Design And Evaluation Of Prostate Cancer Targeting Prodrugs

Chemotherapy is the most widely used approach to combat advanced prostate cancer(PCa),but its therapeutic effect is often unsatisfactory due to lack of specificity and toxicity.Paclitaxel(PTX),a mitotic inhibitor,is the first line chemotherapy for PCa treatment.However,the clinical efficiency of PTX was greatly restricted by the significant side effects,which would be attributed to the non-specific biodistribution in the body.Prostate-specific membrane antigen(PSMA)is overexpressed on the surface of nearly all PCa cells.We utilized DUPA as the targeting ligand to deliver PTX actively for treatment of PSMA expressing PCa.PTX-SS-DUPA was developed by conjugating DUPA with PTX,using a reduction-sensitive disulfide bond as a linkage to facilitate a rapid release of PTX in tumor cells.An ester prodrug(PTX-DUPA)was synthesized as the non-sensitive conjugate.The tumor-targeting drug delivery and tumor-specific drug release would lead to improved antitumor efficacy and reduced toxicity.Firstly,PTX-SS-DUPA was developed by conjugating DUPA with PTX,using a reduction-sensitive disulfide bond as a linkage.An ester prodrug(PTX-DUPA)was synthesized as the non-sensitive conjugate.The structure of PTX-SS-DUPA and PTX-DUPA was verified by 1H NMR and HRMS.The release results suggested that the drug release from ester prodrug(PTX-DUPA)was extremely slow,and PTX-SS-DUPA had a remarkable redox-responsive drug release.To investigate the mechanism of DTT-triggered drug release,the change of molecule weight was analyzed by HPLC and HRMS.All PTX-SS-DUPA prodrugs were reduced within 2 h after addition of DTT.The breakage of the disulfide bond generated a thiolcontaining intermediate(PTX-SH)which would eventually release free PTX.PTX-SS-DUPA(IC50=121.1 nM)was less potent than PTX(IC50=14.25 nM)in 22RV1 cells,possibly because of the delayed release of PTX.The high efficacy of PTX-SS-DUPA was quantitatively inhibited by excess PMPA,which suggested that cytotoxicity required PSMA receptors.By contrast,PTX-DUPA(IC50=563.3 nM)exhibited poor cytotoxicity within the studied range of drug concentrations,due to the extremely slow hydrolysis rate of PTX.PTXSS-DUPA exhibited inferior cytotoxicity in PSMA negative PC-3 cells,showing no significant difference compared with PTX-DUPA at the concentration of 500 nM.PSMA receptor was robustly expressed by 22RV1 cells and complete absence of staining observed in PC-3 cells.To confirm the contribution of PSMA to the transport of the targeted DUPA-drug conjugates,PSMA-positive 22RV1 cells were used to evaluate the cellular uptake efficiency.PTX concentration in PCa cells was determined by UPLC-MS/MS.The PTX concentration in cells derived from PTX-SS-DUPA and free PTX solution was markedly higher than PTX-DUPA.Besides,the cellular uptake of PTX-SS-DUPA was quantitatively inhibited by excess PMPA,indicating PMPA effectively prevented PTX-SS-DUPA from binding to PSMA,thereby blocking the cellular uptake of PTX-SS-DUPA.The content of PTX in major organs was determined using UPLC-MS/MS.The content of PTX in tumors of PTX-SS-DUPA group was higher than that in other organs.The potential value of cancer targeting by PTX-SS-DUPA would be derived from its lack of toxicity in other organs,which might make it a less toxic anticancer drug than PTX.The PSMA-targeting moiety DUPA significantly changed the biodistribution and leaded to much higher accumulation of drug in tumor compared with other major organs due to the selective uptake by PCa cells.We further investigated the in vivo anti-tumor activities of PTX-SS-DUPA in mice xenograft models bearing 22RV1 tumors.The mice treated with PTX-SS-DUPA displayed a significant decrease in tumor volume,showing no significant difference compared with PTX.PTX-SS-DUPA treated mice had a significant reduction in PSA levels.A robust and significant increase in cell apoptosis was observed in tumor samples treated with PTX and PTX-SS-DUPA,.Additionally,there was no significant change observed in body weight or in the hematological parameters between treated and control groups.Furthermore,no noticeable histological abnormalities were observed in H&E staining of major organ sections,indicating that the DUPA conjugates treatments were well tolerated.Increased MAOA expression is associated with PCa progression and metastasis.MAOA inhibition dramatically restricts metastasis and extended survival in PCa xenograft mouse models.NIRF heptamethine cyanine dye(MHI-148)is utilized as a useful tool for personalized oncology to simultaneously target cancer and noninvasively image tumors without chemical conjugation.Given these results,we synthesized a novel compound that combines a NIRF dye and an MAO A inhibitor to selectively accumulate in PCa cells.The NIRF dye MHI-148 was conjugated with the terminal amino group of isoniazid(INH).NIR-INH has PCa targeting,imaging and high anticancer effectiveness,suggesting it is a potentially valuable image-guided anti-tumor strategy.The conjugation of MHI-148 with INH did not change the optical properties of MHI-148,and the fluorescence intensity of MHI-148 and NIR-INH maintained at a stable level for 12 h.The anticancer activity of INH,NIR-INH,and MHI-148 against PC-3 cells was investigated by an MTT assay.Evaluation of IC50 values indicated that the conjugate NIR-INH(IC50=33.81μM)had 4.2-fold higher cytotoxicity than the MHI-148 dye(IC50=141.46 μM)in inhibiting PC-3 cells growth.NIR-INH treatment caused a robust increase in apoptotic cells,as compared to MHI-148 and INH treatment.To investigate subcellular localization,PC-3 cells were costained with MitoTracker and MHI-148/NIR-INH conjugates.The CLSM images indicated that MHI-148 and NIR-INH accumulated in PC-3 cells and localized in cellular mitochondrias,as measured by colocalized with MitoTracker.The uptake of NIR-INH and MHI-148 were markedly decreased when incubated at 4℃ and the cell uptake is mainly mediated by OATPs.In vivo distribution of INH,MHI-148 and NIR-INH was examined in PC-3 tumor bearing nude mice.The mean fluorescence intensity of tumors was stronger than other organs except lung at 48 h.The selective tumor accumulation supports the use of NIR-INH for tumor localization,and thus,it could provide a precise imaging-guided cancer therapy.NIR-INH treated mice had a significant reduction in PSA and MAOA level compared with INH.A robust and significant increase in cell apoptosis was observed in tumor samples treated with NIR-INH,as compared to both INH and MHI-148 treatment.Additionally,there was no significant change observed in body weight or in the hematological parameters between treated and control groups.Furthermore,no noticeable histological abnormalities were observed in H&E staining of major organ sections.This confirms that the NIR-INH conjugate has potent antitumor activity and low treatment-induced toxicity.