Study On Intestinal Permeability Of Conjugated Estrogen In Vivo And Vitro Intestinal Models

Objective:1.Using in vivo and in vitro models,to investigate the absorption characteristics of conjugated estrogen（CE）in the intestines of rats with main active components of sodium equilin sulfate（Eq S）and sodium estrone sulfate（ES）as representatives.The absorption characteristics are compared with the permeability of the standard reference drug to determine the permeability classification level of CE.2.The SPIP in rat model is used to study the permeability of ES in active pharmaceutical ingredient（API）and ES standards at the same concentration,and analyze whether the multi-component environment has an effect on the permeability of a single substance.Methods:1.High performance liquid chromatography（HPLC）methods were established for the determination of rats intestinal perfusion samples of Eq S、ES of main active ingredients in CE and 4 model drugs（high permeability:metoprolol,minoxidil,medium permeability:atenolol,amiloride）,and Eq S、ES and 2 model drugs（high permeability:metoprolol,medium permeability:atenolol）of the Ussing Chamber model samples.2.Using SPIP in rat and Ussing Chamber small intestine in rat model,standard reference drug as an internal standard and collect the intestinal sac fluid and receiving fluid at different time points of Eq S+ES and standard reference drug.After HPLC detection,calculate effective permeability(P_eff)and apparent permeability(P_app)and compare with standard reference drug.3.Use SPIP in rat model to take the same concentration of ES standard and CE in API respectively to infuse,take samples at different time points and calculate P_eff,and analyze with statistical software.Results:1.The HPLC analysis methods of Eq S,ES and standard reference drugs in the two models are reliable,and their specificity,linearity,intra-day and inter-precision,accuracy,extraction recovery,and stability meet the requirements of biological sample analysis methodology.2.In SPIP in rat model,CE is absorbed in the jejunum and ileum without intestinal segment dependence.Its P_effis lower than metoprolol,minoxidil,amiloride,and comparable to atenolol,there was no significant difference between CE and atenolol（p>0.05）;In the Ussing Chamber small intestine in rat model,CE is absorbed in all intestinal segments of rats,P_appis the smallest in the duodenum and the largest in the colon;P_appin all intestinal segments is less than metoprolol,There is a statistical difference（p<0.05）;the P_appof duodenum and jejunum is smaller than atenolol,and it is equivalent to atenolol in the ileum and colon,and there is no statistical difference（p>0.05）.Except for the colon,the P_appof the three intestinal segments CE was between 1.0×10^-6cm·s^-1～10×10^-6cm·s^-1.3.At the same concentration in the jejunum and ileum,the P_effof ES in the API was slightly lower than that of the ES standard,and there was no statistical difference（p>0.05）.Conclusion:1.The established HPLC analysis method is accurate and reliable,and can be used for the detection and content determination of in situ single-pass intestinal perfusions model in rat samples and Ussing Chamber small intestine in rat model samples.2.The results obtained from the SPIP in rat model and the Ussing Chamber small intestine in rat model are basically the same.The permeability of CE is less than that of the high permeability drug,and the permeability in specific intestinal segments is less than or equivalent to atenolol,suggesting CE may be medium permeability drugs.3.The permeability of ES is not affected by the multi-component environment,suggesting that in a multi-component environment,the permeability of a single component may not be changed by the environment.