Influencing Factors Of Drug Permeability Determination By In Vivo And In Vitro Models As Well As The Effects Of Quercetin On Drug Permeability

The Biopharmaceutics Classification System(BCS)is a drug classification and prediction system based on the determination of drug solubility and intestinal permeability.Therefore,accurate determination of the intestinal permeability of drugs is a prerequisite for the scientific classification of drugs,and it is also an important parameter predicts the absorption of drugs in animals.The BCS guidelines issued by the US FDA recommend the use of CaCO-2 cells and rat intestinal perfusion to determine the permeability of drugs and specify a series of internal reference drugs that can be used as criteria for permeability determination.However,the current in vitro methods for the veterinary drug BCS classification and veterinary drug permeability measurement have not yet been developed.This experiment was the first to use the metoprolol,a highly permeable internal reference drug recommended by the FDA,to establish an intestine perfusion model in chickens and explore the effects of different pH,different intestinal segments,and different perfusate concentrations of drugs on the intestinal permeability of the drug.At the same time,MDCK and MDCK-chAbcb1 cells were selected for metoprolol permeability determination to investigate the effect of pH,transporter drug concentration,and transporter protein on cell model determination of drug permeability.Several optimized perfusion and cell models were used to perform several assays.The determination of the permeability of commonly used drugs,and finally studied the effect of quercetin on metoprolol transport.The results of the study can lay a foundation for the establishment of osmotic measurement methods for veterinary drugs BCS classification and provide a reference method for the study of drug-drug interactions.Firstly,healthy 3-week-old AA white broiler chickens were selected to study the effective permeability of metoprolol in chickens at different pH,intestinal segments and concentrations to establish single-pass intestinal perfusion method.With 400 ?g/ml metoprolol perfusion,the ileal effective permeability(Peff)calculated at physiological pH was significantly higher than that of duodenum and jejunum(P<0.01).Duodenum,perfusion fluid pH=5,the effective permeability of the intestinal tract was significantly higher than the intestinal permeability at pH=6(P<0.01);was significantly higher than the infiltration of metoprolol pH=7(P<0.05).As the pH value increases,the effective permeability of metoprolol decreases.In the jejunum,the permeability of metoprolol is the lowest at pH=6,pH=5 is slightly lower than pH=7,and different pH values.The difference in metoprolol permeability was not significant(P>0.05).In the ileum,the permeability of metoprolol was increased with increasing pH,and pH=7 was significantly higher than pH=5(P<0.05).It is suggested that different pH have a certain influence on the permeability of each intestinal segment,pH has a greater effect on the duodenum and ileum,and has little effect on the jejunum.There were no significant changes in the permeability of metoprolol at the same pH in different intestinal segments;at pH 5,the permeability from the proximal to distal end of the small intestine decreased,but the difference was not significant;At pH=6 and pH=7,the permeability of the small intestine from the proximal to the distal intestine has a certain tendency to increase,but the difference is not significant.In addition,metoprolol has a certain concentration-dependent permeability in the jejunum.With the increase in the concentration of metoprolol in the perfusate,the permeability of metoprolol shows a significant upward trend.The metoprolol concentration of 400?g/ml was significantly higher than the permeability of 40 ?g/ml and 4 ?g/ml(P<0.01).Drugs were mainly absorbed in the duodenum and jejunum,the jejunum segment was smooth,the drug had a long residence time in the ileum,and the jejunum had no significant changes in permeability at different pH conditions.A unidirectional in vivo perfusion model of chicken intestine was established under the condition of jejunum pH=7.Using the established perfusion method,the intestinal permeability of several commonly used antibacterial agents of chickens:clindamycin,ivermectin,ciprofloxacin,doxycycline,and