network-pharmacology-vitro-cell-experimental-study-for-exploring

Network Pharmacology and <em>In Vitro</em> Cell Experimental Study for Exploring the Therapeutic Potential of Thiazolone Derivatives in Rhabdomyosarcoma

Rhabdomyosarcoma (RD) is a malignant tumor originating from rhabdomyoblasts, which poses a serious threat to the health of children and adolescents. Based ...

Shanghai University of Sport

: AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside) was reported as the first pharmacological AMPK (adenosine 5'-monophosphate (AMP)-activated protein kinase) activator, and it has been confirmed to exhibit a significant endurance enhancement effect and prohibited for doping by the World Anti-Doping Agency. Due to the fact that the human body can produce such substances, in order to ensure fairness in sports competition, methods for rapid detection and multi-type identification of AICAR drugs taken orally should be established. : to assess AICAR levels, a new rapid, sensitive, efficient, and selective method was reported for the quantitative detection of AICAR in urine using LC-MS/MS. The method was validated for quantitative purposes based on the elemental selectivity, intra- (1.0-15.6%) and inter-day precision (1.3-16.3%), accuracy (99.9-112.8%), matrix effects (88.9-103.6%), recovery (87.4-106.5%), and stability at four different concentrations. The calibration curve was linear over a wide concentration range of 10-10,000 ng mL with a high coefficient of determination (  > 0.998). The limit of detection (LOD) and limit of quantification (LOQ) for the experiment were determined to be 1 and 10 ng mL, respectively. Simultaneously, metabolomics analysis was used to obtain the metabolic fingerprint of different populations and biomarkers to distinguish administration cases through partial least squares discriminant analysis (PLS-DA) and a receiver operating characteristic (ROC) curve. : the method enables easy quantitation for LC-MS/MS analysis with the best recovery yield maintained, and the method was applied to 122 Asian biological samples with an average concentration of 1310.5 ± 1031.4 ng mL. Through drug metabolism research, 734 and 294 variables were extracted for data analysis respectively in the positive and negative ion modes, and more than 100 metabolites with significant up- and down-regulation were found after the test. : this research developed a fast, precise, effective, and specific approach for the qualitative and quantitative identification of AICAR in urine. Meanwhile, administration metabolism studies found that there were significant changes in AICAR levels and other compounds, such as PC types PC(18:1/16:0), PC(16:0/18:0), and PC(16:0/16:0), PE types PE(18:0/20:4), and LPE-type 18:1, which could better distinguish samples before and after AICAR administration. The analysis provides a multi-perspective reference for WADA to determine a positive criterion.

Quantification of AICAR and study of metabolic markers after administration.

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside), as a metabolic modulator, is classified in the S4 category by the World Anti-Doping Agency (WADA). Carbon Isotope Ratio Mass Spectrometry (CIR) is the mainstream method for distinguishing the endogenous and exogenous sources of AICAR in urine due to the significant individual difference in the concentration. The purpose of this study is to establish a gas chromatography combustion Isotope Ratio Mass Spectrometry (GC/C/IRMS) method for AICAR based on efficient two-dimensional liquid chromatography (2D-HPLC) separation.

Two-Dimensional Liquid Chromatography Purified GC/C/IRMS Doping Control Method: Analysis of Endogenous and Exogenous Sources in Urine Samples from Asian Subjects Administered a Low Dose of AICAR.

Chlorphenesin carbamate is a skeletal muscle relaxant, and one of its metabolites is 4-chlorophenoxyacetic acid (4-CPA), which is included on the Prohibited List of the World Anti-Doping Agency (WADA). The non-prohibited substance, chlorphenesin carbamate, as a potential source of 4-CPA, needs to be identified more strongly in doping control protocols. In this paper, the metabolism of chlorphenesin carbamate in human urine was studied. Ten volunteers were recruited to take chlorphenesin carbamate tablets orally, and urine samples were collected before and after being tested. The urine samples were detected by liquid chromatography-Q Exactive HF orbitrap mass spectrometry (LC-Q Exactive HF-MS) in full MS and full MS-ddMS scanning modes. Based on accurate molecular formulae determination and fragmentation pattern analysis by mass spectrometry, a total of 29 chlorphenesin carbamate metabolites were found, comprising 18 newly identified metabolites and 11 previously reported metabolites. There were five potential metabolic processes listed: hydroxylation, amide hydrolysis, C-oxidation, O-glucuronidation, sulfation, and their combinations. Chlorphenesin carbamate and the 29 metabolites were compared for their detection windows, and the findings indicated that the two recently reported metabolites, M9 and M10, had longer detection windows than the metabolites documented by the WADA. These metabolites are anticipated to be long-lasting biomarkers for the detection of chlorphenesin carbamate intake. By analyzing the results of metabolomic profiling, it was found that the metabolites with significant changes were mainly related to histidine metabolism and β-alanine metabolism pathways. This paper provides a comprehensive report on the metabolic profile of urinary chlorphenesin carbamate and reveals a point of view the changes in the metabolic in the human body, which is conducive to supporting the detection of chlorphenesin carbamate and meclofenoxate for doping control, as well as a better understanding of the mechanism of action of chlorphenesin carbamate as a drug.

Detection and Identification of New Chlorphenesin Carbamate Metabolites in Human Urine and Metabolomic Characterization Using Liquid Chromatography-Q Exactive-HF-Orbitrap-Mass Spectrometry.

Using a single qualitative and quantitative test for ethanol consumption sometimes fails to reflect the objective facts at the beginning of the incident for a variety of reasons, such as metabolism, diffusion, collection contamination, storage losses, and decomposition. Stable isotope signatures, because they are natural properties of the substance itself, have the ability to describe the origin of the substance and therefore have the potential to directly trace ethanol. Previous studies have shown that discrimination of ethanol origins can be achieved to a certain extent by combining the measurement of δC values with the calculation of likelihood ratios. In this study, we analyzed 99 alcohol consumption samples (group A), 77 contamination samples (group B), and 14 decomposition samples (group C) by a gas chromatography-isotope ratio mass spectrometry method. We obtained δC values, δO values, and δH values for all three sample types and analyzed the data by a likelihood ratio calculation or a k-means clustering analysis-ROC curve method and compared them to 5 authentic samples to determine whether they were derived from alcohol consumption. The accuracy of the likelihood ratio method was 80 %, and the accuracy of the k-means cluster analysis-ROC curve system was 100 %. These findings demonstrated that increasing the dimensionality of the data can improve the accuracy of the likelihood rate method and that the k-means cluster analysis-ROC curve system was able to distinguish with good accuracy whether ethanol in blood samples was derived from alcohol consumption.

Article	Year
Quantification of AICAR and study of metabolic markers after administration. RSC advances\| PubMed ID: 38873554	2024
Two-Dimensional Liquid Chromatography Purified GC/C/IRMS Doping Control Method: Analysis of Endogenous and Exogenous Sources in Urine Samples from Asian Subjects Administered a Low Dose of A... Journal of chromatography. A\| PubMed ID: 39232419	2024
Detection and Identification of New Chlorphenesin Carbamate Metabolites in Human Urine and Metabolomic Characterization Using Liquid Chromatography-Q Exactive-HF-Orbitrap-Mass Spectrometry. Rapid communications in mass spectrometry : RCM\| PubMed ID: 39620316	2025
K-means clustering analysis of C/C, O/O and H/H isotope ratios for identification of alcohol origin. Forensic science international\| PubMed ID: 40592125	2025

Yue Zhuo

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