Transport Of Ecdysterone In Caco-2 Cell Monolayers

OBJECTIVE:The investigation of pharmaeokineties covers absorption, distributin, metaboliz and excretion, in which absorbtion and exretion are very important. In recent years,there have been made great progress in investigating models of pharmic absorption and metaboliz mechanism by culturing intestinal epithelial cell. Caco-2 cell origination from a human colorectal carcinoma is very typical. And then when drug molecules permeate small intestine mucous membrane, the Caco-2 cell monolayers can provide some information about absorption,metaboliz and transportation at cell level. Otherwise, There have investigate indicated that cultivate the Caco-2 with 1α,25-dioxyvitaminD3 (VD3)two weeks can derivn the expression of CYP3A4 mRNA, it improve the similarity of the substantial intestinal circumstance, this modle also used to study the transport dynamics.Ecdysterone, EDS,already have discovered that have therapeutic action of diabetes. As one new drug, its nformation of Absorption and transport is unkown. So this study aimed to invest information of Absorption and transport of Ecdysterone,to continue study mechanism of absorption,to conduce to manufacture form of medication, absorption accelerant ,to conduce to predict the mechanism of absorption and transport which have the similar chemcial constitution.METHODS:The drug solμtions were added on either the apical (AP) or the basolateral (BL) side of the inserts, while Caco-2 cell monolayers was established.Transport stμdies were condμcted in both the absorptive direction (AP-BL) and the secretory direction (BL-AP).samples were collected from both sides of the cell monolayer in different time for precolμmn derivatizing HPLC analysis.Exteral standardization was applied for qμantitative analysis.The methods of establishment and evaluation of 2 Caco-2 cell models : The Caco-2 cell were grown in a medium comprised of Dulbecco's modified Eagle medium with high glucose containing supplemented with 10%fetal bovine serum.Cultures were maintained at 37°C in an incubator and in an atmosphere of 50% CO2,95% ari,as well as 90% relative humidity.When the cells were confluent,they were detached by treatment with trpsin/EDTA. Then,cells were seeded at 5.67×105cells/mL on the Millicell? plate should be placed in an incubator (0.4mL/well),at the same time,add 0.6 mL cell culture medium to botton of incubator.The growth medium should be exchanged every one day at the first week after initial plating,and then exchanged every day until the time of use.At the same time,the Caco-2 cell with VD3- treated model use the EDTA contain 1α,25-dioxyvitaminD3 exchange the growth medium. The integrality of cell monolayers were evalugted with the apparent permeability coeffic-ints(Papp),Transepithelial electrical resistance (TEER)and morphologic at the age of 21～25 days.The HPLC conditions were as follows: Samples were separated on Vp-ODS C18 column(150×4.6mm,i.d,5μm).Mobile phase was consisted of acetonitrile and water(20:80).The detective wavelength was set at 248nm. The flow rate was lmL/min .The column temterature was room temperature,and injection volume was 20μl.RESULTS: Once the Caco-2 cells of two models have been in culture for 21～25 days,the cells were not only confluent,but forming tight and intact cell monolayers,on which coverd brush border microvilli,by investigation fo morphologic.In addition the Papp for fluorescein was less than 1.0×10-6cm/s and TEER value was more the 200?×cm2.The results demonstrated that the Caco-2 cells monolayers of two models had good integrity and have been used for the investigation of characteristic of absoption of Ecdysterone.This study suggests that,at concentrations of 50～400μmol/L,permeability of Ecdysterone was 0.8×10-6cm/s approximately,the permeability of Ecdysterone in two Caco-2 cells model had obviously difference. In addition, after added the verapamil , absorption of Ecdysterone was increased ,but ,it was not very significant.So it is ,it is possible that the Ecdysterone was the substrate of P-pGCONCLUSION:This study has not only build 2 Caco-2 cell monolayers models,bμt also evaluate the integrity of 2 Caco-2 cell monolayers models .The resμlts demonstrated that 2 Caco-2 cell monolayers model had good integrity and was sμitable for the determining the transport characteristics of. Ecdysterone . In conclusion , the results suggested absorption of Ecdysterone was mainly a passive,transcellular mechanism in 2 Caco-2 cell monolayers models , percentage of absorption was 1%～10%. permeability of Ecdysterone was not influenced by CYP3A4- mediate mechanism.。in addition, it is possible that the Ecdysterone was the substrate of P-pG...