利用Caco-2細胞體外試驗評估Genistein磷酸酯衍生物之吸收

Abstract

Genistein為已知12種大豆異黃酮的成分之一，其化學結構與人體雌激素相似，具類雌激素的生物活性，被認為是植物來源的雌激素，有預防骨質疏鬆、心血管疾病、乳癌及前列腺癌等生理活性。然而，根據Merck Index及眾多文獻記載，genistein幾乎不溶於水，生物可利用率 (bioavailability, BA) 不佳。本實驗室先前已發展藉由微生物轉換的方式，可將genistein轉化生成genistein 7-O-phosphate (G7-P)。在製藥工業上常利用磷酸化手段提升化合物水溶性，進而提升BA。由於已有一些磷酸酯衍生化前驅藥物 (phosphate ester prodrug) 的成功前例，因此，本研究的主要目的在於利用體外試驗比較G7-P及genistein之穿透，藉以評估G7-P作為genistein prodrug的潛力。試驗首先進行G7-P之物化性質探討，發現其水溶性為285 mM，大於genistein約10萬倍；在模擬胃液 (simulated gastric fluid, SGF) 與模擬腸液 (simulated intestinal fluid, SIF) 中，G7-P與genistein於4 h內皆有良好之安定性；溶離試驗顯示G7-P的溶離速率顯著高於genistein，於5 min內即近100% 溶離；對Caco-2細胞之毒性試驗結果顯示，G7-P及genistein在100 uM下皆未對細胞造成毒性傷害。在HBSS緩衝溶液系統之穿透試驗，比較10 uM溶解態genistein (Gsol, 溶解於0.2% v/v DMSO)、懸浮態genistein (Gsus, 懸浮於0.5% CMC w/v) 以及G7-P三者之穿透比率，結果發現Gsol與G7-P兩者之穿透比率及表觀穿透係數皆高於Gsus，顯示化合物溶解程度影響穿透程度；在SIF緩衝溶液系統進行穿透試驗的結果中，G7-P之穿透比率在投藥60分鐘後即顯著高於genistein，且將投藥濃度提升至50 uM時兩者間差異更為顯著。相較HBSS溶液而言，SIF之模擬條件更接近真實小腸環境，G7-P能夠顯著提升genistein之吸收。此外，本研究比較Caco-2細胞ALP對fosphenytoin (phosphate prodrug之正控制組) 和G7-P之水解效率，結果顯示ALP對G7-P水解效率較佳。然而，我們認為Caco-2細胞株之ALP活性不如真實小腸是造成本研究穿透試驗結果中G7-P與genistein間穿透比率差距未如預期的主要因素。

Genistein, one of soy isoflavones, possessing estrogenic activtity because of the similar chemical structure to that of 17-beta-estradiol, has gained great attentions for its benefits in the prevention of osteoporosis, hypercholesterolemia, breast and prostate cancers. However, according to Merck Index and literatures, genistein shows poor bioavailability due to its extremely low aqueous solubility, which could result in poor dissolution behavior in gastrointestinal fluid. Earlier we published a biotransformation process exhibited that genistein could be converted into a highly soluble phosphate conjugate, namely genistein 7-O-phosphate (G7-P). On the basis phosphate prodrug concept, G7-P was supposed to be superior in intestinal permeability in contrast to genistein based on the increased aqueous solubility. The aim of this work was to investigate the capability of G7-P as an alternative of genistein by means of determining the dissolution characteristics and intestinal permeability of G7-P and genistein. First, we revealed that G7-P displayed 100-thousand times higher in aqueous solubility than genistein, and whether the presence of solubilizers or not, the dissolution rates of G7-P in all media used in this study were much higher than that of genistein during 60-min incubation. In addition, either G7-P or genistein remained stable in simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) during 240-min incubation. Moreover, both G7-P and genistein showed non-cytotoxic effect toward Caco-2 cells under the concentration range of 0 to 100 uM. In in vitro transport study, G7-P and genistein were dissolved or suspended in HBSS buffer, the results showed G7-P group exhibited higher permeation ratio and apparent permeability coefficient (Papp) than genistein suspension group, indicated that the practically dissolved amount of tested samples could be highly correlated to its permeability. Similarly, G7-P performed a better permeability than genistein in both low dose (10 uM) group and high dose (50 uM) group under SIF buffer. In contrast with the effect of low dosage treatment on the permeability, the high dosage treatment increased the difference in permeable amount of genistein in tested G7-P group. Furthermore, alkaline phosphatase (ALP) activity of Caco-2 cells was evaluated by treating G7-P and an ALP-sensitive phosphate ester prodrug, fosphenytoin, as the substrates to compare the dephosphorylation efficiency with each other. The results revealed that G7-P was more sensitive in dephosphorylation by Caco-2 ALP than fosphenytoin. Therefore, we considered that ALP activity of Caco-2 cells would be crucial in the formation of concentration gradient of genistein on the apical membrane of Caco-2 cells, which would affect greatly in successive permeability of genistein when G7-P was treated. In conclusion, we considered G7-P could be a promising alternative of genistein with better absorption.