Design, Synthesis And Activity Evaluation Of Tumor-targeted Camptothecin Prodrugs Based On Prostate Membrane Antigen

Background and objective:Cancer is one of the major diseases which severely threat human life and health,its pathogenic factors are complex,morbidity and mortality are high.Chemotherapy is still one of the main methods in the clinical treatment of malignant tumors.Most of the conventional chemotherapeutic drugs have the defects of poor targeting,toxic and side effects,low bioavailability and drug resistance,which leads to poor clinical efficacy and poor quality of life of patients.Therefore,it is of great clinical value and significance to find new targeted antitumor drugs.Camptothecin is a natural antitumor alkaloid isolated from Camptotheca acuminata,which demonstrated remarkable antitumor effect on gastric cancer,colorectal cancer,bladder cancer and other malignant tumors in vitro and in vivo.A variety of widely used broad-spectrum anticancer drugs have been successfully developed based on camptothecin.However,these drugs have poor targeting and often cause a series of toxic and side effects such as vomiting,diarrhea,bone marrow suppression and hemorrhagic cystitis.In addition,these drugs also have the defects of poor water solubility and poor stability.Prostate specific membrane antigen(PSMA)is a type II transmembrane glycoprotein located on the surface of prostate epithelial cells.Studies have shown that it is specifically expressed in prostate tissues and a variety of non-prostate solid tumors vascular endothelial cells,but not in normal tissues.In addition,PSMA also has the activities of folate hydrolase(FOLH),glutamate carboxypeptidase ?(GCPII)and N-acetylated a-linked acid dipeptidas(NAALA-Dase).PSMA is a good target and research hotspot for tumor diagnosis and anti-vascular targeted therapy.In this study,under the guidance of the principle of prodrug,a series of tumor-targeted camptothecin prodrugs were designed and synthesized for the first time using the specific hydrolyzed polypeptide substrate of PSMA as tumor-targeted ligand.The antitumor activity,targeting and toxicity of prodrugs in vitro and in vivo were systematically evaluated by using tumor cell model in vitro,recombinant human PSMA and xenograft tumor model in nude mice in vivo.This study aims to discover new camptothecin lead compounds with high efficiency,low toxicity,strong targeting,clear mechanism of action and prospect of clinical application.We hope our study can provide a new idea for the secondary development of other cytotoxic drugs.Research content:Based on the principle of prodrug,twelve water-soluble tumor targeting camptothecin prodrug CPT-X were elaborately designed and synthesized by coupling tumor-specific targeting ligands,PSMA hydrolyzing substrate(Asp-Glu*Glu*Glu*Glu(WT-H),Asp-Glu*Glu*Asp-Glu(HT-H)and Glu*Glu*Glu*Asp-Glu(HT-J)("*" mean the y-glutamyl linkage;"-" mean the a-glutamyl linkage),to the 20-OH of CPT through different ligation fragments.To better study the PSMA-targeting ability of the prodrug CPT-X,the PSMA hydrolysate CPT-MX were prepared as well;The PSMA-expressing human prostate cancer cell LNCaP-FGC and non-PSMA-expressing cells(MCF-7,HepG2,Hela,PC-3,DU145)and normal hepatocytes(L02)were used to evaluate the cytotoxicity and PSMA targeting of CPT-X and CPT-MX;Meanwhile,the selective cytotoxic and PSMA-targeted activity of the prodrug CPT-W12were further verified by flow cytometry and laser confocal microscopy;The in vitro targeting release and in vivo targeting distribution characteristics were evaluated on the recombinant human PSMA and human breast cancer MCF-7cells in nude mice by the high performance liquid chromatography(HPLC)and small animal imaging system;The pharmacokinetics of CPT-W12in mice was evaluated on the BALB/c mice;The antitumor activity and safety of CPT-W12in vivo were systematically evaluated by using the xenograft tumor model of human breast cancer cell line MCF-7in nude mice;To explore the possible anti-tumor mechanism of the prodrug CPT-W12,immunohistochemical staining was used to evaluate the effect of CPT-W12on the expression of Ki-67,Caspase3,Bcl-2and MMP-2.Results:(1)Twelve new water-soluble tumor-targeted camptothecin prodrugs CPT-X and twelve PSMA hydrolysates CPT-MX were successfully designed and synthesized.