Screening And Mechanism Study Of Florfenicol Absorption Enhancers Based On PepT1

Florfenicol（FF）was extensively used in veterinary.Due to the hard dissolved in water,many studies have focused only on the method of solubilization,pharmacokinetics and tissue distribution in different animals.Some have shown that it’s well absorbed by oral administration,but there is still a lack of studies on the intestinal absorption mechanism of FF.Oligopeptide transporter 1（PepT1）is a transporter closely related to peptide transport in intestinal mucosa,FF structure has a structure similar to peptide bond.This study studied the interaction between PepT1 and FF during intestinal absorption,aiming to explore the intestinal transport mechanism of FF based on PepT1.In this study,the relationship between FF and PepT1 was further verified at the cellular level,and PepT1 was taken as a target to screen oral absorption enhancer that could promote the expression of PepT1 at both the cellular and molecular levels,so as to increase the intake of FF in the intestine.1.Direction of FF absorption across the monolayer of Caco-2 cells:Caco-2 cell model was used to conduct the transmembrane transport experiment of FF,and the transmembrane resistance value and the ratio of alkaline phosphatase on both sides were used to verify the successful establishment of the single-layer cell model.After 21 days of Transwell plate culture,the transmembrane resistance of each pore was greater than 300Ω/cm²,and the ratio of alkaline phosphatase activity on the AP side and BL side was close to 3:1,which verified that the cells were well connected,polarized and differentiated,and formed a complete monolayer of cells,which could be used for transport experiments.The K_aand P_appvalues of the AP-BL side were（2.01±0.03）×10^-2,8.02±0.14×10^-4,and the K_aand P_appvalues of the BL-AP side were（1.01±0.08）×10^-2,(4.36±0.34×10^-4),respectively.The Ka value of the AP-BL side is greater than that of the BL-AP side,so it can also be inferred that the presence of transporters on the side of intestinal epithelium facing the intestinal lumen increases the FF transfer volume on the AP-BL side.When the concentration was<200μg/m L,the intake of FF slowly increased with the increase of the concentration,no significant difference was found（P>0.05）.When the concentration continued to increase to 200μg/m L,the intake of FF increased sharply,and there was a significant difference with the low concentration（P<0.05）.When the concentration continued to increase to>200μg/m L,the intake of FF increased sharply.Their intake did not change significantly with increasing concentration,At low concentration,the factor limiting FF uptake is the concentration of FF,that is,the amount of transporter in Caco-2 is much larger than FF molecule;when the concentration increases to a certain range,the amount of transporter in Caco-2 is restricted,that is,the transporter has reached saturation,that is,the transport of FF in the intestine is not only passive diffusion.2.Factors affecting FF uptake:Using Caco-2 cells to construct a compact intestinal epithelial monolay model on the surface of the support material,the effects of different molecular lactam bonds,different concentrations of Gly-Sar and verapamil on uptake of FF cells were investigated with the percentage of uptake of FF cells at 2 h.Flubenicolamide（FFA）,the metabolite of FF,has no molecular lactam bond in its molecular structural formula.The intake of FFA at low concentration（25-100μg/m L）in Caco-2 cells increases with the increase of the concentration,and the factor affecting the uptake of FFA is the concentration of FFA.The intake of FFA in Caco-2 cells at different concentrations was significantly lower than that of FF,indicating that FF transport in the intestine was significantly superior to FFA,and molecular lactam bonds promoted the transport of FF in the intestine.After the co-incubation of Gly-Sar and FF,the intake of the group without Gly-Sar co-competition was significantly higher than that of the group with Gly-Sar,and the intake of FF decreased significantly after the addition of Gly-Sar,and there was a significant difference between them（P<0.05）.In addition,there was a significant difference between the high concentration Gly-Sar group and the low concentration Gly-Sar group（P<0.05）,indicating that the higher concentration Gly-Sar co-competed with the lower concentration of PepT1 site,resulting in the decrease of the contact frequency and intake of FF and PepT1.After the addition of verapamil,FF intake in the intestinal tract showed an obvious upward trend,and high concentration of verapamil had a more obvious promoting effect.P-gp had an obvious efflux effect on the absorption of FF in the intestinal tract,that is,P-gp participated in the transport of FF in the intestinal tract.3.Screening of FF oral absorption enhancers:Caco-2 cells were used to construct a compact intestinal epithelium single layer model on the surface of the supporting substance,and the percentage of FF cell uptake at 2 h was used as the index to screen the effects of different absorption enhancers on FF uptake.The results showed that all the selected absorption enhancers could significantly increase the uptake of FF,and there was no significant difference between different concentrations of enhancers（P>0.05）.The results indicated that low concentration（25μg/m L）of promoter could affect the uptake of FF.Fluorescence quantitative results showed that CS,CMS-Na,EDTA-2Na,PEG6000 andβ-CD could all up-regulate PepT1,among which,only CMS-Na andβ-CD down-regulated P-gp,PepT1was the protein that FF entered the intestinal side,the up-regulation of PepT1 could significantly increase the uptake of FF,P-gp was the efflorescent protein.Down-regulating the expression of P-gp can reduce the efflux of FF,thus increasing the amount of FF entering the body.Therefore,screening excipients that can up-regulate PepT1 while down-regulate P-gp can improve FF intake.According to the results of fluorescence quantitative PCR and Western-blot tests,the expression of P-gp decreased while the expression of PepT1 was up-regulated by CMS-Na andβ-CD.CMS-Na andβ-CD could be used as oral absorption enhancers for FF for follow-up pharmacokinetic experiments.4.In vitro absorption characteristics of FF and oral absorption enhancers:The effects of FF and oral absorption enhancers on transintestinal absorption of rats were investigated by in vivo enteral perfusion.The experimental animals were divided into three groups:original drug group（FF）and different oral absorption enhancers group（CMS-Na andβ-CD）.The three groups were given in vivo enteral perfusion for 2 h,respectively,and the concentrations of FF and phenol red in the circulating fluid were measured every 15 min.The FF concentration was obtained by correcting the change of circulating liquid volume with the change of phenol red concentration.After perfusion with different promoter groups,there was a significant difference in P_effbetween the absorption promoter group and the original drug group（P<0.05）,indicating that the absorption promoter could significantly increase the uptake of FF in the intestine.5.Study on oral pharmacokinetics of FF and oral absorption enhancers:Different absorption accelerator groups were given orally by intragastric administration.Venous blood was collected from the posterior orbital venous plexus at different time points within 24 h after administration.FF concentration in blood was determined by High Performance Liquid Chromatography（HPLC）for pharmacokinetic analysis.The results showed that the pharmacokinetic data of the active drug group,the CMS-Na group and theβ-CD group were consistent with the two-compartment model.There were significant differences in T_1/2Kabetween the active drug group,CMS-Na andβ-CD groups,and the absorption promoter group was higher than the active drug group,indicating that the absorption promoter promoted the oral absorption of FF,and the promoting effect of CMS-Na was greater than that ofβ-CD.AUC_0-12and AUC_0-∞also showed the same result,absorption enhancers increase the oral absorption of FF.The results of this study indicated that the intestinal absorption of FF interacts with the intestinal epithelial transporter PepT1,and the oral absorption promoters were screened to increase the uptake of FF in the intestinal epithelium.The results showed that the screened CMS-Na andβ-CD could significantly increase the uptake of FF in the intestinal epithelium,up-regulate the expression of PepT1 and increase the absorption of FF in rats.