Study On The Absorption Mechanism And Intestinal Permeability Of Conjugated Estrogen By Caco-2 And MDCK Cell Model

Objective:Based on Caco-2 and MDCK scell monolayer model,the intestinal absorption mechanism of CE was studied,and the permeability of absorption direction of four model drugs（metoprolol,minoxidil,atenolol,amiloride）was compared to determine the permeability classification grade of CE,so as to provide basis for BCS classification of CE.Methods:1.HPLC methods were established for the determination of two main components（ES and Eq S）and four model drugs in CE.2.The establishment of monolayer cell model was evaluated by observing the cell morphology,measuring the transmembrane resistance（TEER）value,and measuring the specific transmission of sodium fluorescein.3.MTT method was used to explore the safe concentration range of CE and four model drugs in two kinds of cells.4.The permeation mechanism of CE was studied by monolayer cell model.Papp and ER were calculated by bi-directional transport experiment.In addition,the effects of time,concentration,P-gp inhibitors（verapamil and cyclosporine A）on the transmembrane transport of CE in monolayer cell model were investigated.Give ES and Eq S standard solution,compare the permeability of the two main components.5.Compare the Papp of CE and four model drugs in the absorption direction of Caco-2 cells,compare the Papp of CE and atenolol in the absorption direction of MDCK cells,and determine the permeability classification grade of CE by comprehensive analysis.Results:1.The system applicability,specificity,linearity,precision,stability and extraction recovery of HPLC detection method for the established CE and four model drug cell osmotic solution samples all meet the requirements of Pharmacopoeia 2015.2.Under the inverted optical microscope,the cells of Caco-2 and MDCK were closely arranged without gap;the TEER of Caco-2 were more than 600Ω·cm²and MDCK were more than 1000Ω·cm²,and the Papp value of fluorescein sodium was（0.59±0.08）×10^-6cm/sec.3.The results of MTT showed that the concentration of CE in Caco-2 cells ranged from 1-1600μg/m L,the concentration of four model drugs ranged from 1-800μg/m L,the concentration of CE in MDCK ranged from 1-1600μg/m L,and the concentration of atenolol ranged from 1-800μg/m L is safe concentration range.4.In Caco-2 monolayer cell model,in the bi-direction transport test,the Efflux Ratio of CE=2.60,P-gp inhibitor（100μM verapamil or 10μM cyclosporine A）increased the absorption direction,decreased the secretion direction,and decreased the efflux ratio to0.21（verapamil）and 0.43（cyclosporine A）,indicating that CE is indeed the substrate of P-glycoprotein.The results of time and concentration showed that:the cumulative transmission of CE increased with time;the Papp of different concentration groups increased with the concentration of drug administration,and there was no transport saturation phenomenon.It can be inferred that CE is mainly passive transport,and there is P-glycoprotein-mediated efflux.The results of single compound experiment showed that the permeability of CE powder was significantly lower than ES+Eq S,and the permeability of Eq S was significantly higher than the other three groups.In MDCK monolayer model,the Efflux Ratio of CE was 3.30,and the P-gp inhibitor（100μM verapamil）increased the absorption direction and inhibited the secretion direction.The ER decreased from 3.30 to0.46,indicating that CE is indeed the substrate of P-glycoprotein.The results of single compound experiment showed that the permeability of CE powder was lower than that of ES+Eqs,there was no significant difference among the three groups,it shows that the permeability of Eq S and ES in MDCK was similar.5.There was no significant difference between CE and atenolol in Caco-2 cell model,p>0.05.There was significant difference between CE and metoprolol,minoxidil and amiloride,p<0.05,and Papp of CE was lower than the three model drugs.In MDCK cell model,there was no significant difference between CE and atenolol,p>0.05,indicating that CE and atenolol had similar permeability.Conclusion:1.The established HPLC method is accurate and reliable,and can be used for the detection of transport samples.2.The evaluation indexes of Caco-2and MDCK cell model meet the requirements,which can meet the requirements of the follow-up study of drug absorption characteristics.3.The concentrations of CE and four model drugs were within the safe range.4.The permeation mechanism of CE is mainly passive transport,and there is P-gp mediated efflux.5.The absorption to Papp of CE was lower than that of metoprolol,minoxidil and amiloride,which was similar to that of atenolol.