Development of an hollow fiber solid phase microextraction method for the analysis of unbound fraction of imatinib and N-desmethyl imatinib in human plasma.

Therapeutic drug monitoring (TDM) of imatinib (IM) in cancer therapy offers the potential to improve treatment efficacy while minimizing toxicity. There was a significant correlation between unbound concentration and clinical response and toxicity, compared with total plasma concentrations, and the quantification of unbound IM and its metabolite, N-desmethyl imatinib (NDI) are of interest for TDM. However, traditional unbound drug separation methods have shortcomings, especially are susceptible to non-specific binding (NSB) of drugs to the polymer-constructed components of filter membranes, which are difficult to avoid at present. Hence it is necessary to developed a reliable separation method for the analysis of the unbound fraction of IM and NDI in TDM. We developed and validated an hollow fiber solid phase microextraction (HF-SPME) method coupled with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) that to measure unbound IM and NDI concentration in human plasma. It used the NSB phenomenon and solve the NSB problem. The preparation procedure only involves a common vortex and ultrasonication without dilution of samples and modification of membrane. A total of 50 chronic myeloid leukemia (CML) patients were enrolled in our study. The relationship between the unbound and total concentrations for IM and NDI, as well as the concentration ratios of NDI to IM in 50 clinical plasma samples were investigated. The extraction recovery is high to 95.5-106 % with validation parameters for the methodological results were all excellent. There were both a poor linear relationship between the unbound and total concentrations for IM (r2=0.504) and NDI (r2=0.201) in 50 clinical plasma samples. The unbound concentration ratios of NDI to IM varied widely in CML patients. The determination of unbound IM and NDI concentration is meaningful and necessary. The developed HF-SPME method is simple, accurate and precise that could be used to measure unbound IM and NDI concentration in clinical TDM.

Circ_0007611 modulates the miR-34c-5p/LPAR2 cascade to suppress proliferation and enhance apoptosis of HTR-8/SVneo cells.

INTRODUCTION: The aberrant biological behaviors of trophoblast cells actively take part in the pathogenesis of preeclampsia (PE). Herein, we defined the action of the circular RNA (circRNA) circ_0007611 on trophoblast cell apoptosis and growth to understand its role in PE. METHODS: Expression of circ_0007611, miR-34c-5p and lysophosphatidic acid receptor 2 (LPAR2) mRNA was analyzed by qPCR. LPAR2 protein was determined by western blotting. Cell proliferation was analyzed by EdU assay. We assessed apoptosis through flow cytometry and analysis of caspase3 activity and apoptosis-related marker proteins. The binding of miR-34c-5p and circ_0007611 or LPAR2 was verified by dual-luciferase and pull-down assays. RESULTS: Circ_0007611 and LPAR2 levels were augmented, while miR-34c-5p was diminished in blood samples of PE. Circ_0007611 deficiency repressed cell apoptosis and enhanced the growth of HTR-8/SVneo cells. Circ_0007611 interacted with miR-34c-5p, and miR-34c-5p depletion reversed circ_0007611 deficiency-induced HTR-8/SVneo cell apoptotic inhibition and growth enhancement. MiR-34c-5p targeted LPAR2, and circ_0007611 affected LPAR2 expression via miR-34c-5p competition. Circ_0007611 deficiency-induced HHTR-8/SVneo cell apoptotic inhibition and growth enhancement were also counteracted by LPAR2 overexpression. DISCUSSION: Circ_0007611 modulates the miR-34c-5p/LPAR2 cascade to enhance apoptosis and inhibit proliferation in HTR-8/SVneo cells, thereby contributing to the progression of PE.