| Drug-drug interactions with P-glycoprotein (P-gp) are known to play a significant role in early drug discovery. Combinatorial chemistry and other advances in synthetic chemistry have led to a tremendous inflow of new chemical entities (NCEs) being fed into the screens for high throughput, high efficiency and high accuracy.There are diverse screens including in vivo and in vitro that are set-up in order to predict the interactions between P-glycoprotein and compounds.This dissertation consists of two parts. Firstly, we established Caco-2 cell line model, in vitro everted gut sacs and in situ perfusion method. A set of well-characterized and well-studied compounds were selected to test the validity of the assay.Model Construction:The Caco-2 cell monolayer is similar with the small intestine epithelium, which expresses P-gp highly and makes it an attractive model for P-gp substrate screening. In the transport experiment, the Caco-2 cells are seeded in the Millicell firstly, and 21 days later the cells complete the differentiation and can be used for transport studies. The bi-directional permeability studies were initiated by adding an appropriate volume of buffer containing test compound to either the apical (for apical to basolateral transport; A to B) or basolateral (for basolateral to apical transport; B to A) side of the monolayer. The concentrations of test compound were analyzed by LC-MS or LC-MS/MS method. Then apparent permeability (Papp) was calculated.P-gp inhibition studies were conducted using fluorescent dye Rhodamine123 and Calcein-AM as the probe. Accumulation of intracellular calcein fluorescence was measured using a Spectrofluorophotometer or flow cytometry. P-gp inhibitors significantly increased intracellular calcein fluorescence in Caco-2 cells. Using cultured Caco-2 monolayers on 96 well and measured with a Spectrofluorophotometer is a useful tool in high-throughput screening. Importantly, using Caco-2 cells in suspension combined with flow cytometry analysis improve reliability, specificity, and sensitivity of the assay.We established multi-drug resistance reversal model for P-gp, which was one of major causes for multidrug resistance (MDR).The determination of inhibitory concentrations (IC50 values) allowed to differentiate cytostatics into P-gp or non-P-gp inhibitors.This assay was improved as a high-throughput screening-capable assay. P-gp induction model: induction of P-gp expression in Caco-2 cells treated with compounds was measured using Western blot analysis. Our results showed that this model couble be used to the research of P-gp induction.The effects of compounds on rat intestinal P-gp function were studied on everted gut sacs and in situ intestinal perfusion models. The absorption of drugs were studied by the means of the everted gut scas and predict absorption trend in different intestines.Model Application:Permeability of two series new peptide drug candidates synthesized by our institute was evaluated in Caco-2 cell monolayer. Results from Caco-2 model demonstrated that if the N atom in free amino groups at N terminal ureido of the linear decapeptide substituted by alkyl group, the stability of the peptide increased signicantly. The H atom in free amino groups at N terminal ureido of the linear decapeptide substituted by alkyl group, the modified peptide shows high permeability.We used Caco-2 cells to screen compounds for their permeability characteristics and P-glycoprotein interactions on Bi-directional transport method. Compounds such as R-8021, S-8021 and thiophenorphine were P-gp and MRP2 substrates,thus Agm, SP-8, RS-8021, C36D2 and HS-1 were not. This result explained lower bioavailability of R-8021 comparing to RS-8021. Fluorescence assay was used to screen 17 componds'interaction with P-gp. It shows that LSY-001, LSY-002, LSY-003 and LSY-004 upregulated intracellular calcein up to 18.4%, 27.2%, 18.5% and 30.5% respectively. The result indicated that these four compounds were P-gp inhibitors.The permeability characteristics of Traditional Chinese Medicine prescription Dang-gui Shao-yao San (DSS) were carried on Caco-2 cell monolayer. A new method was designed, in which the internal liquid obtained from everted gut sacs was added to Caco-2 cell incubation culture. By using this assay, it may overcome defects such as constituents complex and thick in Traditional Chinese Medicine prescription. The results indicated that Papp(A→B) of vanillic acid and albiflorin after compound filtering and the internal liquid obtained from everted gut sacs was significantly higher than that of the monomers or mixture of monomers in DSS. It shows that in the presence of other herb couple components, the influx permeability (A to B) of vanillic acid and albiflorin significantly increased promote transport. Otherwise, Compound A shows just the opposite result.In summary, we had successfully established integated rapid Drug-drug interactions with P-glycoprotein models. It could be used to screen compounds associated with P-glycoprotein in vitro, in vivo and in situ. The screening models of discovery compounds for their intestinal permeability characteristics and P-glycoprotein interactions are well established and used in pharmaceutical industries. Furthermore identification of compounds that are P-gp substrates, inhibitors, and inducers can aid drug candidate selection and optimization and offer reference to the research of other transport proteins. |