amoxicillin was determined and calculated.The effective permeability of the jejunum is compared with the permeability of metoprolol.The results showed that among the five drugs,the permeability of doxycycline hydrochloride and ciprofloxacin hydrochloride was the highest,followed by clindamycin and amoxicillin,and ivermectin was the lowest.We further investigated the effect of the highly expressed efflux pump transporter P-gp in the intestine of chickens on the determination of drug permeability.For this purpose,we selected MDCK cells and MDCK-chAbcbl cells constructed by our laboratory to highly express chicken-derived P-gp.The effect of different pH,different transporting concentrations(4,40,and 400 ?g/mL)and transporter inhibitors on the metoprolol permeability(Papp)of the internal reference drug was measured using a monolayer biphasic transport assay.The results showed that the net efflux rate of metoprolol(NER)was?2,and that the P-gp inhibitor verapamil reversed the Papp of MDCK-chAbcbl against metoprolol BL-AP,confirming that metoprolol was substrate of P-gp,increasing the concentration of metoprolol,can evade the efflux action of P-gp.Metoprolol was found to be concentration-dependent on the AP-BL side in both MDCK and MDCK-chAbcbl cells.The Papp measured at 40 ?g/mL and 400?g/mL was significantly higher than 4 ?g.Papp/mL(P<0.01);however,Papp on the BL-AP side did not show a significant concentration dependence,suggesting that the measurement of Papp for P-gp substrate drugs should not be performed at low concentrations.Different pH had an effect on the determination of metoprolol Papp in MDCK and MDCK-chAbcbl cells.As the pH increased,Papp on the AP-BL side showed an upward trend.Finally,we investigated the effect of quercetin on metoprolol transport in vitro using in vitro cell lines and chicken intestine perfusion models.First,MDCK and MDCK-chAbcbl cells were pretreated with different concentrations of quercetin(10,50,100 ?M)for 24 h,and then their effects on metoprolol Papp were measured.The results showed that when MDCK cells transported 4 ?g/ml metoprolol,AP-BL permeability increased significantly after treatment with 10 ?M and 100 ?M quercetin(P<0.01).Compared with the control group,BL-AP 10,50,100 ?M treatment group increased significantly(P<0.01);when the metoprolol was transported at 40 ?g/ml,AP-BL and BL-AP permeability did not change significantly,but the efflux rate increased significantly.(P<0.01).When MDCK-chAbcbl cells transported 4 jig/ml metoprolol,there was no significant change in the permeability of AP-BL by quercetin treatment,but the BL-AP significantly increased when quercetin reached 100 ?M(P<0.05),both sides of the outer row rate compared to the control group were significantly increased(P<0.01);40 vg/ml,its AP-BL side permeability in the 10?M and 50 ?M quercetin treatment group increased significantly(P<0.05),both sides of the outer row rate significantly increased(P<0.01).When the chickens were continuously orally fed with 15 mg/kg and 60 mg/kg of quercetin for 5 d and 10 d,respectively,unidirectional single-pass intestinal perfusion of jejunum with 400 ?g/ml metoprolol was performed.The results showed that the treated group of cells Metoprolol permeability was significantly lower than that of the control group(P<0.01).The jejunum permeability of metoprolol treatment group was lower than 5 days after treatment of 10 days;the metoprolol permeability of quercetin 15 mg/kg treatment group was lower than that of 60 mg/kg group after 5 days and 10 days.Effects of quercetin on the intestinal permeability of ivermectin The results showed that after oral administration of different doses of quercetin(15 kg/mg and 60 mg/kg)for 5 d and 15 d,400 ?g/ml metoprolol was used.Although quercetin can increase the concentration of ivermectin jejunal intestine perfusate,there is no significant difference in permeability(P>0.05).In summary,we have initially established a unidirectional single-pass intestinal perfusion model and cell models for jejunal intestinal tract in chickens.Both should consider the concentration of the test drug,pH,and the influence of transporters when determining the drug permeability.The validation of the two models suggests that quercetin can affect the transport of metoprolol and metoprolol,the P-gp substrate,and mediate the interaction between drugs.