Synthesis Of Glycosylated Paclitaxel And Its Targeting Effect And Mechanism To Cancer Cells Via GLUT-1 Interaction

Paclitaxel(Paclitaxel,PTX)is a natural secondary metabolite compound which isolated from the bark of Taxus baccata Linn.It is widely used as a first-line anti-tumor natural drug for series of cancers such as breast cancer,ovarian cancer and non-small cell lung cancer,etc.PTX and its derivatives,docetaxel and cabataxel,are the only type of tubulin polymerization agents currently in clinical use,which have unique anti-tumor mechanisms by inducing and stabilizing polymerization of tubulin.This mechanism causes the mitotic process of tumor cells to be bound in the G2/M phase,thereby inducing tumor cells to undergo apoptosis.However,water solubility of PTX is extremely poor,which leads to low bioavailability for oral administration and poor compliance.It is indispensable to use a toxic solubilizing agent to dissolve the drug before clinical practices.This results in a high incidence of adverse reactions in patients,which seriously affects the quality of life of patients.The low aqueous solubility of formulation and the lack of tumor selection are two key issues affecting drug use and efficacy of PTX.Currently,in addition to the traditional cosolvent-type PTX injection,human albumin-type PTX and PTX liposomes are used in clinical practises,both improve aqueous solubility of PTX and reduce the toxicity while maintaining pharmacological activity.However,the preparation process of the two is complicated,the source of raw materials is narrow,and the price remains high for a long time and has not been included in the National Medical Insurance Catalogue of China.The clinical treatment cost is extremely high,which causes a huge economic burden on the patient.Therefore,the traditional solvent-based PTX injection is still the most common PTX preparation in China.In recent years,the academic community has proposed a strategy for chemical modification of paclitaxel to improve solubility,such as glycosylation at side chain of PTX,and related research has progressed,but currently limited to the physicochemical properties of the solubility of glycoconjugates,no new derivatives of paclitaxel have been found.Abnormally boost of glucose metabolism is one of the most obvious physiological characteristics of tumor cells.In order to uptake more glucose,glucose transporter-1(GLUT-1)on the surface of tumor cells is overexpressed,reduction of GLUT-1 expression can inhibit tumor proliferation and invasion.Accordingly,this study proposes glycosylation modification of PTX,which can enhance the uptake of drugs by tumor cells through interaction with glucose transporters,and glycosylation conjugate on the other handimprove water solubility on the one hand.Thus,the glucose conjugate TXGDs modified by different sites of PTX were designed,TXGD-A,a single glycosylated conjugate at 2'-OH of C-13 side chain.In addition,TXGD-B,a double glycosylated conjugate at both 2'-OH of C-13 side chain and 7-OH of the Baccatin structure.Both the conjugates used succinate ester as linkers.Subsequently,the in vitro antitumor activity,solubility,lipid-water partition coefficient and other aspects of the synthesized TXGDs were studied.The results showed that TXGD-A may have better potential for drug development.Therefore,the further determination of TXGD-A tumor cell uptake rate and its interaction with GLUT-1 protein,the results of the research are roughly as follows:In this study,two PTX glucose conjugates,TXGDs were synthesized,including the single-site substituted conjugate TXGD-A and the double-site substituted conjugate TXGD-B.The glucose ring modification sites were C-1 hydroxyl groups,and the linkers were succinate esters,1H NMR,13C NMR and HRMS were used for identification of the target compounds,which can confirm that the target product has been successfully synthesized.Subsequently,the in vitro antitumor activity and related drug design parameters of TXGDs were determined.The results of in vitro tumor cell inhibition test showed that although inhibition against human breast cancer cells and ovarian cancer cells of TXGDS were weaker than the original drug PTX.The cytotoxicity of TXGDs were still significant and the IC50 can be maintained on nM level.The IC50 value of TXGD-A against human breast cancer cells and ovarian cancer cells were 148.75 nM and 1077.15 nM,respectively.IC50 values of TXGDs were lower than TXGD-B,suggesting that the inhibition of tumor cells by single-site substituted PTX glucose conjugates is stronger than that of double.Tumor cell apoptosis test results showed that TXGD-A induced human breast cancer cell MDA-MB-231 cells to apoptosis,21.23%of tumor cells are in apoptotic state,which promotes apoptosis.Apoptosis inducing effect of TXGD-A is significantly stronger than TXGD-B,suggesting that the single-point-substituted glucose conjugate has a stronger pro-apoptotic effect on tumor cells than the two-site-substituted glucose conjugate.TXGDs have less inhibitory effect on human normal mammary epithelial cells than PTX,which suggests that the glycosylation strategy of PTX will not cause additional toxicity to normal cells.The results of tubulin kinetics indicated that TXGDs have obvious tubulin-promoting effects.After different doses of TXGDs were administered,the OD values were significantly higher than that of the blank control group,suggesting that the glycosylation strategy can maintain similar anti-tumor mechanism to PTX,and the promotion of tubulin polymerization by PTX glucose conjugates substituted with different concentrations of single-site points in vitro.Compared to the PTX glucose conjugate substituted by the same concentration of double site,the OD value of the TXGD-A administrating groups could be increased to 0.391-0.507,which were significantly higher than the same concentration of TXGD-B.Based on the results of in vitro anti-tumor experiments,it can be suggested that the