Analysis of Drug Permeation Mechanism across Oral Mucosa Using Cultured Stratified Cell Layers

为了阐明药物经口腔粘膜渗透的机制，确定了HaCaT细胞的最佳培养条件，并对葡萄糖转运系统进行了各种基础研究，从增殖活性的角度选择了DMEM EGF（+）或DMEM+补充EGF（+）作为HaCaT细胞的培养条件。从组织学角度来看，选择DMEM EGF（+）。从跨粘膜电阻来看，DMEM：Ham 'sF-12 EGF（+）的效果最好。综合考虑细胞活力、分层、跨粘膜电阻和葡萄糖转运等因素，选择以DMEM EGF（+）培养3 ~ 4周为HaCaT细胞层的最佳培养条件。提示HaCaT细胞中立体选择性葡萄糖转运蛋白的表达。Western blot分析显示，SGLT、GLUTs 1、2和3的存在。然而，D-葡萄糖跨细胞层的转运速率与L-葡萄糖没有显著差异。选择甘露醇、褪黑素和雌二醇作为被动转运药物，测定Papp值。三种药物的Papp值的结果与其亲脂性相关，表明培养的HaCaT细胞层可有效用于评价被动转运药物的口腔粘膜渗透性。

In order to clarify the drug permeation mechanism across oral mucosa, the optimal culture condition for the stratified cell layer using HaCaT cells was determined and the various basic studies were performed on glucose transport systems.As to the culture condition for HaCaT cells, DMEM EGF(+) or DMEM+Supplement EGF(+) was selected from the point of proliferative activity. From histological view point, DMEM EGF(+) was selected. From the transmucosal electrical resistance, DMEM : Ham's F-12 EGF(+) was the best. Taking factors such as cell viability, stratification, transmucosal electrical resistance and glucose transport into consideration, the best condition for HaCaT cell layers was selected to be the culture for 3 to 4 weeks using DMEM EGF(+).The result of glucose transport experiments using HaCaT cells showed that the uptake of D-glucose into cells was much faster than that of L-glucose, suggesting that the expression of stereo-selective glucose transporters in HaCaT cells. Western blot analysis showed that the presence of SGLT, GLUTs 1,2 and 3. Nevertheless, the transport rate of D-glucose across the cell layer was not significantly different from that of L-glucose. The reason for this observation is remaining unsolved.As the passively transported drugs, mannitol, melatonin and estradiol were selected, and the Papp values were measured. The results of Papp values for the three drugs were correlated with their lipophilicity, suggesting that the cultured HaCaT cell layers are effective for the evaluation of the oral-mucosal permeability to passively transported drugs.