Transport Model Of Drug In Vitro And The Study Of Transport Kinetics Of Propranolol Enantiomers

ObjectiveThey are a lot of chiral biological macromolecules in the bitsystem,so different drugs will interact with different macromolecules according to their stereochemical structure,which will bring about different biological response and display different pharmacokinetics and pharmacodynamics.So the study based on the bidogical behavior differens of chiral drugs is of much significant value.The investigation of pharmaeokineties covers absorption,distribution,metaboliz and excretion,in which absorption and excretion 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 originating 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.Furthermore, the observed results in Caco-2 monolayers agree very well with existing and published in vivo data about small intestinal absorption of oral drug.So this study choose Caco-2 cell monolayers as cell model of absorption .Racemic propranolol as chiral drug, is in common used in clinic.But some study showed that propranolol existed some prominent chiral pharmacological characteristics and was a very typical chiral drug. At present,we have not seen some reports regarding propranolol as chiral drug enantiomers ,determining the transport characteristics of enantiomer of propranolol across Caco-2 cell monolayers at one time. So this study aimed to choose propranolol as chiral model drug, to choose Caco-2 cell monolayers as a classical model system .And the study focused on the absorption kinetics characteristics of chiral drug in this vitro model system.MethodsThe drug solutions were added on either the apical (AP) or the basolateral (BL) side of the inserts, while Caco-2 cell monolayer was established.Transport studies were conducted 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 precolumn derivatizing HPLC analysis.Exteral standardization was applied for quantitative analysis.The methods of establishment and evaluation of cell model:The Caco- -2 cells 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 atmos- phere of 5% CO₂, 95% air, as well as 90% relative humidity. When the cells were confluent, they were detached by treatment with trypsin/ 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 0.6mL cell culture medium to bottom 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. The integrality of cell monolayers were evaluated with The apparent permeability coeffic- ients (Papp),Transepithelial electrical resistance(TEER)and morphologic at the age of 21–25 days.The HPLC conditions were as follows: Samples were separated on a Shimadzu Vp-ODS C18 column (4.6×150mm i.d,5μm). Mobile phase was consisted of methanol and 20mmol·L^-1 of NaH2PO4 buffer (75:25). The detective wavelength was set at 220nm. the flow rate was 1mL·min^-1. the column temperature was room temperature, and injection volume was 20μL.The sample of chiral derivatization methods:After 150μl of samples were evaporated to dryness in vacuum trunk, 20μl of methanol, and 400μl of GITC (480μmol/L in acetonitrile) were added. The mixture was vortexed for 1 min and the reaction was carried out at 80℃for 50 min. When the chiral derivatization was complete, the reaction mixture was evaporated to dryness and the residue was reconstituted with 100μl of methanol. The mixture was vortexed for 1 min and then centrifuged at 5000 rpm for 10 min.Finally,an supernatant of 20μl was injected into the HPLC system.ResultsOnce the Caco-2 cells have been in culture for 21-25 days, the cells were not only confluent ,but forming tight and intact cell monolayers,on which covered brush border microvilli, by investigation of morphologic.In addition , the Papp for fluorescein was less than 1.0×10^-6 cm/s and TEER value was more than 200Ω×cm² .The results demonstrated that Caco-2 cell monolayers had good integrity and have been used for the investigation of chiral characteristic of absorption kinetics of propranolol . This study suggests that , at concentrations of 20.0–240.0μmol/L, enantioselective permeability of propranolol was not observed across Caco-2 cell monolayers during the absorption.During the process of detecting chiral drug- propranolol, peak areas presented good linear relationship with concentration in wider ranges, correlation coefficients were more than 0.9998.Precision (RSD) was less than 15% and recoveris were between 94% and 110%. The limits of detection (LOD) were 0.1μmol·L^-1 for S/R-propranolol.ConclusionThis study has not only established Caco-2 cell monolayers,but also evaluated the integrity of Caco-2 cell monolayers ,which demonstrated that Caco-2 cell monolayers had good integrity and was suitable for the determining the transport characteristics of drug.So this study investigated chiral characteristics of absorption kinetics of propranolol with Caco-2 cell monolayers ,the results suggested absorption of propranolol was mainly a passive, transcellular mechanism and has no enantioselectivity as lipophilic drug. Furthermore,a precolumn derivatizing RP-HPLC method was established, which could separate and determine the propranolol enantiomers, within 20 minutes. The method is simple, specific, reliable.