Study On The Absorption Mechanism Of Halofuginone Hydrobromine Encapsulated By TPGS Polymer Micelles And Its Preventive Effect On Chicken Eimeria Tenella

Chicken coccidiosis has caused huge economic losses to chicken industry worldwide.Nowdays,the prevention and control of chicken coccidiosis still mainly depends on chemical drugs due to limitation of the production and efficacy of chicken coccidiosis vaccine.Due to the increasing resistance of coccidium to clinically commonly used anticoccidial drugs,and the shortcomings of low permeability of existing anticoccidial drugs,it requires a larger dose to reach an effective concentration in intestinal epithelial cells.Therefore,there is an urgent need to use new formulation technology to improve the solubility and permeability of anticoccidial drugs for promoting their efficiency of entry into the cell and increasing drug utilization.Halofuginone hydrobromide（HF）exhibit the excellent anti-coccidial effect and is not easily produced drug resistance.However,its high production cost,poor water solubility,and bitter taste greatly limited clinical application.Therefore,it is of great significance for its clinical application to develop highly efficient preparations for reducing the use amount.Our laboratory has successfully developed halofuginone hydrobromide encapsulated by TPGS polymer micelles（HTPMs）to effectively improve the low solubility of halofuginone hydrobromide,it’s expected to be developed into a high-efficiency preparation for oral administration via drinking water of drugs.Based on this,the in situ unidirectional perfusion of chicken small intestine and the Caco-2 cell monolayer model were used to study the absorption mechanism of HTPMs in this study.Then,this study conducted prophylaxis effect of HTPMs on Eimeria tenella in order to study whether it has an enhanced anti-coccidial effect,to explore the relationship between intracellular delivery of halofuginone hydrobromide and anticoccidial effect.The specific research is as follows.1 Intestinal absorption mechanism of HTPMsThe effect of TPGS polymer micelles on the permeability of HF was studied by in situ unidirectional perfusion of chicken small intestine and Caco-2 cell monolayer model,and the absorption and transport mechanism of HTPMs on monolayer of Caco-2 cells was described.Firstly,the permeability of HTPMs and HF was studied by in situ unidirectional perfusion of chicken small intestine.The results showed that TPGS polymer micelles can improve the effective permeability(P_eff)of HF in small intestine.Then Caco-2 cells were cultured in the Transwell chamber for 21 days,and the TEER value reached 400Ω·cm²,the apparent permeability coefficient(P_app)of lucifer yellow in the monolayer of Caco-2 cells was<0.50×10^-6 cm/s,and the P_app value of propranolol also met the requirements of≥10×10^-6cm/s.The results showed that the integrity and permeability of Caco-2 cell monolayer met the test requirements,indicating Caco-2 cell monolayer model was successfully established.Furthermore,the CCK-8 method was used to screen suitable concentrations of HF and HTPMs（2μg/m L）,and the permeability of HTPMs and HF and the transmembrane transport mechanism of HTPMs were studied by single and double direction transport experiments.The results showed that TPGS polymer micelles could enhance the permeability of HF in Caco-2 cell monolayer,reduce the efflux of P-gp,and improve the intracellular uptake of HF.In addition,it was found that HTPMs could enter Caco-2 cells mainly through an energy-dependent active transport mode;further studies have shown that HTPMs enter Caco-2 cells mainly through caveolin/lipid raft-mediated endocytic pathways.2 Prevention effect of HTPMs on Eimeria tenella infection in chickensThis experiment was conducted to evaluate the preventive effect of drinking water administration of HTPMs on Eimeria tenella infection in chickens.Chickens were artificially infected with Eimeria tenella at 14 days of age,and were administered using HTPMs at three doses of high,medium and low（3 mg/L,1.5 mg/L and 0.75 mg/L）and HF premix at dose of 3mg/kg 2 days before infection.According to the survival rate,relative weight gain rate,cecal lesion value and oocyst value,the anti-coccidial efficacy evaluation indexes were obtained.The results showed that,the ACI of HF premix group was 200.97,the ACI of HTPMs at high-dose and medium-dose groups were 195.51 and 208.38,and the ACI of HTPMs at low-dose group was also 194.59.According to ACI evaluation criteria,all treatment achieved high anti-coccidial efficacy,indicating that HTPMs has a good preventive effect on Eimeria tenella infection in chicken.