Preparation Of Targeted Drug GnRH-MTX Conjugate And Its Antitumor Activity Of Prostate Cancer In Vitro And In Vivo

ObjectiveThis study take ligand-receptor specific binding as a theoretical basis, according to the prostate cancer cell line PC-3 cell was overexpressed GnRH receptor, while the normal prostate tissue was no expressed GnRH receptor, we designed and synthesized a novel anti-tumor targeted drugs. GnRH ligand was conjugated to methotrexate via a covalently bond, the product was GnRH-MTX conjugate. The anti-tumor activity of GnRH-MTX conjugate was evaluated in vivo and in vitro, we also examined the biodistribution of GnRH-MTX conjugate in animal tissues by choosing tumor (PC-3) bearing mice as models. Our research can lay a foundation for seeking good curative effect and low toxicity targeted drug for prostate cancer.Methods1. First, the GnRH-MTX conjugate was synthesized using methotrexate and GnRH polypeptides as starting material by two-step reactions. And then the structure of GnRH-MTX conjugate was identified by HNMR and LC-MS.2. The cytotoxicity of GnRH-MTX conjugate and free methotrexate to human prostate cancer PC-3 cell and human prostate fibroblast cell was evaluated by CCK8 kit and flow cytometry.3. PC-3 cells were injected subcutaneously into BALB/c nude mice, and then a tumor bearing nude mice xenograft model was established. The anti-tumor activity of GnRH-MTX conjugate was evaluated by the tumor bearing nude mice xenograft model.4. A HPLC method was established for the quantitative determination of MTX in different tissues. The tumor bearing nude mice xenograft model was used as animal model, the tumor bearing nude mice was intravenously administrated of MTX and GnRH-MTX conjugate respectively. Heart, liver, spleen, lung, kidney and tumor were taken out after 4 hours, to removed protein by added organic solvent. The supernatant was collected after centrifugation. The concentration of MTX in different tissues was determined by the HPLC method. To compare the concentration of MTX in different tissues of MTX groups with the GnRH-MTX groups, the concentration of MTX was used to evaluate whether GnRH-MTX conjugate have targeting-tumor capacity.Results1. The GnRH-MTX conjugate was successfully synthesized using methotrexate and GnRH polypeptides as starting material by two-step reactions. The first step is to generate active esters and the reaction conditions are as follows:The molar ratio of reactants of MTX:EDC.HCL:NHS:DMAP=2:6:6:1, the reaction temperature is 25℃, the reaction under substantially anhydrous and anaerobic and dark conditions last 24 hours. The reaction was terminated and the reaction solution was treated simply. The compound was purified by silica gel column chromatography using ethyl acetate and petroleum ether as eluent in 72.36% yield. The second step is to form GnRH-MTX conjugate and the reaction conditions are as follows:The molar ratio of reactants of MTX active esters:GnRH polypeptides=1.5:1, the reaction last for 24 hours at 25 ℃, ether was added to terminate the reaction. The reaction solution was treated simply, and then the GnRH-MTX conjugate crude sample was purified by reversed phase preparative chromatography in 88.98% yield. The desired products of GnRH-MTX conjugate purity of 98.35%. The molecular weight of compound is detected by by mass spectrometry, the MW is 1632, the structure of GnRH-MTX conjugate was identified by HNMR, All the experimental result agrees well with the theoretical predictions.2. The CCK8 kit experimental results show that the IC50 of MTX in PC-3 cell was 1.36μmol/L after 24 hours of treatment and the IC50 of GnRH-MTX conjugate in PC-3 cell was 0.41 μmol/L after 24 hours of treatment; The CCK8 kit experimental results also show that, the HPrF cell was treated with free MTX and GnRH-MTX conjugate separately for 24 hours, the IC50 of MTX is 147.19 μmol/L, the IC50 of GnRH-MTX conjugate is 93.63 μmol/L. MTX and its conjugate act on PC-3 cell 24 hours respectively, and then the apoptosis rate was determined by flow cytometry. In the MTX groups, the apoptosis rate of the blank was 5.71%; the apoptosis rate was 10.56% when the concentration of MTX was 0.24 μmol/L, the apoptosis rate was 62.21% when the concentration of MTX was 0.98 μmol/L, the apoptosis rate was 70.33% when the concentration of MTX was 3.91 μmol/L; In the