Study On Preparation And In Vitro Evaluation Of Paclitaxel Small Molecule Prodrug And Its Oral Absorption Effect

Paclitaxel as a broad-spectrum antitumor drug,is used alone or in combination with other antitumor drugs in clinical chemotherapy.Due to its low water solubility(0.3-0.5 μg/mL),currently PTX preparations are mostly used for injection in clinical applications,and the excipients added to the formula will cause serious allergic reactions,increase the burden on patients,and have poor patient tolerance.Oral administration is safer and more convenient than injection administration,and is more acceptable to patients.Chemical modification of the drug substance to prepare a small molecule prodrug can effectively avoid the toxic and side effects brought about by the large molecule carrier material or auxiliary materials.In order to improve the water solubility and oral bioavailability of PTX,this project uses succinic anhydride as a linker to introduce cystamine to prepare PTX small molecule prodrug PTX-Cys and small molecule prodrug PTX-SS-Val based on intestinal transporter(PEPT1)targeted transport,and its physical and chemical properties in vitro,antitumor activity,intestinal absorption effect in vivo,and pharmacokinetic characteristics were investigated.Project research methods and results include the following five parts:1.Synthesis and characterization of PTX prodrugsFirst,the 2’-OH of PTX and succinic anhydride(SA)form an ester bond to obtain PTX-COOH through an esterification reaction,and then an amide reaction is used to link the amino group of cystamine(Cys)with the carboxyl group of PTX-COOH to obtain PTX-Cys,and finally introduced L-Valine(L-Val)on the basis of PTX-Cys with an amide bond.The synthesis was verified by NMR hydrogen spectroscopy(1H-NMR)、Fourier transform infrared spectroscopy(FT-IR)and mass spectrometry(MS).2.Study on the aggregation state of PTX prodrugs and investigation of its in vitro propertiesThe aggregation state of PTX-Cys and PTX-SS-Val was prepared by the nano-precipitation method,and the appearance morphology、particle size distribution and Zeta potential of the nanoparticles were examined using transmission microscopy(TEM)and dynamic light scattering methods(DLS).The PTX-Cys and PTX-SS-Val solubility were measured by a shake flask method.The high-performance liquid chromatography(High Performance Liquid Chromatography,HPLC)and the dialysis bag method were used to investigate the chemical stability of PTX-Cys and PTX-SS-Val and their stability in artificial gastric fluid and artificial intestinal fluid,respectively.The experimental results show that the PTX-Cys and PTX-SS-Val can self-assemble into nanoparticles in aqueous solution,the appearance is spherical,and the particle size distribution is uniform.The solubility of PTX-Cys and PTX-SS-Val is 2.1592μg/mL and 16.5938μg/mL,respectively.The release of PTX in both buffer solutions at pH=6.4 and pH=7.4 was less than 15%,and the release of PTX in artificial gastric juice and artificial intestinal juice was less than 4%,and the structural stability was high.3.In vitro antitumor evaluation of PTX small molecule prodrugsFirst,human breast cancer cells(MCF-7)were used as the cell model,and the cytotoxicity of PTX-Cys and PTX-SS-Val was examined by the thiazole blue method(MTT).The results showed that both of them had strong cytotoxicity.Then,the prodrug nanoparticles containing coumarin(C6),a fluorescent substance,were studied under inverted fluorescence microscopy and flow cytometry to confirm that cancer cells have a certain uptake capacity for PTX-Cys and PTX-SS-Val.Finally,the ability of PTX-Cys and PTX-SS-Val to induce cell apoptosis was investigated using an AnnexinV-FITC/PI kit under an inverted fluorescence microscope and a flow cytometer.Compared with PTX-SS-Val,PTX-Cys has stronger cytotoxicity,cell uptake ability and ability to induce cancer cell apoptosis in vitro cancer cells.4.In vivo one-way intestinal circulation experiment of PTX small molecule prodrugIn this part,the oral absorption of PTX-Cys and PTX-SS-Val is primarily investigated by the rat in vivo intestinal circulation method.In order to explore the effect of pH on the jejunal absorption,the intestinal absorption in two kinds of perfusate pH=6.5 and pH=7.4 was investigated.The results showed that PTX-Cys and PTX-SS-Val have stronger absorption capacity in the perfusate with pH=7.4.Glysar,a substrate of the transporter(PEPT1),was used to further confirm the targeted transport of PEPT1 to PTX-SS-Val.The concentration gradient method was used to study the absorption mechanism of PTX-Cys and PTX-SS-Val.The results showed that PTX-Cys showed first-order absorption kinetics,and PTX-SS-Val showed a ligand-receptor targeted transmembrane transport.The experimental results also confirmed that PTX-Cys and PTX-SS-Val were also effectively absorbed in the duodenal and ileal segments.Compared with PTX-Cys,due to the targeted transport of PTX-SS-Val by PEPT1,its intestinal cell absorption capacity is stronger.5.Oral pharmacokinetics of PTX small molecule prodrugBased on the results of in vitro stability studies and rat intestinal circulation experiments in vivo,the oral pharmacokinetic characteristics of PTX-SS-Val were investigated.After intravenous injection of Taxol,intragastric administration of Taxol and PTX-SS-Val,the PTX blood concentration data at predetermined time points were measured and processed to obtain various pharmacokinetic parameters.The results showed that the area under the drug curve(AUC)of the PTX-SS-Val group was 1.28 times and 1.56 times of the intravenous Taxol group and the intragastric Taxol group,respectively,and the absolute bioavailability increased from 19.31%to 30.61%.Tissue safety has further confirmed that the prodrug PTX-SS-Val has better safety in vivo.