| Summary: | 碩士 === 國防醫學院 === 藥學研究所 === 94 === Resistance against chemotherapeutic regimen has hampered the treatment of malignancies. Several mechanisms of cellular resistance have been incriminated. A major one results from reduced intracellular accumulation of structurally unrelated anticancer drugs and usually involves over expression of drug efflux pumps. P-glycoprotein (P-gp) is the first identified of these transporters. More recently, export pumps belonging to the multidrug resistance protein (MRP) subfamily, such as MRP1 and MRP2, have also been demonstrated to confer multidrug resistance on tumor.
The expression and function of the MRP subfamily might impact on the rate and extent of efflux of the substrates for the MRP subfamily. Elevated expression of P-gp, MRP have been reported in many cancers and has been shown to be associated with poor treatment response, indicating that P-gp and MRP are probably a predictive factor for the outcome of treatment of malignancies. The regulation of the activity of MRP may be a feasible direction to improve the treatment of malignancies with multidrug resistance.
The purpose of this study is to develop the MRP1 modulators. A simple and reliable in vitro screening method was setup and characterized using over expressed MRP1 HL60/ADR cell lines to study the effect of Chinese herbal enhancers (CHEs) on MRP1-mediated transport of a model substrate. A specific MRP1 substrate carboxyfluorescein (CF) was used as a fluorescent marker after hydrolysis from CF diacetate (CFDA) in the cell. The functional activity of MRP1 was evaluated by measuring CF retention/efflux in the presence of CHEs and MK517 used as a positive control. Intracellular accumulation of CF was measured by flow cytometry. The validation of the screening platform included accumulation kinetic under different temperatures, efflux kinetics, generation of the cell on efflux activity, and hydrolysis ability of CFDA in the cells. 83 pure constituents in CHEs has been completed up-to-now. The results indicated that the efflux of MRP1 was inhibited by some of the CHEs. Intracellular retention of CF increased 20-46 fold with No. 80, 65, 70, 09, 77, 03, 26, 68, and 61 CHEs at 100 M as compared with control (p＜0.001). As such as the same, increased 10~19 fold with No. 81, 10, 71, 79, 34, 02 (p＜0.001). Based on the in vitro screening studies has been done, top 20 CHEs would be further evaluated to study affinity. Function evaluation of rMRP1 on S.D. rat red blood cells in the present of CHEs have be conducted in vitro first. The effect of potent CHEs on the modulation of MRP1 function in vivo in rats have be further carried out by use IV administration of CHEs (HUCHE 61, 03, 68) combined with CFDA. The result show that only HUCHE 61 make the amount of CFDA uptake on RBCs increased 1.8 fold (p＜0.05), but not change with other pharmacokinetic parameters. Although it doesn’t successfully demonstrate the significant result, it may improve the individual variation of drug kinetic profile. It is still worth to develop these pure constituents in futher study.
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