Preliminary Study On The Characteristics Of Intestinal Absorption Based On Harmine And Its Derivatives In Situ And Vitro Model

Objective:On the basis of the determination of the HM and its derivatives(HM-Y-A and HM-Y-B),the different influencing factors of the absorption mechanism of the three drugs were studied by single-pass intestinal perfusionsrat in rats and MDCK cell model.Methods:①Using single-pass intestinal perfusionsrat model,establish the HPLC method of HM,HM-Y-A and HM-Y-B.To investigate the effects of different absorption sites,concentrations,pH and two inhibitors of P-glycoprotein(verapamil and cyclosporine A)in rats,calculate the absorption rate constant(Ka)and apparent permeability coefficient(Papp);②Building MDCK monolayer cell model,measurement of resistance value(TEER)of upper and lower chambers of transwell plate to evaluate monolayer cell model;Use MTT method to explore the range of safe administration of HM,HM-Y-A and HM-Y-B in MDCK cells;③Using a MDCK cell model,to establish the HPLC method of HM、HM-Y-A and HM-Y-B in cell osmotic fluid.To investigate the effects of concentrations,directionn and two inhibitors of P-glycoproteins(verapamil and cyclosporine A)in cell monolayer models,calculated cumulative absorption(Q)and apparent permeability coefficient(Papp).Results:① The results of single-pass intestinal perfusion model,showed that the absorption of HM and HM-Y-A in four intestinal segments of rats was duodenum≈jejunum>ileum≈colon,HM-Y-B duodenum>jejunum≈ileum≈colon;The HM duodenum increased with concentration(P<0.05),but there was no difference between the concentrations in jejunum(P>0.05),and the absorption parameters of HM-Y-A and HM-Y-B in duodenum and jejunum decreased with the increase of concentration(P<0.05);The absorption parameters in HM and HM-Y-B in duodenum and jejunum increased with pH(P<0.05),HM-Y-A largest is pH6.4,the second is pH7.4;The HM absorption parameters increased significantly after the addition of inhibitors(P<0.05),but there was no significant difference between HM-Y-A and HM-Y-B absorption parameters(P>0.05).②Determination of transmembrane resistance within 2-7 days of the MDCK model,Resistance>600Ω·cm2,about 5 days demonstrate successful modeling;The safety concentrations of MTT of HM,HM-Y-A and HM-Y-B were 1.87～7.77,2.54～40.63,and 2.13～136.50 μg·mL-1 respectively.③The results of the two-way transport experiments show that,The efflux ratios of HM,HM-Y-A and HM-Y-B were 2.62,1.07,1.13,With the inhibitor,HM absorption direction Papp is increased;the Papp of HM-Y-A and HM-Y-B did not increase in absorption direction;At different concentrations,The cumulative transmittance Q(μg)and Papp in the HM within 120 min increased with the concentration,and the Papp of low have statistical difference with others;The cumulative transmittance Q(μg)in the HM-Y-A within 120 min increases with concentration,there was no statistical difference with Papp(P>0.05);The Q(μg)in the HM-Y-B within 120min increases with concentration,the Papp of low have statistical difference with others.Conclusion:①In one-way perfusion model of rats showed that,HM,HM-Y-A and HM-Y-B have good absorption in all intestinal segments,the main absorption sites were duodenum and jejunum,HM and HM-Y-B have good absorption when the pH value is 7.4,HM-Y-A is 6.4.HM could be a P-gp substrate,HM-Y-A and HM-Y-B are not P-gp substrates.②MDCK cell models show,MTT test toxicity from high to low is HM>HM-Y-A>HM-Y-B.The three drugs also show good absorption properties in cells,HM could be a P-gp substrate;HM-Y-A and HM-Y-B are not P-gp substrates.③The HPLC method established by this method is stable and reliable,revealing the characteristics of the absorption of three drugs in cell and animal models,It provides an important reference for further research and development and dosage form transformation in the future.