Effects Of Pluronic~?F68 And Labrasol~? On The Permeability Of Four Drugs And Efflux Function Of P-Glycoprotein

Oral administration is the most important form of group administration in veterinary clinic,and the absorption of oral drug into systemic circulation through the intestinal cell membrane is the premise of its efficacy.It has been recognised that many transporters expressed in intestinal epithelial cells,e.g.ABC family transports,can mediate drug efflux,thus restricting intestinal absorption or reabsorption after biliary excretion,which is considered to be the main factor of poor absorption and variability for oral drugs.P-glycoprotein（P-gp）is a major group of the transporter family,which is expressed in intestinal epithelial cells and the body barrier system.The factors affecting the expression and function of P-gp may impact the absorption of a drug in the small intestine.Recently,several studies have shown that some pharmaceutical excipients may improve oral absorption of drugs by inhibiting the activity of P-gp,thus improving the efficacy of drugs in human medicine.However,researches related to the role of pharmaceutical excipients from food animals have been rarely reported.In this thesis,we evaluated the role of Pluronic^?F68（F68）and Labrasol^?（Lab）on the permeability of commonly used drugs in chickens and the P-gp transport function by using MDCK-chAbcbl cell line which overexpresses chicken P-gp and the single-pass intestinal perfusion method of chicken small intestine.The results might provide new ideas for the research and development of veterinary drugs with improved oral bioavailability.The research contents are as follows:I Roles of F68 and Lab on permeability to four drugs and their impact to P-gp by cell monolayer modelThis research aims to explore the roles of pharmaceutical excipients F68 and Lab on P-gp efflux transport function.Drug accumulation experiments and transport experiments were used to determine the impact of F68 and Lab to permeability of metoprolol,the reference drug for the high-permeability drug metoprolol presenting by FDA,and ciprofloxacin,enozafloxacin,tilmicosin and sulfadiazine,the commonly used drugs for chickens,and the chicken P-gp function by MDCK/MDCK-chAbcb1 cell model.In accumulation experiment,we found that the accumulation of Rho 123（classical substrate of P-gp）,ciprofloxacin and enrofloxacin was significantly lower in MDCK-chAbcbl cells than that in MDCK cells.However,the accumulation of Rho 123,ciprofloxacin and enrofloxacin was significantly increased in MDCK-chAbcb1 cells,when treated with F68 or Lab within 15 min in MDCK-chAbcbl cells（P<0.05）.The results indicated that F68 and Lab could promote the accumulation of ciprofloxacin and enrofloxacin by inhibiting the efflux of P-gp to these drugs.In the bidirectional transport experiments,the net efflux rate（NER）of metoprolol was greater than 2.0,as judged by FDA for P-gp substrates,in MDCK-chAbcbl cells.After treated with different concentrations（5,50,500 μg/mL）of F68 and Lab,the NER value of metoprolol could be reduced to about 1.0,as similar as the effect of P-gp inhibitor verapamil.Moreover,F68 and Lab could significantly promote the metoprolol transport from the apical side to the basal side（P<0.01）,Papp（AP→BL）,and reduce it from the basal side to the apical side（P<0.01）,P_{app（BL→AP）}.No significant changes observed to corresponding MDCK cells.In the transport experiments of ciprofloxacin,enozafloxacin,tilmicosin and sulfadiazine,the NER values were 3.29±0.31,2.12±0.14,2.18±0.02,and 2.47±0.11,respectively,without the addition of F68 or Lab in MDCK-chAbcb1 cells.While the NER values all decreased to less 2 with F68 and Lab treatment.The results suggested that the four drugs were P-gp substrates,and F68 and Lab could inhibit the function of P-gp.Specially,F68 treatment significantly inhibited the efflux of ciprofloxacin,enrofloxacin and tilmicosin（P<0.05）,while Lab treatment significantly inhibited on the efflux of ciprofloxacin,tilmicosin and sulfadiazine（P<0.05）.These results suggested that F68 and Lab could inhibit the efflux of P-gp and promote the permeability of drug.II Effects of F68 and Lab on the small intestine permeability to four drugs by chicken in situ single-pass intestinal perfusion modelA chicken in situ single-pass perfusion model was chosen to further investigate the effects of F68 and Lab on drug permeability and P-gp efflux function.The permeability of the reference drug metoprolol and ciprofloxacin,enrofloxacin,tilmicosin and sulfadiazine were treated without or with low concentrations（50 μg/mL）and high concentrations（500μg/mL）of F68 or Lab.The results showed that the P_eff value of metoprolol was significantly increased by about 1.15-folds in low concentration of F68 treatment（P<0.05）,while the P_eff values were significantly increased by 1.19-and 1.16-folds in low and high concentrations of Lab treatment,respectively（P<0.05）.Meanwhile,the efflux of metoprolol in the jejunum were reduced after the treatment of different concentrations of F68 and Lab.In addition,the inhibition of the efflux to ciprofloxacin,enrofloxacin and sulfadiazine at low concentration excipient was more strongly than that at high concentration of both F68 and Lab in the jejunum perfusion test.However,some variations were observed:the effective permeability(P_eff)to ciprofloxacin,tilmicosin and sulfadiazine was significantly increased at both low and high concentrations of F68（2.2-,4.0-,1.9-and 2.2-,8.3-,2.3-folds,respectively）（P<0.01）;the P_eff to enrofloxacin was significantly increased at low concentration of F68（1.2-folds）（P<0.05）,while there had no significant changes on the permeability at high concentration of F68.Both the low and high concentrations of Lab had a significant effect on the P_eff of tilmicosin and sulfadiazine（P<0.05）,and the low concentration Lab had a significant effect on P_eff of ciprofloxacin and enrofloxacin（2.5-and 1.3-folds,respectively）（P<0.05）,but the high concentration Lab did not the permeability of ciprofloxacin and enrofloxacin（P>0.05）.The results of this study confirmed that pharmaceutical excipients F68 and Lab could improve the permeability in the chicken small intestine of ciprofloxacin,enrofloxacin,tilmicosin and sulfadiazine.In summary,our results showed that excipients F68 and Lab could inhibit P-gp efflux function to improve the transportion and absorption of commonly used chicken drugs as revealed by cell model and perfusion small intestine model.This study provided a basis for the identification of P-gp inhibitors from pharmaceutical excipients and the development of the oral veterinary drugs with improved bioavailability.Moreover,it can also provide a reliable method for the detection of intestinal permeability for commonly used drugs in chicken,and lay a foundation on research BCS in veterinary drugs.