Comparision Of The Apparent Permeabilities Of Several Antimicrobials Determined In Mdck Cells With Overexpressed Different Transporters

The Biopharmaceutical Classification System(BCS)is a drug classification and prediction system based on the determination of drug solubility and permeability.It is currently approved by the FDA,WHO,EMA,and other authoritative organizations and applied to exempt human in vivo bioequivalence studies from its use in drugs.The R&D process plays an important role.It is imperative to apply the BCS concept to the research and development of veterinary drug preparations in view of research and development costs and profitability of veterinary drugs.However,there are large differences between humans and animals in terms of anatomy,physiology,and types of drugs used clinically.Therefore,it cannot be performed.Direct interspecies extrapolation requires further study to resolve limitations and methodological problems before they can be applied.Permeability measurement is an important step in the BCS classification system.The FDA has used Caco-2 cells as a guide for the determination of human in vitro drug permeation,and based on the measured drug permeability,the drugs were classified by BCS to further guide the drug's Research and development process.The study found that Caco-2 cells express high amounts of human ABC transporter P-glycoprotein(P-gp)and breast cancer resistance protein(BCRP),but P-gp and BCRP in different species of animals have significant differences with humans.The substrate spectrum also has certain differences,so the use of Caco-2 cells for chicken drug permeability determination may have a certain impact.In this experiment,MDCK cells that overexpress and simultaneously over-express chicken P-gp and BCRP were used to determine the permeability of the four antibiotics and to compare the results.The results of the study can lay a foundation for the establishment of in vitro methods for the determination of the permeability of poultry drugs,provide reference for the development of new veterinary drugs and better guide the veterinary clinical drug use,and therefore have important theoretical and practical values.To investigate the effect of P-gp and BCRP over-expressing cell lines on the determination of antibacterial drug permeability.We determined the permeability of the four drugs using the MDCK-chAbcbl,MDCK-chAbcg2 and MDCK-chAbcb1/Abcg2 overexpressing cell lines and wild-type MDCK cells constructed in this experiment.First,we established a four-cell compact monolayer model on the TranswellTM monolayer membrane.The integrity of the cell membrane was verified by electrical resistance measurements.The results showed that the transmembrane resistance values of the four cells were greater than 1 000 ? after 6 days of culture.Consistent with the need for compactness of transport cell monolayer model.Then the safe concentration of the four drugs on the cells was detected by MTT assay to ensure that the drug had no effect on the integrity of the monolayer cell model at the working concentration,and the high-pressure liquid phase detection method for the four drugs was optimized.Finally,the apparent permeability(Papp)of the four antibiotics was measured using biphasic transport experiments,and the net efflux rate(NER)of each transporter against four antimicrobial agents was calculated.Based on NER>2,the drug was identified as a transporter substrate..The results showed that tetracycline was a common substrate for chicken BCRP and P-gp(P-gp:NER50?M=13.94,NER100?M=8.77,BCRP:NERs0?M=2.23,NER100?M=2.74),and sulfonamide.M-dimethoxypyrimidine is the substrate for chicken P-gp(NER50?M=2.24;NER50?M=2.33).Compared to the MDCK cell line,tetracycline,a common substrate of only two transporters in the MDCK-chAbcbl/Abcg2 cell line,showed a clear efflux effect.The net efflux rate of tetracycline in the double expressing cell line was NER50?M=8.94;NER50?M=9.1 2.To further compare the differences in drug permeability data between the four cell lines,we performed AP-to-BL apparent penneability data and MDCK cell data for the four drugs in three overexpressing transporter cell lines.compare research.For the antibacterial drug tetracycline,compared with the results in MDCK cells,in the MDCK-chAbcg2 cells,APapp-to-BL Papp decreased by 9.81-fold and 3.47-fold at 50 ?M and 100 ?M,respectively;In MDCK-chAbcbl cells,APapp-to-BL Papp decreased by 13.95-fold and 3.07-fold at 50?M and 100 ?M,respectively;and AP in tetracycline 50 ?M and 100 ?M in MDCK-chAbcbl/Abcg2 cells.The Papp of AP to BL side was reduced by 22.08-fold and 13.87-fold,respectively.For sulfamonomethoxine sodium,compared with MDCK cells,the Papp of AP to BL in MDCK-chAbcg2 cells was decreased by 2.15 times and 2.70 times at 50 ?M and 100?M,respectively.In the MDCK-Abcb1 cells,AP-to-BL Papp did not change significantly at 50 ?M,whereas AP-to-BL Papp decreased 1.45-fold at 100 ?M;in MDCK-Abcg2/Abcb1 cells,at 50 ?M and 100 ?M,the Papp of AP to BL decreased 1.71-fold and 1.97-fold,respectively.For the antibacterial drug sulfachloropyrazine sodium,compared with MDCK cells,MDCK-cliAbcg2 cells showed no significant changes in Papp from AP to BL at 50?M,but decreased by 1.38 times at 100?M.In the MDCK-chAbcbl,AP-to-BL Papp decreased by 1.98-fold and 1.78-fold at 50 and 100 ?M,respectively,and AP-to-BL at 50?M and 100 ?M in MDCK-chAbcbl/Abcg2 Papp decreased by 2.49 and 2.58 times,respectively.Compared with the results in MDCK cells,the Papp(AP to BL)of sulfamethazine in MDCK-chAbcg2 cells was increased by 1.52 and 2.16 times at 50 ?M and 100?M,respectively.In MDCK-chAbcb1,the Papp of AP to BL decreased by 1.41 at 50 ?M,and increased significantly by 2.98 times at 100 ?M.In MDCK-chAbcb1/Abcg2,there was no significant change in the AP-to-BL apparent permeability at 50 ?M,while it can be significantly increased 1.91 times than that of in MDCK at 100 ?M.To sum up,the efflux pump transporters in animal body has an effect on determining the drug permeability,especially on a variety of transporter substrates.Therefore,this factor is considered when the cell model is established for drug permeability determination,and it is best to choose the cells expressing the transporters.And the concentration of drugs should be able to overcome the efflux transport proteins.The conditions for cell model determination should be further optimized.