Synthesis And Evaluation Of Oral Prodrugs Of Zanamivir PepTides Targeting Intestinal Oligopeptide Transporters(PepT1)

Objective: To design and improve drugs according to clinical needs is the future direction of drug research and development in recent years.Oral drugs need to be absorbed from the intestine into the blood after digestion by the human body.The crosscooperation between clinicians and drug developers is the original design intention of this study to transform drugs that cannot be taken orally into oral drugs that can be recognized and absorbed into the blood by intestinal transporters.Oral medication is the most convenient way to administer it.Perioperative children,tracheal intubation patients and other high-risk groups of influenza virus have an urgent need for oral zanamivir dosage form.Zanamivir is a potent inhibitor of the flu virus and is on the government stockpile.However,zanamivir has low oral bioavailability and its clinical application is limited because it cannot be taken orally.Based on the previous studies,this study designed and synthesized zanamivir amino acid ester peptide-like prodrug with intestinal epithelial cell oligopeptide-transporter PepT1 as the transport target for this defect.The stability,transport status,transport targets and toxic side effects of the prodrug in gastrointestinal tract were investigated through cell and animal models,and the possibility of zanamivir peptide-like prodrug becoming an oral preparation of zanamivir was initially discussed.Methods: 1.Designed and synthesized 8 kinds of zanamivir-like peptide prodrugs by chemical synthesis methods such as hydrogenation,catalysis,condensation and esterification,and tested the purity,molecular weight and structure of 8 kinds of prodrugs by means of mass spectrometry,nuclear magnetic resonance(carbon spectrum,hydrogen spectrum)and liquid phase analysis.2.In vitro,different media were used to simulate the stability of the prodrug in human gastrointestinal tract and blood,including chemical stability and enzyme stability.In the study,rat gastric juice,rat intestinal juice,plasma,hydrochloric acid solution,phosphate buffer solution with different pH values,artificial gastric juice(containing pepsin),artificial intestinal juice(containing pancreatic enzyme)and other media were used.3.The membrane permeability of various prodrugs was investigated in vitro through cell models to simulate the transport and absorption of prodrugs in the intestine,and the prodrugs with better transport and absorption were selected for further study;The mechanism of absorption and uptake of prodrugs in intestinal epithelial cells and the competitive inhibition of PepT1 classical substrates were investigated in the cell model with high PepT1 expression in vitro,and whether prodrugs were targeted by PepT1 as transport and absorption targets was investigated.4.The oral pharmacokinetics of zanamivir peptidoides in animals were investigated using rats as animal models,and the effects of designed and synthesized zanamivir peptidoides on oral bioavailability of zanamivir were discussed.The toxic and side effects of zanamivir on liver and immune system were studied in zebrafish with liver and neutrophil fluorescence as animal models.Results: 1.Eight new zanamivir peptide prodrugs were designed and synthesized,namely Zan-R-ALA,Zan-S-ALA,Zan-R-D-VAL,Zan-S-D-VAL,Zan-R-L-VAL,Zan-S-L-VAL,Zan-R-ILE,Zan-S-ILE,Zan-S-ILE.The purity was more than 95%,and the molecular weight and structure of the target compound were consistent with that of mass spectrometry,nuclear magnetic resonance carbon spectrum and hydrogen spectrum analysis.2.Except that Zan-R-ALA may be decomposed in advance by enzymes in gastric and intestinal fluid,the remaining 7 prodrugs showed ideal chemical and enzyme stability.3.Zan-R-L-VAL had the highest membrane permeability and intestinal absorption and transport,which was 11.8 times that of Zanamivir,followed by Zan-S-L-VAL.In the study on the uptake mechanism of zanamivir and its peptidoid prodrug,the uptake rate of8 prodrugs in PEPT1-MDCK cells was higher than that of the mother drug,and the highest uptake rate was found in Zan-R-L-VAL,and the prodrug Zan-R-L-VAL could competitively inhibit the classical substrate Gly-Sar(glycyl sarine)of PepT1.4.A HPLC-MS/MS method for the determination of zanamivir in plasma was established.Compared with the mother drug Zanamivir,the oral bioavailability of the peptidogenic prodrug Zan-R-L-VAL was significantly improved.Studies on the toxic and side effects of Zan-R-L-VAL on the liver and immune system of Zebrafish showed that ZAN-R-L-VAL had no obvious toxic and side effects on the liver and immune system.Conclusions: Eight new amino acid ester precursor drugs of zanamivir were synthesized in this study.Through the membrane permeability study,it was found that the translocation and absorption of the peptidogenic prodrug of zanamivir were better than that of the mother drug.The absorption mechanism showed that prodrug was transported and absorbed by intestinal oligopeptide transporter PepT1.The stability of zanamivir peptide prodrug was investigated in vitro,and the stability of Zanamivir prodrug in gastric and intestinal fluid was confirmed.Animal pharmacokinetic study found that the oral bioavailability of prodrug was higher than that of mother drug zanamivir,and had no obvious toxic and side effects on liver and immune system.In conclusion,zanamivir peptidoid prodrug synthesized in this study can be transported and absorbed into the blood through intestinal oligopeptide transporter PepT1,and has the potential to become an oral preparation of zanamivir.