Design And Synthesis Of Tumor-targeting Doxorubicinprodrugs And Research On Their Activity

AimMalignant tumor is a severe threatens to human healthy and survival, which is a main cause of disease leading death. Doxorubicin is a broad-spectrum antitumor drug of anthracyclines, which is often an essential component of treatment of breast cancer, children solid tumor, soft tissue sarcomas, and aggressive lymphomas. However, the clinical use of is limited by such serious problems as the development of resistance in tumor cells or toxicity and reverse side effect in healthy organs during the treatment, for example: chronic cardimyopathy and congestive heart failure (CHF), kidney damage, myelosuppression and so on. Our research chose doxorubicin as pristine drug, based on design strategy of prodrugs, synthesis a series of tumor-targeting doxorubicin-prodrugs which were in combination with polymer or small molecular compounds showed tumor-targeting effect; the molecular structure and their characteristic in vitro were identified by NMR, IR, MS, HPLC etc; the tumor targeting activity and their antitumor effect in vivo and vitro were investigated by choosing tumor bearing mice and tumor cells as models, and because of the fluorescent feature of doxorubicin, using fluorescent microscope and flow cytometry as analytical methods; investigated the pharmokenitics charatistics and biodistribution of all the three prodrugs by HPLC/MS/MS, our research could apply experiment and theory foundation for increasing therapeutic effect and reducing toxicity of doxorubicin, and also open a new idea for the further clinical use of doxorubicin.MethodsWe have synthesized a series of DOX prodrugs under nitrogen protection, monitored the reaction and products by TLC, and observed the react progress and the purity of products by UV lights with silica gel plates. Polyethylene glycol 6000 (PEG) was oxidized to polyethylene glycol 6000 dioic acid(compound 2) through KMnO4;α-linoleic acid or polyethylene glycol 6000 dioic acid was conjugated to doxorubicin structure via amino group at C-3'with EDCI or DCC/DMAP as catalyzer to obtain DOX-LNA or PEG-DOX amid conjugate(compound 1 and 3); polyethylene glycol 6000 dioic acid was reacted with BOC protected hydrazine to produce polyethylene glycol 6000 dioic acid hydrazide(with BOC protected)(compound 4) catalyzed by EDCI, and then got rid of BOC group to obtain polyethylene glycol 6000 dioic acid hydrazide(compound 5); PEG-DOX hydrazone(compound 6) conjugate was obtained with addition of hydrazone bond at C-13 carbonyl group catalyzed by EDCI too; melting points of each products were detected by melting point meter, and characterization were undertook by NMR, MS,IR, HPLC methods respectively to see if target compounds were obtained. Set up HPLC conditions, analyzed the doxorubicin content of prodrugs from the quantity of aglycone, hydrolyzed product of DOX, and meanwhile investigated the in vitro release performance.HepG-2(hepatoma carcinoma cell), MCF-7 and MDA-MB-231(breast carcinoma cell ) cell lines were cultured and incubated determinate time with DOX, DOX-LNA, PEG-DOX amid and PEG-DOX hydrazone at different concentration, for the significant fluorescence feature of DOX, the intracellular distribution of drugs were studied using fluorescence microscope and the uptake efficiency of DOX and its prodrugs by tumor cells were semiquantitatively analyzed by flow cytometry, established HPLC-MS-MS analysis methods of DOX for biological samples, then detected the difference in uptake of different prodrugs between different cells; the IC50 values of different drugs to the three selected cell lines were calculated in combination with the formula of cell growth inhibition rate by MTT methods, thereby evaluated the difference of in vivo antitumor activity between the prodrugs and free DOX; the apoptosis induced by drugs were evaluated by transmission electron microscope through morphology change; inoculated the MDA-MB-231 cell with logarithmic growth phase to athymic mice under armpit to establish tumor-bearing models, treated the animals with DOX(positive group), DOX-LNA and PEG-DOX hydrazone( high/middle/low dose and chemoprevention groups), recorded the weight change and tumor growth of those athymic mice, the tumor volume were calculated according to the formula; when the experiment finished, the mice were sacrificed, the tumor tissues were all removed and weighted, evaluated the tumor growth inhibition by formula, recorded the survival time and amount at the same time, thus evaluate the antitumor activity of our synthesized prodrugs in vivo. Dupulicated tumor-bearing models, animals were given DOX, DOX-LNA, PEG-DOX hydrazone respectively by iv and executed them after 2,8,24h, detected the content of DOX in tumor and healthy tissues, then evaluated the tumor targeting of those prodrugs.SD rat were given DOX, DOX-LNA, PEG-DOX amid, PEG-DOX hydrazone 5mg/kg by iv administration, Blood samples were collected(with heparin) from the vena abdominalis under light ether anesthesia at different times(0.08,0.17,0.5,1,2,4,8,12,24h) after iv. The samples were immediately centrifuged for 10 min at 3000 rpm to separated plasma. Meanwhile, organ including heart, liver, spleen, lung, kidney were obtained and weighted, homogenated for analysis. We developed a high-throughput and sensitive bioanalytical method using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) equipped with an electrospray ionization interface used to generate negative ions [M-H]– and selected multiple reaction monitoring (MRM) to detect the concentration-time relationship in blood and tissues after iv administration, and calculated the pharmacokinetic parameters by 3p97 software.ResultsAccording