| Background and Objective:Epirubicin(EPI) is a clinically classic anthracycline-based broad-spectrum antitumor drugs, and was limited widespread clinically application for its greater cardiotoxicity. In recent years, the methods employed pharmaceutical chemistry to change the chemical structure of drugs eventually reduces its toxic effects, which become more and more popular. Meantime, other methods, for example, the enzyme-activated targeting antitumor prodrug design strategy also develop maturely.There are large quantity of research data from domestic and foreign shows that fibroblast activative protein alpha(FAPα) expressed in tumor-associated fibroblasts, and tumor genesis and development were closely associated with tumor fibroblasts. FAPα Enzyme-activated targeting antitumor prodrug design strategy take advantage of the FAPα around tumor cells as a breakthrough that will alter the parent drug with greater side effects to the prodrug with almost no side effects and the prodrug is exclusively hydrolyzed by FAPα specific enzyme. As lack of the necessary tools--FAPα enzyme, Z-GP-EPI can’t play cytotoxic effects when reaching the normal tissue in vivo, which achieve the desired purposes of reducing the side effect. When arriving directly or circulating in tumor tissue in vivo, Prodrug will be hydrolyzed to release the parent drug and play the anti-tumor effect.In the early experimental study, we have successfully applied this FAPα Enzymeactivated targeting antitumor prodrug design strategy to Doxorubicin(DOX), which was validated viability through systems medicine studies. In recent years, the toxicity particularly cardiotoxicity of EPI remains unresolved and the EPI, has more wide application potential compared to DOX, is DOX’s cis-trans isomer, only one chiral carbon atom of a different conformation. Thus, this enzyme-activated targeting antitumor prodrug design strategy will be applied to EPI as a meaningful effort. Early experiments have been modified the parent drug EPI to Z-GP-EPI, however, the ability to achieve targeted anti-tumor effect as well as reduce cytotoxicity, still need to experiment to investigate.Therefore, this experiment investigates the targeting antitumor efficacy and cardiotoxicity of prodrug Z-GP-EPI to evaluate its druggable initially. Experimental Method: 1 Targeting antitumor efficacy of Z-GP-EPI 1.1 Antitumor activity in vitroUse of non-tumor cells(HEK-293, NIH-3T3), tumor cells(4T1) and transfected stable high expression FAPα cells(NIH-3T3/FAPα+, 4T1/FAPα+), to the parent drug as a control, the use of MTT method study the cytotoxicity of Z-GP-EPI at a concentration of 0.15- 50 μM.Based on our previous experimental basis, confirming HPLC chromatography detected the Z-GP-EPI and parent drug EPI at the same time.(Mobile phase: Acetonitrile(A)- water(containing 0.1% TFA)(B), gradient: 0-1min, A is 30%; 1-5min, A is 30%-70%; 5-11 min, A is 70%; flow rate: 1 m L / min; UVD wavelength: 495 nm).Confirming the enzymolysis activity of recombinant human FAPα through making use of Z-GP-DOX based on our previous experimental basis. Further investigating whether the Z-GP-EPI can be specific enzymolyzed by rh FAPα or not. Z-GP-EPI(15 μg / m L) and rh FAPα(1 μg / m L) were incubated at 37°C for 2 h, 4 h, 8 h, 12 h, 24 h and 36 h then determinated the Z-GP-EPI and EPI simultaneously in supernatant via HPLC method. 1.2 Antitumor efficacy in vivoConstructing Bal b/c tumor-bearing mice were treated with breast carcinoma 4T1 cells, the model animals were randomly divided into Z-GP-EPI high and low dose groups(3 mg/Kg and 1.5 mg/Kg) and equimolar dose EPI high and low dose groups(2 mg/Kg and 1 mg/Kg) and Control group(10 m L/Kg) total five groups of six, is administered by intravenous injection every other day for 3 weeks. Observing and measuring tumor size on the every other day, making tumor growth curve, calculating relative tumor inhibitory rate. 22 d after the first administration the mice were killed by cervical dislocation method, stripping tumor masses, weighed to calculate the inhibition rate. 2 preliminary study Z-GP-EPI cardiotoxicityThe Bal b/c mice were randomly divided into Z-GP-EPI high and low dose groups(15 mg/Kg and 7.5 mg/Kg), EPI group(5 mg/Kg) and Control Group(20 m L/Kg) total four groups of eight, is administered by intraperitoneal injection every other day for 2 weeks. Observing mice survival status, recording the deaths in mice and calculating the survival rate, measuring body weight and making growth curve. On 14 d after first administration, mice were sacrificed by cervical dislocation method after femoral artery blood approximately 1 m L, centrifuging and testing myocardial enzyme. The cardiac tissues were examinated by HE staining, Massion staining and 3-nitrotyrosine immunohistochemical staining. Experimental Results: 1 Targeting antitumor efficacy of Z-GP-EPI 1.1 Antitumor activity in vitroAccording to the cells survival data of 48 h, the IC50 of Z-GP-EPI to non-tumor cells(HEK-293, NIH-3T3) was more than 50 μM, while the IC50 of EPI was less than 1 μM. To stable high expression FAPα cells(NIH-3T3/FAPα+, 4T1/FAPα+), the IC50 of Z-GP-EPI and EPI were nearly close(about 0.2 μM). Z-GP-EPI specific enzymolysis results showed detectable of free EPI in 2 h and about 40% Z-GP-EPI was enzymolyzed at 24 h while about 95% at 36 h. 1.2 Antitumor efficacy in vivoThe tumor volume of Z-GP-EPI high and low dose groups have statistically significant compared with Control group. And relative tumor inhibition rate were less than 40%, and Z-GP-EPI low dose group was with a similar inhibitory rate(about 55%) compared to equimolar dose EPI group. 2 preliminary study Z-GP-EPI cardiotoxicityThe Z-GP-EPI high and low dose groups showed no mice died, no body weight decrease significantly. Several mice in EPI group died on 10 d since first administration.To the administration end, survival rate of EPI group was 50%, and body weight decrease significantly. The results of myocardial enzyme test showed that there was no significant difference in the Z-GP-EPI high, low dose groups compared to Control group, while EPI group had a significant difference compared to Control group. Cardiac HE examination, Massion inspection and 3-nitrotyrosine immunohistochemical findings showed, Z-GP-EPI high and low dose groups found no significant lesions, while EPI group found that myocardial morphological changes, dissolution and other obvious lesions. In conclusion: 1. Z-GP-EPI has almost no toxic effects to normal cells, and could be enzymolyzed by FAPα specific enzymes to release parent drug EPI so exert cytotoxic effects, and has a similar resistance compared to parent drug in an animal model used in this tumor efficacy study. 2. Z-GP-EPI has clearly improved cardiotoxicity compared to parent drug. To sum up, it can be indicated that the FAPα enzyme-activated targeting prodrug anti-tumor design strategy was modified to prodrug Z-GP-EPI, retains the anti-tumor efficacy of the parent drug, while significantly improved cardiac toxicity, which has been reached the intended purpose of the policy modification. Therefore, Z-GP-EPI, as like Z-GP-DOX, is expected to become the new FAPα enzyme-activated targeting anti-tumor candidate compounds, entering preclinical research and development next. |
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