The Absorption Enhancing Effects And Mechanism Of PAG On The Poorly Absorbable Drug

In addition to intravascular administration,drug must go through the process of absorption after application to the body.But the process is influenced by many factors(such as drug solubility,degree of dissociation,dissolution,mucosal permeability,first-pass effect and other physiological factors),which is usually low bioavailability compared to intravenous.And the most simple and effective way to enhance drug plasma concentration and bioavailability is to use the absorption enhancers.PAMAM dendrimer is a new synthetic polymer,a unique three-dimensional configuration and good biocompatibility,permeability and stability so that it can improve the bioavailability of the drugs.We design in this paper a new drug codendrimer PAM A M-co-OEG(PAG)by decorated fourth-generation PAMAM(G4)with the hydrophilic second-generation OEG dendron(G2).It not only reduces the cationic toxicity on the surface of the PAMAM-NH2 dendrimer to the body,but also improves the solubility of the entire molecule.This topic selects doxorubicin as model drug,using in vivo and in vitro model to investigate the toxicity of PAG,absorption promoting effect and mechanism of action,and DNA microarray chip technology is also introduced into the study of the mechanism of penetrating membrane of PAG.This research has three levels involving animals,cells and molecules in order to seek an efficient and low toxicity,widely used and mechanism of clear absorption enhancers.Specific studies are as follows:1.Established and verified Caco-2 cell model.In vitro cell culture method is adopted to establish the model of the cell.Observe the cell morphology under the microscope,drawing the cell growth curve,measuring TEER to detect the integrity of cell.Experimental results show that the study of the cultured cells with normal morphology,the TEER greater than 500? in the cell experiments,showing good cellular integrity.In this experiment condition,this method is fast,reliable and simple.2.Evaluated PAG cytotoxicity to determine the concentration of PAG in transport experiments.MTT assay was used,making absorbance value of sedimentary water insoluble crystal violet formazan in cells as detection index deposit in the cell water-insoluble formazan crystal violet absorbance detection index value,setting up the negative control groups which cell survival rate was 100%,the positive control groups(3%of TritonX-100),PAG groups,ordinary PAMAM dendrimers(-NH2/-COOH/-OH on the surface)groups.Experimental results showed that the cell survival rate of Caco-2 cells of the concentration of 0.1%(w/v)was 95.3±5.13%and compared with survival rate of cells of positive control group,a 3%TritonX-100,it did not show any significant impact.Therefore,the experiment used 0.1%(w/v)concentration as the suitable concentration of drug delivery.3.Evaluated PAG effects on transport of doxorubicin in vitro.With Caco-2 cell culture,removed a certain volume of the solution from the BL side at different time points in the doxorubicin transported experiments,calculated the amount of doxorubicin and the transport Papp value by the standard curve,compared the drug group and blank group PaPP value size.The results showed that Compared with the control group which did not join the PAG,the PAG promotion rate was 2.33.on Caco-2 cells of doxorubicin4.Evaluated PAG in vivo absorption promoting effect of doxorubicin.Experimental animal models were rats,giving with or without 0.I%PAG solution in the intestine,using intestinal loops in situ method,blooding at different times from the rat jugular or femoral vein,with three times amount of plasma to precipitate protein with methanol.Measured the plasma concentration of doxorubicin at different time points and drawed the medicine-time curve.After the blooding,isolated small intestines and collected lavage fluid to test the total protein content and the activity of LDH to detect the toxcity of PAG to the intestines.The results showed that:0.1%PAG has absorption promoting effect of doxorubicin on rat small intestines and the ratio of absorption promote is 2.13 times.There was no significant increase in the total protein content and the activity of LDH compared with cnntrol group.5.DNA microarray studied PAG mechanism.Used gene chip microarray method to test the changes of Caco-2 cells expressing genes between 0.1%PAG groups and the blank groups.Among them,organic cation transporters(OCT1)and the new organic cation transporters(OCTN2),clathrin light chain A and B,muti-drug resistance-associated protection(MRP),muti-drug resistance protection(MDR)are involved in 0.1%of PAG across the plasma membrane.