to the synthesis route we designed beforehand, we have successfully synthesized three DOX prodrugs, target compound 1 was a dark red powder, which melting point was 91oC, and the yield was 73%. The data of 1H NMR spectra wasδ7.8 (2H, d, CH-2 and CH-4), 7.4 (1H, d, CH-3), 5.50 (1H, s, CH-1'), 4.08 (3H, s,H3C-O-C-1),δ5.28–5.49 (6H, m,CH-9,10,12,13,15 and 16), 2.81–2.78 (4H, t, CH2-11 and CH2-14); Mass spectrometric analysis showed the stable molecular ion peak was 802.8([M-H]-), and the infrared spectrum of the product was in agreement with the designed structure. Target compound 3 was a dark red powder, which melting point was 82oC, and the yield was 87%, the DOX content was 3.9%. The data of 1H NMR spectra wasδ7.8 (2H, d, CH-2 and CH-4), 7.2 (1H, d, CH-3), 5.40 (1H, s, CH-1'), 4.10 (3H, s,H3C-O-C-1),δ3.61 (PEG bone structure); and the infrared spectrum of the product was in agreement with the designed structure. Target compound 6 was a dark red powder, which melting point was 78oC, and the yield was 82.5%, the DOX content was 10.64%. The data of 1H NMR spectra wasδ7.1 (2H, d, CH-2 and CH-4), 6.8 (1H, d, CH-3), 5.30 (1H, s, CH-1'), 4.08 (3H, s,H3C-O-C-1),δ3.61 (PEG bone structure); and the infrared spectrum of the product was in agreement with the designed structure. We set up a HPLC analysis method for DOX and its prodrugs, the chromatographic peak of DOX showed a good peak shape in selected HPLC conditions without interfere of impurity and solvent, the retention time of DOX was 3.79min and blank solvent shared no special absorption peak in these conditions, the chromatographic analysis method was feasibility, reproducibility, stable and reliable. The linear range was 0.5-100μg/mL, and the lowest detectable limit was 0.1μg/mL, intra and inter day precision all no more than 15%. The results of research on release of DOX showed that PEG-DOX hydrazone was acid sensitive which could release more DOX when pH was lower and seemed be more stable when under neutral environment, however, PEG-DOX amid did not show a acid-sensitive activity, and the release ability was not as well as PEG-DOX hydrazone.The experiment on fluorescence microscope indicated that there were only a few of DOX could enter the cells after incubation, however, intracellular distribution of all the three DOX prodrugs were higher than free DOX, among of the three prodrugs, the intracellular amount of PEG-DOX amid was lowest, while the amount DOX-LNA and PEG-DOX hydrazone were higher; in addition, different cells gave different sensitivity to drugs, which MCF-7 was least sensitive while HepG-2 cells made strongest fluorescence signals. The uptake experiment indicated that prodrugs could increase the uptake efficiency, and showed a time-depend manner. The peak value of uptake efficiency by HepG-2 was highest, when MCF-7 was lowest. After 24, 48, 72h incubation with cells, Polymer prodrugs were found the uptake efficiency show time and dose-depend manner. MTT results hinted all these three prodrugs have determinate cyctotoxicity to three tumor cells, and were a good dose-depend manner. The IC50 value of DOX-LNA were lower than DOX, on contrary, The IC50 value of PEG-DOX amid and hydrazone were higher; besides, the cytotoxicity of polymer prodrugs also had a time-depend manner, the longer was the incubation, the lower was the IC50, the better was the antitumor activity. TEM results indicated all the prodrugs show some apoptosis activity on HepG-2, MCF-7 and MDA-MB-231 cell. However, compared with DOX, the induction of DOX-LNA was much higher even though PEG-DOX amid and PEG-DOX hydrazone were lower.We set up a tumor-bearing athymic mice model successfully, and found a inverse ratio relationship between the weight change and given dose both of DOX-LNA and PEG-DOX hydrazone, the mice weight from the chemoprevention groups of the two prodrugs were most obviously increased; prodrugs could inhibit the growth of tumor, prolong the survival time and show antitumor activity, which possessed dose-depend manner, the antitumor activity of chemoprevention group was better than those of therapy, while DOX-LNA was more effective than PEG-DOX hydrazone.Both DOX-LNA and PEG-DOX hydrazone could not only increase the biodistribution in tumor, but also reduced their content in healthy organ especially for heart, which showed tumor-targeting effect and in accordance with our design.Pharmacokinetics and biodistribution data testified prodrugs could prolong their half-life, improve bioavailability, reduce the content of DOX in healthy tissues, increase safety, and among those prodrugs, PEG-DOX amid and PEG-DOX hydrazone had slow-release characteristics.ConclusionsWe have successfully synthesized three DOX-prodrugs and identified their structure were in accordance with our design. The two polymer-progrugs could release DOX with a moderate speed and long duration, PEG-DOX hydrazone conjugate showed acid-sensitive activity. Prodrugs could increase the intracellular distribution of DOX, improve the uptake of DOX by all these three tumor cells; the cyctotoxicity of DOX-LNA was higher than free DOX, on contrary, PEG-DOX amid and PEG-DOX hydrazone were less toxic than DOX. Prodrugs could also induce tumor cells apoptosis, participate the growth regulation of those cells. Compared with DOX, DOX-LNA and PEG-DOX hydrazone could inhibit the growth of tumor, prolong the survival days of tumor bearing mice, be tumor- targeting, reduce cardiotoxicity and decrease the influence on weight change caused by DOX; the antitumor activity exhibited a dose-dependent manner.DOX-LNA, PEG-DOX amid and PEG-DOX hydrazone all could prolong half-life, improve bioavailability, lower toxicity; while the high-molecule prodrugs were slow-released.