Evaluation Of Anti-Tumor Efficacy Of FAP?-Activated Prodrugs Of BF211 In Vitro And In Vivo

Research Background:Chemotherapy is one of the most important means of cancer treatment,but many clinical anti-tumor drugs will cause the severe toxicities to normal tissues despite its potent antitumor activity,which are attributed to to the lack of selectivity toward tumor.These factors greatly limit the clinical application of these chemotherapeutic drugs.With continuous clarify of the pathogenesis of cancer,tumor-targeted therapy has gradually become a new direction of cancer treatment.Finding a suitable drug target is critical for tumor-targeted therapy.Tumor-associated fibroblasts belong to the interstitial cells of the tumor and can promote tumor development by direct or indirect means.The fibroblast activating protein a(FAPa)is a membrane serine protease expressed by activated tumor-associated fibroblasts,almost not expressed in tumor cells or normal tissues.FAPa has endopeptidase and collagenase activity,dissolving gelatin and collage.FAPa plays an important role in tumor microenvironment remodeling,participating in the process of tumorigenesis,development,invasion and metastasis.Therefore,tumor-associated fibroblasts(TAFs)and fibroblast activating proteins a are attracting attention as a target for tumor targeted therapy.Tumor-targeting methods based on FAPa are mainly about immunotherapy,enzyme activity inhibitors and FAPa-activated prodrugs.Small molecule modificated with bnzyptide(Z-Gly-Pro)which FAPa reads specifically and slices selectively can effectively target the tumor site and reduce the adverse effects of drug treatment,because cytotoxicity could be largely reduced through the decoration of Z-Gly-Pro and its potential cytotoxicity could be restored by FAPa-posotive tumor.BF211,a derivative of bufadienolides,exhibits a strong an-titumor effect in preclinical studies.This compound showed potent anti-cancer activities to a broad spectrum of tumor cell lines with nano molar level IC50 values,bring the phenomenon of complete disappearance of tumor in many transplanted tumor models.BF211 as a molecules with high anti-tumor activity have severe cardiac toxicity and neurotoxicity,so the transformation of BF211 for tumor targeting is necessary for its drug developing.According to this,we collaborated with the researcher of the Shanghai Institute of Materia Medica,Chinese Academy of Sciences,to synthesize BF211 series prodrug by using the N-terminal blocked glycine proline dipeptide sequence(Z-GP),which can be specifically hydrolyzed by FAPa,in order to find a compound which can effectively target the tumor site and improve the toxicity side effect of BF211 treatment.Research purposes:The aim of this study is to find a compound with high FAPa,-enzymatic efficiency and big hydrolytic differences between tumor and normal tissue homogenate by screening 14 FAPa-activated prodrugs by using rhFAP and tissue homogenate.In vitro and in vivo anti-tumor pharmacodynamic evaluation will be carried out focusing on the pilot compounds.Research methods:1.FAPa-activated BF211 series of prodrugs were evaluated by rhFAPa digestion and tumor/cardiac homogenate incubation in the aim of finding a compound that could be specifically and highly hydrolyzed by tumor tissue.2.HPLC method was used to detect compound's content and stability in plasma,tissue homogenate,cell culture fluid.3.MTT assay was used to detect the inhibitory effect of the compounds on the growth of tumor cells and cardiomyocytes.4.The Construction of FAPa-overexpressling cell lines by retroviral method.5.The anti-tunmor activity of the compounds in vivo was evaluated using the HCT-116 xenograft mode.Experimental results:1.The results of enzymatic digestion of rhFAPa showed that BF211-03 have the highest hydrolyzed efficiency in the 14 compounds and the hydrolyzed efficiency increased in a time-dependent manner,indicating that BF211-03 was a good cleavage substrate for FAP?.2.In the tumor and cardiac tissue homogenate incubation experiments,BF211-03's lysis rate in the tumor homogenate is very high(80%to 100%),however,just a little cracking(30%)occur in heart homogenate.BF211-03's hydrolyzed efficiency difference between the tumor and heart Homogenate was the most obvious.BF211-03 is the most selective substrate for FAP.3.BF211-03 show good stability in the plasma and FAPa-negative normal tissue homogenate(except for the liver).4.Co,pared with BF211,the inhibitory effect of BF211-03,obtained by modification of BF211 by dipeptide,was significantly attenuated.In FAP?-negative tumor cell lines(HCT-116,MGC-803),a small amount of BF211-03 was cleaved to release BF211,BF211-03 in these two cells showed lower cytotoxic than BF211(30 to 40 Times difference).For the FAPa-positive cell line MDA-MB-435,nearly half of BF211-03 can be cleaved to release BF211,cytotoxic difference between BF211 and BF2111-03 was 4 times.These results indicate that BF211-03 is more toxic to FAP-positive cell lines.5.rhFAP,stable-contructed FAPa cell line or tumor homogenate incubation can restore the potential toxicity of BF211-03.6.Anti-tumor efficacy evaluation in vivo showed that BF211-03's anti-tumor effect was similar to BF211 with the double dose of BF211,but BF211-03's the toxic side effects was more slight than BF211.Conclusion:The results of this study show that the toxicity of BF-211 is effectively hidden after being modified to BF211-03,and BF211-03's potential cells toxicity can be effectively restored through releasing the original drug BF211 under FAPa selective enzymolysis;BF211-03 showed good stability in normal organ tissue homogenate,and most of BF211-03 was activated into BF211 in the tumor tissue homogenate;In vivo anti-tumor efficacy evaluation,BF211-03 can achieve similar effect of tumor inhibition but it present reduced side effect.This study provides a study of FAPa-activated prodrugs and provides experimental evidence for the feasibility of FAPa-based targeted therapy strategies.