Synthesis, Pharmacokinetics And Pharmacological Effects On Cancer Metastasis Of Metapristone

Purpose:There are a lot of new discoveries and knowledge in circulating tumor cells,tumor metastasis and theoretical approach,long-term use of oral contraceptives significantly reducing the incidence of cancer.We assume that metaprisone,the major metabolite of the oral contraceptive mifepristone,may be one of drug candidates for preventing tumor metastasis.In the present study,we aimed at 1)synthetizing and characterizing metapristone;2)investigating pharmacokinetics and the intervention of tumor cell metastasis of metapristone;3)illuminating the potential molecular targets of intervention from the molecular and cellular pharmacology perspective;and 4)exploring a safe,effective and innovative way to prevent long-term tumor metastasis.Methods:Metapristone was synthesized,using mifepristone as the starting material.Chemical structure of metapristone was characterized by UV-vis,IR,1H-NMR and MS.Determination and pharmacokinetics were studied using UPLC-MS/MS method.Long-term toxic effect in mice was administered by gavage for 35 days.The effect of metapristone and mifepristone on the viability of different cancer cell lines was investigated using MTT assay.The colony formation assay was studied using microscope.Metapristone-induced morphological changes were assessed by light microscope and fluorescence microscope after DAPI staining.The apoptosis was detected by Annexin V-PE/7-AAD staining,and DNA cell cycle was detected by propidium iodide(PI)analysis.The anti-migration and anti-adhesion effects were investigated by cell scratch,Transwell and MTT methods,and the expression of cell adhesion molecules were detected using flow cytometry,Western Blotting and RT-PCR.The experimental metastasis assays were performed in CT26 colorectal cancer metastasis model for BALB/c mice and in B16F10 melanoma lung metastasis model for C57BL/6 mice.Results:Metapristone was successfully synthesized by mono-demethylating mifepristone,and its structure was confirmed by UV-vis,IR,MS and 1H-NMR.Metapristone(IC50=57.4±4.3-98.5±4.2 ?M)had modest cytostatic effect on cancer cell lines and normal cells compared with RU486(IC50=33.7±3.8-68.5±1.8 ?M).After the HT-29/B16F10 cells treated by metapristone(0-80 ?M),cells appeared apoptotic morphological changes and it decreased mitochondrial membrane potential,promoted apoptosis/necrosis and arrested cell cycle in G0/G1 phase.Metapristone could inhibit colony formation(P<0.01).This newly developed method,UPLC-MS,was successfully applied to study the pharmacokinetics of metapristone and there was a significant difference in pharmacokinetic profile between genders.Metapristone(0-40 ?M)interfered the adhesion of HT-29 cells to HUVECs in a dose-dependent manner,inhibited the expression of ICAM-1,E-slectin,Sialyl-Lewis' and Integrin ?1,and it(50 mg/kg)inhibited CT26 cell metastasis in a BALB/c mouse model.Metapristone(0-40 ?M)suppressed cell migration by down-regulating the expression of MMP-2,intervented adhesion of B16F10 cells to FN by decreasing the expression of integrin a4 and it(2.5-50 mg/kg)inhibited B16F10 cell metastasis in a C57BL/6 mouse model.In addition,long-term administration of metapristone(<50 mg/kg)in an effective concentration had no significant side effects.Conclusion:Comparative studies of metapristone and mifepristone were comprehensively investigated from medicinal chemistry,pharmacokinetics,toxicology and pharmacology.Metapristone had lower toxicity and was suitablte for long-term use.Metapristone intervented the expression of adhesion molecules on cell surface and had anti-metastasis effects in vivo and in vitro.It is expected to be one of drug candidates to prevent cancer metastasis and this opens up a new field of metastasis prevention.