anti-tumor activity of TXGD-A is better than TXGD-B in promoting apoptosis of tumor cells and promoting the polymerization of tubulin.It may have superior potential for drug development than TXGD-B.The glucose metabolism activity of tumor cells is significantly stronger than that of normal cells,but its metabolic efficiency is extremely poor.The reduced glycolysis rate needs to be compensated by a large amount of glucose uptake,so the tumor cells have physiological characteristics of overexpression of glucose transporter GLUT-1.Beiing compared with passive uptake of cellular uptake,transporter-mediated cellular uptake is more specific and has a higher uptake rate.Therefore,GLUT-1 protein may be a potential anti-tumor therapeutic target.Thus,the tumor cell uptake rate and the degree of interaction with the glucose transporter GLUT-1 were measured in this study,with TXGD-A.Results of SPRi assay showed that TXGD-A could form a complex with GLUT-1 and its interaction degree was significantly better than that of the original drug PTX under different protein concentration environments.This result suggested that the interaction between the drug and GLUT-1 protein can be achieved after PTX was modified by glycosylation.Confocal microscopy was utilized to observe the localization of TXGD-A and PTX in human breast cancer cells MDA-MB-231.The morphological observation exhibited that both TXGD-A and PTX can be taken up by the tumor cells and mostly localized in the cytoplasm.The fluorescence area of TXGD-A was larger than that of PTX,suggesting that the uptake rate of TXGD-A by tumor cells was higher than that of PTX.The results of uptake rate determination exhibited that human breast cancer cells uptake more TXGD-A than the original drug PTX.The uptake rate of TXGD-A by MDA-MB-231 was higher than that of PTX.After 24 h of administration,the uptake rate of TXGD-A by cells was 35.03%,which was significantly higher than that of PTX.In order to investigate whether the uptake rate of TXGD-A by tumor cells is related to the expression of GLUT-1 protein,the cells were regulated by GLUT-1 protein expression level and the uptake rate of TXGD-A was measured.The results showed that the uptake rate of TXGD-A is affected by GLUT-1 protein level.After upregulation/downregulation of GLUT-1 protein expressiing level,uptake rate of TXGD-A could be increased/decreased to 60.52%/14.85%.The binding site of TXGD-A and GLUT-1 can be calculated by computer simulation.The main binding site may be the glutamine residue at position 161 of GLUT-1 protein(GLN161).And the glutamic acid residue at position 380(GLU380),the binding mode is hydrogen bonding.In addition,a variety of physical and chemical properties were determined for the synthesized TXGDs.The results showed that the solubility of TXGDs in pure water and different clinical use injection media was significantly better than that of PTX.The solubility of TXGD-A in pure water and clinical injection medium reached 44.6-49.8 times of original drug PTX.The solubility of TXGD-B in the three media can be increased to 86.9-97.4 times of the original drug PTX,indicating that the glycosylation strategy can significantly improve the solubility of PTX.Compared to PTX,TXGDs showed lower lipid water distribution/distribution coefficient.After glycosylation modification,the lipid water distribution/distribution coefficient Log P and Log D of TXGDs fell to the range of 0-3,which were in adequate ranges of drug formation,indicating that glycosylation strategy can significantly improve the hydrophilicity of PTX.It is more suitable for the development of new drugs.TXGDs can partially release the original drug PTX in fetal bovine serum and rabbit serum,showing certain prodrug characteristics.Comparing the two products,TXGD-A released 17.57%and 17.87%PTX in fetal bovine serum and rabbit serum after 24 h,and the PTX release rate was higher than TXGD-B.The calculation results of TXGDs showed that the drug design parameters can be optimized after glycosylation of PTX.The fit quality of the the ligand-receptor conformity(FQ)of TXGD-A and TXGD-B for human breast cancer cells can be increased to 0.225 and 0.246,respectively,and the FQ for human ovarian cancer cells is increased to 0.314 and 0.296,respectively.From the perspective of lipophilicity and ligand affinity,the lipophilic ligand efficiency of TXGD-A and TXGD-B against human breast cancer cells can be reduced to 72.860 and 57.144,respectively,and LELP for human ovarian cancer cells is reduced to 52.200 and 47.616 is also superior to the original drug PTX.The solubility of TXGD-A in pure water and clinical injection media is affected by pH,its solubility is improved in the environment of pH 9-10,but it can be maintained in the environment of pH 1-8.In the interval of about 16-20 ?g/mL,there is no obvious fluctuation,suggesting that TXGD-A does not cause abnormal increase of drug concentration in a certain tissue in different tissues due to different environmental pH in different tissues.Results of solvent system screening showed that the solubility of TXGD-a was 806.67 ?g/mL in the 2.5%alcohol-free solvent system of Crmophor EL,indicated that the solubility of TXGD-A can be well solubilized in the alcohol-free system with low Cremophor EL ratio,and TXGD-A has potential for clinical application.It can reduce the proportion of the use of toxic surfactants,reduction of the probability of adverse reactions caused by the toxicity of the solvent,and exhibited certain clinical development and application value.In summary,the glycosylation strategy can improve solubility,hydrophilicity and drug design parameters of PTX,and is a feasible modification strategy for PTX.TXGD-A is not only enhancing the interaction between PTX and GLUT-1 protein,thereby promoting the uptake of drugs by tumor cells,but also effectively reduces the amount of toxic solubilizer in the solvent,suggesting that TXGD-A has the prospect for secondary development of PTX.