GnRH-MTX groups, the apoptosis rate of the blank was 4.12%; the apoptosis rate was 20.31% when the concentration of MTX was 0.24 μmol/L, the apoptosis rate was 77.31% when the concentration of MTX was 0.98 μmol/L, the apoptosis rate was 98.71% when the concentration of MTX was 3.91 μmol/L. The apoptosis rate increased along with the increasing concentration of drugs.3. The anti-tumor activity of GnRH-MTX conjugate was evaluated in vivo, when the administration of drugs were finished, the relative tumor growth rate was calculated, the relative tumor growth rate of bicalutamide groups, MTX groups, GnRH polypeptide groups, Low dose groups of GnRH-MTX conjugate, medium dose groups of GnRH-MTX conjugate and high dose groups of GnRH-MTX conjugate were 25.67%,36.85%,95.77%,40.67%,33.47% and 30.84% respectively. At the same time the inhibition rate of tumor was also investigated, the inhibition rate of tumor of bicalutamide groups, MTX groups, GnRH polypeptide groups, Low dose groups of GnRH-MTX conjugate, medium dose groups of GnRH-MTX conjugate and high dose groups of GnRH-MTX conjugate were 79.47%,69.54%,9.27%,67.55%,76.82% and 79.47% respectively.4. A HPLC method was established for the quantitative determination of MTX in different tissues, MTX can be effectively separated with other interfering substances in sample, which was fast, sensitive and specific. The tumor bearing nude mice was intravenously administrated of MTX and GnRH-MTX conjugate after 4 hours, the concentration of MTX in different tissues was determined by the HPLC method. The concentration of MTX in tumor tissue of free MTX groups is 4.27 mg/kg, while the concentration of MTX in tumor tissue of GnRH-MTX conjugate groups is 13.61 mg/kg, these data showed that the targeting-tumor capacity of GnRH-MTX conjugate was more better than the targeting-tumor capacity of free MTX.Conclusions1. The GnRH-MTX conjugate was successfully synthesized using methotrexate and GnRH polypeptides as starting material by two-step reactions, the reaction operation is very simple, the reaction is fast, the reaction condition is gentle and the yield is high.2. In vitro pharmacodynamics models were used to evaluate the anti-tumor activity of drug, the experiment datas shown that the anti-tumor activity of GnRH-MTX conjugate on PC-3 cell was stronger than the anti-tumor activity of free MTX conjugate on PC-3 cell because the PC-3cell was high expressed GnRH receptor.3. GnRH-MTX conjugates can inhibit the crazy growth of tumor which is human prostate cancer PC-3 xenografts in nude mice, The anti-tumor activity of GnRH-MTX conjugate was better than the anti-tumor activity of free MTX in vivo.4. GnRH-MTX conjugate have better targeting-tumor capacity than the free MTX, the biodistribution of GnRH-MTX conjugate have also been changed.In summary, GnRH ligand can be used as targeted drug carrier which can be delivery drugs to tumor cell of high expression of GnRH receptor, to enhance the curative effect and reduced the toxicity and to achieve tumor targeted therapy.Moreover, micafungin sodium was also been investigated in this study, an isocratic HPLC method for separation and determination of the related substances of micafungin sodium was developed. The chromatographic separation was achieved on Agilent Zorbax SB-C18 column (250×4.6 mm,5μm). The mobile phase composition was pH 2.9 buffer (1.20 g of sodium dihydrogen phosphate and 6.15 g of sodium perchlorate in 1,000 mL of water and adjusted to pH 2.9 with phosphoric acid) and acetonitrile in the ratio of 62:38 (v/v). The flow rate was kept constant at 1 mL/min and the column was maintained at 45℃. The detection was performed at 210 nm using a diode array detector and the injection volume was 10μL. The performance of the method was validated according to the present ICH guidelines for specificity, linearity, accuracy, precision and robustness. The results showed that the method is specific, sensitive, linear, precise and rugged. Forced degradation studies were performed on the drug substance to show the stability-indicating nature of the method. The drug was found to be quite unstable to base degradation and oxidation degradation; the drug was found to be moderately unstable to acid degradation and photolytic degradation; the drug was found to be slightly unstable to water degradation and thermal degradation.