(2)The cytotoxic activity and PSMA targeting study in vitro showed that the prodrug CPT-X showed moderate to potent cytotoxicity against all the tested tumor cell lines;However,their cytotoxicity was much lower than the parent compound CPT;Most of the prodrug CPT-X exhibited significantly stronger anti-proliferative activities against the PSMA-expressing cell line LNCaP-FGC than the non-PSMA-expressing cell lines MCF-7,PC-3,DU145,HepG2and Hela,but they exhibited low cytotoxicity towards human normal hepatocytes L02,showing excellent cytotoxicity selectivity and PSMA targeting activity;Flow cytometry and laser confocal fluorescence observation further confirmed the good cytotoxicity selectivity and PSMA targeting of prodrug CPT-W12,which exhibited significantly higher apoptosis-inducing activity against PSMA-expressing LNCaP-FGC cells than the non-PSMA-expressing HepG2 cells;Meanwhile,CPT-W12 could effectively accumulated in LNCaP-FGC(PSMA+)meanwhile minimized in HepG2(PSMA-)cells.(3)The stability,targeting in vitro and in vivo and pharmacokinetics experiments demonstrated that the prodrug CPT-W12had good PSMA sensitivity and buffer salt stability,which could be specifically hydrolyzed by rh PSMAAnd CPT-W12 also exhibited good targeting in vivo,it could effectively accumulate and retain in the tumor site;Meanwhile the half-life of CPT-W12 was significantly longer than that of CPT in vivo.(4)The antitumor activity study showed that the prodrug CPT-W12 exhibited excellent antitumor effect on the transplanted tumor model of human breast cancer MCF-7 cells in nude mice;The tumor inhibition rates of low dose CPT-W12(10mg·kg-1),middle dose CPT-W12(30 mg kg-1)and high dose CPT-W12(60mg kg-1)were 51.67%,57.78%and 65.00%,respectively;The tumor inhibition rate of the middle dose CPT-W12 group was similar to that of the positive drug irinotecan group(10mg kg-1),and the tumor inhibition rate in the high dose CPT-W12 group was better than that of the irinotecan group;Blood biochemical and histopathological studies showed that prodrug CPT-W12 could significantly reduce the nephrotoxicity of camptothecin;Even if the dosage of CPT-W12 was six times of that of irinotecan,no obvious systemic toxicity was found.(5)The study of antitumor mechanism in vivo showed that prodrug CPT-W12could effectively decrease the expression of Ki-67 and Bcl-2protein,increase the expression level of Caspase-3 protein,inhibit the proliferation of tumor cells and promote the apoptosis of tumor cells.In addition,CPT-W12 can also reduce the expression of MMP-2,inhibit tumor metastasis and tumor angiogenesis.Conclusion:In this paper,12 kinds of water-soluble PSMA targeting camptothecin prodrug CPT-X were successfully designed and synthesized based on the principle of prodrug.It showed strong cytotoxicity selectivity,PSMA targeting and stability in vitro.Meanwhile,it demonstrated strong anti-tumor activity,tumor targeting and safety in vivo,which could effectively accumulate in tumor site and act the anti-tumor effect.CPT-W12 showed similar anti-tumor activity to irinotecan without caused obvious systemic toxicity.The prodrug CPT-X successfully overcome the defects of poor water solubility,stability and targeting of camptothecin,and significantly prolong the half-life of camptothecin.This study proves that the tumor-targeted drug design strategy based on PSMA is effective and feasible.This study is expected to provide a new idea for the development of traditional cytotoxic drugs,especially some insoluble cytotoxic drugs in traditional Chinese medicine.At the same time,it is expected to provide a new molecular targeted drug for the treatment of solid tumors such as breast cancer.