis. A selective liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for cellular PK in co-culture models. Results CAP had little anti-proliferative effect on the five monolayer CRC cell lines (SW480, LoVo, HCT-8, HCT-116 and SW620) or the hepatocarcinoma cell line (HepG2). However, CAP exerted marked anti-tumor activities on each of the CRC cell lines in the co-culture models containing both CRC and hepatocarcinoma cell lines, although its effect on the five CRC cell lines varied. Moreover, after pre-incubation of CAP with HepG2 cells, the culture media containing the active metabolites of CAP also showed an anti-tumor effect on the five CRC cell lines, indicating the crucial role of hepatic cells in the activation of CAP. Conclusion The simple and cost‑effective co-culture models with both CRC and hepatocarcinoma cells could mimic the in vivo process of a prodrug dependent on metabolic conversion to active metabolites in the liver, providing a valuable strategy for evaluating the PK and PD characteristics of CAP-like prodrugs in vitro at the early stage of drug development.
Fuzhuan brick tea, a distinctive dark tea fermented by microorganisms, is a traditional beverage in China throughout history. Recently, it has attracted considerable attention owing to its unique quality characteristics and potential health benefits. The aim of this study was to establish a method for the quality control of Fuzhuan brick tea for stable production. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was used to identify Fuzhuan brick tea, and the major components were chosen for further quantitative analysis. Subsequently, a quantification method was developed using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry, and its reliability was verified through methodological validation. Finally, a total of 30 compounds were identified, including catechins, flavonoids, alkaloids, and fatty acids. The established method was reliable for methodological validation and was applied to the quantitative analysis of Fuzhuan brick tea. This study provides a fundamental basis for the quality control and further studies on the component analysis of Fuzhuan brick tea.
Background: CPUL1, a phenazine analog, has demonstrated potent antitumor properties against hepatocellular carcinoma (HCC) and indicates a promising prospect in pharmaceutical development. However, the underlying mechanisms remain largely obscure. Methods: Multiple HCC cell lines were used to investigate the in vitro effects of CPUL1. The antineoplastic properties of CPUL1 were assessed in vivo by establishing a xenograft nude mice model. After that, metabolomics, transcriptomics, and bioinformatics were integrated to elucidate the mechanisms underlying the therapeutic efficacy of CPUL1, highlighting an unanticipated involvement of autophagy dysregulation. Results: CPUL1 suppressed HCC cell proliferation in vitro and in vivo, thereby endorsing the potential as a leading agent for HCC therapy. Integrative omics characterized a deteriorating scenario of metabolic debilitation with CPUL1, presenting an issue in the autophagy contribution of autophagy. Subsequent observations indicated that CPUL1 treatment could impede autophagic flow by suppressing autophagosome degradation rather than its formation, which supposedly exacerbated cellular damage triggered by metabolic impairment. Moreover, the observed late autophagosome degradation may be attributed to lysosome dysfunction, which is essential for the final stage of autophagy and cargo disposal. Conclusions: Our study comprehensively profiled the anti-hepatoma characteristics and molecular mechanisms of CPUL1, highlighting the implications of progressive metabolic failure. This could partially be ascribed to autophagy blockage, which supposedly conveyed nutritional deprivation and intensified cellular vulnerability to stress.
An active substance of pyrano[3,2‐a]phenazine, also called CPUL1, is a synthesized phenazine derivative and displays broad‐spectrum anticancer activities. Quantitative assessment of CPUL1 in biological samples has not been well established, hindering pharmaceutical development and application. According to international guidelines, a sensitive and selective liquid chromatography‐tandem mass spectrometry method in negative ion mode was developed and validated for quantification of CPUL1 in human plasma, colorectal cancer cell lines, and rat plasma, whereby linearity and accuracy were demonstrated for the range of 1–1000 ng/ml. The validated liquid chromatography‐tandem mass spectrometry method was successfully employed in pharmacokinetic studies of CPUL1 in vitro and in vivo. Notably, the cellular pharmacokinetic behavior of CPUL1 varies in colorectal cancer cell lines. Regarding the pharmacokinetic processes in vivo, oral absorption was less effective than an injection, with a bioavailability of 23.66%. CPUL1 was linearly eliminated after a single administration; however, it could accumulate in tissues (heart, liver, spleen, lung, and kidney) after multiple injections. In summary, this study established a capable bioanalytical method for CPUL1 and provided exploratory pharmacokinetic data, paving the way for use of this promising derivative in disease models.
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