Evaluation Of The Tumor Targeting Of FAPα-activated Prodrug Z-GP-Dox

Study Background and ObjectivePresently, in clinical chemotherapy of cancer, the majority of anti-tumor drugs used are cytotoxic agents. In spite of the potent antitumor activity, those therapeutic drugs are usually restricted in clinical application due to the toxic side effects. Accordingly, the targeted delivery of antitumor agents is indispensible for reducing adverse drug reaction. Recent studies have demonstrated that fibroblast activation protein-a (FAPa) is a tumor-associated antigen uniquely expressed by reactive stromal fibroblasts, and shows essential functions to regulate the growth and development of tumors. FAPa also possesses endopeptidase activity toward N-blocked glycyl-prolyl aminos acid substrates, such as substrates with N-terminal of Z-Gly-Pro (Z-GP). Hence, our research group previously utilized the specific dipeptide Z-GP to decorate doxorubicin (Dox) for the synthesis of FAPa-based prodrug Z-GP-Dox, with the purpose of improving its tumor targeting and reducing its toxic side effect, especially cardiotoxicity. Previous in-vivo study revealed that Z-GP-Dox produced favorable antitumor efficacy in the FAPa-positive tumor model of 4T1 mouse breast cancer, and also showed low cardic toxicity. Therefore, the aim of current work was to validate the tumor targeting of this FAPa-activated prodrug and to study the antitumor pharmacological mechanism, which would offer a foundation for the application of such FAPa-targeted therapeutic strategy. It also would be helpful to establish a research mode for the targeting evaluation of FAPa-based prodrug candidates.Methods1. FAPa-based release of Dox from Z-GP-DoxZ-GP-Dox was incubated in the buffer with FAPa for 24 h at 37℃. And the release of Dox and the enzymolysis property were analyzed by HPLC. Also, the cleavage of Z-GP-Dox with FAPa-positive 4T1 tumor homogenate and the stability of Z-GP-Dox in mouse plasma or normal tissue homogenates were respectively detected by HPLC.2. Measurement of FAPa-based cytotoxicity of Z-GP-DoxCytotoxicity of Z-GP-Dox or Dox at various drug concentrations from 0.01μM to 10μM was respectively detected by CCK-8. Then, cell viability assay was performed using CCK-8 to measure the antitumor activity of Z-GP-Dox incubated with FAPa.3. Preliminary toxicological assessment of Z-GP-Dox Parallelly compared to Dox with gradual doses from 8.0 mg/kg to 25.3 mg/kg, the acute toxicity of Z-GP-Dox at the maximum dose of 25 mg/kg by intravenous administration was evaluated in KM mice. And the organ toxicities of Z-GP-Dox and Dox were analyzed respectively by pathological study.4. Drug distribution of Z-GP-Dox in heart and tumor tissueThe 4T1 tumor-bearing mouse model was established. Z-GP-Dox and Dox were respectively administered to the model mice (n=5) at the single dose of 13.77μmol/kg via tail vein. Mice were sacrificed, respectively at 1 h,4 h, and 12 h after administration. And tissues including heart and tumor were collected to determine the drug level in tissue by HPLC.Results1. Enzymolysis study implied that FAPαcould selectively cleave Z-GP-Dox to release Dox and this hydrolysis was highly dependent on the concentration of FAPα. Also, Z-GP-Dox incubated in 4T1 tumor homogenate was rapidly cleaved to release Dox, while Z-GP-Dox treated in normal tissue homogenates or mouse plasma showed favorable stability.2. Cytotoxicity assay indicated that Z-GP-Dox demonstrated significantly lower cytotoxic effect on 4T1 cells than Dox. However, Z-GP-Dox could restore its potential antitumor activity by FAPα-based enzymolysis.3. Acute toxicity study showed that the medial lethal dose (LD50) of Dox was 13.4 mg/kg, while no individual death was observed in the group with Z-GP-Dox at maximum dose of 25 mg/kg. Pathological analysis indicated that the pathological effect of Z-GP-Dox (25 mg/kg) on normal organs was obviously lower than Dox (8.0-14.2 mg/kg), suggesting that the toxicity of Dox was obviously reduced through the modification with Z-GP dipeptide.4. Drug distribution study implied that drug level of Z-GP-Dox in heart tissue was significantly lower than Dox during 12h after administration (p<0.01), and Z-GP-Dox also showed higher tumor/hear ratio of drug accumulation than Dox respectively at 1 h,4 h, and 12 h.ConclusionsOur current work suggested that Z-GP-Dox could be specifically cleaved to release Dox by FAPα, and Z-GP-Dox would restore its potential cytotoxicity through FAPα-based enzymolysis. It was also revealed that the specific decoration with Z-GP dipeptide could significantly reduce the toxic effect of Dox on normal organ and improve its tumor targeting.