盐酸氯丙嗪促进药物直肠吸收机制研究

In vitro mechanisms of drug absorption in rectum promoted by chlorpromazine hydrochloride

目的应用Caco-2细胞(the human colon adenocarcinoma cell lines)考察盐酸氯丙嗪对直肠黏膜上皮细胞完整性与稳定性的影响。方法以不同浓度的盐酸氯丙嗪作用Caco-2细胞后,采用Bradford法测定上清液吸光度值[D(595)],计算细胞膜破坏百分率以了解Caco-2细胞膜完整性的变化;应用DPH(1,6-diphenyl-1,3,5-hexatriene)荧光探针法测定细胞膜脂质DPH的荧光强度并计算荧光偏振度,评价细胞膜流动性的变化,以了解其对Caco-2细胞膜稳定性的影响。结果盐酸氯丙嗪浓度>140μmol/L时能影响Caco-2细胞膜完整结构,使细胞内容物释出,且浓度与对膜破坏程度呈现S型曲线。盐酸氯丙嗪浓度<112μmol/L时Caco-2细胞膜脂质荧光偏振度值随浓度的增高及作用时间的延长,呈逐渐下降的趋势(P<0.05)。结论盐酸氯丙嗪在低浓度时能够增大Caco-2细胞膜的流动性,使其结构紊乱,影响膜的稳定性;当超过一定浓度时可破坏细胞膜的完整性,使细胞内容物释出;这些改变很可能是盐酸氯丙嗪促进药物跨膜转运吸收的机制之一。

Objective It was demonstrated from our previous studies that chlorpromazine hydrochloride can increase the relative bioavailabilities of drugs through rectal administration, due to the alternations of cell membrane architecture and permeability caused by the amphipathic structure of chlorpromazine hydrochloride. To prove this mechanism, Caco-2 cell membrane was exploited to investigate the effect of chlorpromazine hydrochloride on the membrance＇ s integrities and stabilities. Methods After the Caco-2 cells were treated with chlorpromazine hydrochloride at different concentrations for different durations, the optical density value （OD- value） of the supernatant was determined by Bradford protein assays. The calculated OD ratios showed the changes of Caco-2 cell membrane integrity. The fluorescent intensity （fluorescence polarization, Pr） was determined by fluorescence spectrophotometer with 1,6-diphenyl-1,3,5-hexatriene （DPH） as a fluorescein probe, the mobility and stability of Caco-2 cell membrane were evaluated by the calculated Pr＇ s data. Results Compared with the control, Caco-2 cell membrane was damaged at different degrees by high-dose chlorpromazine hydrochloride （ 〉 140 μmol/L） , and the intracellular content was poured out. It was observed an S-shape curve between the membrane-damaged percentage vs the concentrations of chlorpromazine hydrochloride. Meanwhile, degression of the fluorescence polarization （Dr） of Caco-2 cell membrane was observed after the Caco-2 cells treated with low-dose chlorpromazine hydroehloride, and Pr values were decreased with the increase of concentration of chlorpromazine hydrochloride and extension of treatment time （ P 〈 0. 05 ）. A significant negative correlation was found between Pr value and duration at the same concentration group （P 〈0. 05）. Conclusion Low dose chlorpromazine hydrochloride increases lipid fluidity and changes the stability of Caco-2 cell membrane, and even damages cell membrane integrity when the concentration is increased or treatment time prolonged, resulting in the increase of cell membrane permeability. The changes of cell membrane may be one of mechanisms for promoting the rectal absorption of drugs by ehlorpromazine hydrochloride.