Establishment Of Hollow Fiber Reactor Using Caco-2Cell For Prediction Of Oral Drug Absorption

With the rapid progress of the pharmaceutical industry, a large number of new compounds has been developed as drug candidates in recent years. Appropriate intestinal absorption ratio and oral bioavailability are important prerequisites for developing oral-administered drugs. Pharmaceutical companies tend to evaluate the nature of drug candidates in preclinical screening stage, using reliable in vitro models which mimic the absorptive function in small intestine and metabolic function in liver. Among these models, Caco-2cell monolayer cultured on semi-permeable membrane insert is most widely used. However, such model with a culture period up to21days is laborious, expensive, complicated and low-throughput. This research try to establish a new model based on hollow fiber membrane in order to promote the growth and differentiation of Caco-2cells. The expected short culture period and full expression of the intestinal function in this new model will guarantee the rapid and accurate prediction of oral drug absorption.For this purpose, we first screened out the polyethersulfone (PES) hollow fiber membrane as the most suitable material for the symmetrical adherent and growth of Caco-2cells on its inner surface. Subsequently, time-dependent morphology observation of cell layers and permeable properties indicated that the Caco-2cells grown on hollow fiber formed confluent monolayer within5days. The brush border enzymes (alkaline phosphatase and y-glutamyltransferase) and efflux proteins (P-glycoprotein and multi-drug resistance protein2) were well differentiated with higher expressed function within7days compared to the traditional model at day21. Therefore, this hollow fiber reactor significantly reduced the culture period compared to traditional model. The culture of Caco-2cells on the PES flat membrane suggested that such fast differentiation was attributed mainly to the specific configuration of hollow fiber compared to flat membrane insert.According to the evaluation of16drugs, the prediction of their oral absorption ratio in this hollow fiber reactor showed comparable accuracy (R2>0.87) with traditional model, suggesting its feasibility on the simulation of intestinal absorption. The efflux of three substrates of transporter proteins were also observed in hollow fiber reactor. Moreover, by employing miniature hepatocyte bioreactor as a component, we also established an intestine-liver conjugated reactor in order to predict oral bioavailability of drugs. The predicted bioavailability values of four drugs were in good accordance to in vivo data.In summary, Caco-2cells cultured on the inner surface of PES hollow fiber membrane formed confluent and full functional monolayer within7days. This bioreactor can be used for the prediction of oral drug absorption in human small intestine. Besides, the establishment of intestine-liver conjugated reactor also provide new method for the prediction of oral bioavailability of drugs.