Studies On N3-O-Toluyl-Fluorouracil Loaded PLGA Microspheres

Drug chemotherapy is one of the major treatment methodologies for malignant tumors. However, many drawbacks such as poor biodistribution, serious side effects and short bio-half-life have limited the clinical use of anti-tumor drugs. Many studies have concentrated on the structure modification of the conventional chemotherapy drugs. Prodrugs, as one of the strategies used to improve the pharmaceutical properties and therapeutic efficiency of the anti-cancer agents, are receiving more and more attentions. The prodrug is a modified form of parent drug which is inactive or less active in pharmacology before administration. After administered, the prodrug can be metabolized into its parent drug in vivo; therefore it can remarkably improve the therapeutic effect by the improved target activity and better stability in vivo as well as the reduced toxicity.N3-O-toluyl-Fluorouracil (TFu) was the new generation of effective prodrugs of 5-Fu which is synthesized and evaluated in our department. Its strong anticancer effect had been proved in GC1901 and NKM-45 cell lines and mice. It is anticipated to exhibit tumor-target therapeutic effect by selectively releasing 5-Fu in tumor tissue for a relatively long time. However, the clinical application of TFu was limited due to its poor aqueous solubility.Many pharmaceutical strategies have been employed to increase the solubility of hydrophobic drugs, including microspheres, nanoparticles, liposomes and micells etc. Among these novel formulations, microspheres could increase the drug stability and solubility, reduce the drug toxicity, control drug release and prolong the treatment time of one dosage. There have been many commercial products on the market, such as Decapeptyl, Vivitrol and Zoladex etc. Especially, microspheres prepared using biodegradable and biocompatible materials, such as PLGA, attracted more interests as a method to solve the problem of solubility as well as control drug release. In the present study, TFu was treated as model drug to be encapsulated into PLGA microspheres by modified emulsion-solvent evaporation method. The particle size and size distribution, drug entrapment efficiency, and drug loading were determined, respectively. Behaviors of the in vitro release and in vivo pharmacokinetics in rats were investigated, which provided experimental bases for clinical use of TFu loaded microspheres(TFu-PLGA-MS) in chemotherapy of malignant tumor. The main methods and results were as follows:Method:The modified emulsion-solvent evaporation method was employed to prepare TFu-PLGA-MS. The single factor experiments and orthogonal design experiments were employed to optimize the parameters of formulation and process parameters. The morphology and size distribution of TFu-PLGA-MS were observed and analyzed by light-microscopy. The concentration of TFu was assayed by HPLC. In vitro release properties of TFu from TFu-PLGA-MS were performed by dialysis method and the release kinetic mode was analyzed. TFu concentrations of plasma in rats were determined by HPLC method. The pharmacokinetics of TFu-PLGA-MS was evaluated in rats after intramscular injection.Results:HPLC method was established to determine TFu concentration. The good linearity of the drug concentration with respect to area of the HPLC peak was discovered within the range of 0.40-20.00μg/mL. The linear equation was A=37318×C-10994, R2=0.9948. The recovery was between 99% and 106% and the variances of the method precision (extra-and inter-day) were less than 2% and 5%, respectively.The optimal formulation was obtained by orthogonal experimental design studies. The drug entrapment efficiency of TFu-PLGA-MS was set as the evaluation index to optimize the influence factors including concentration of PVA, the dosage of PLGA, concentration of PVA in external water phase, and the speed of stirring when forming the emulsions. The optimization formulation was as follows:concentration of PVA was 4%, dosage of PLGA was 130mg, concentration of PVA in disperse water phase was 0.5%, speed of stirring was 1000r/min, and volume of organic phase was 5ml. Three batches of TFu-PLGA-MS were prepared by the optimized formulation, and these microspheres were found as nearly white to very light yellow powder, and spherical particles without adhering under the microscope. The mean diameter of TFu-PLGA-MS was (6.54±0.304) u m. The entrapment efficiency and drug loading were (83.61±1.58)% and (14.11±1.33)%, respectively.In vitro drug release showed that the loaded TFu could be released from TFu-PLGA-MS in a relatively steady speed for about 35days. The in vitro release behavior of TFu-PLGA-MS could be described by bioexponential kinetics equation and could be described by the following equation: 100-Q=81.98e-0.1275t+50.59e-0.0016t, Ra=0.9983, Rb=0.9814.HPLC method was established to investigate the TFu concentrations in plasma and the results showed that endogenous substances did not interfere with TFu determination using the selected chromatographic method. The recovery of drug in blood of rats was between 98 and 101%, the variances of the method precision (extra-and inter-day) were less than 2% and 5%, respectively. There was a good linearity of the drug concentration within the range of 0.4-80μg/ml and the linear equation was A=5467.6C+50223 (R2=0.9969). The minimum quantitation level was 28ng/ml and minimum detection level was 5ng/ml, which could satisfy the need of analysis for biological detection.The results of the in vivo pharmaceutical experiments showed the AUC of TFu-PLGA-MS after administrated is 5.87 times than that of the TFu suspension (TFu-Sus), which indicated TFu-PLGA-MS gained better bioavailability. Drug concentration of the plasma in groupTFu-PLGA-MS could be detected until 28th day after administration, while the TFu-Sus can not be detected after 72 hours. The mean residence time of TFu-PLGA-MS group was 14.72 times than that of the TFu-Sus group. These results showed that TFu-PLGA-MS could release TFu more slower compared with TFu-Sus. Therefore TFu-PLGA-MS could prolong the existing time of TFu in vivo, improve drug efficacy and reduce side effects.Conclusion:In this study, TFu-PLGA-MS had high entrapment efficiency was produced by emulsion-solvent evaporation method with simple, feasible preparation technology and good reproducibility. The in vitro release behaviors of TFu-PLGA-MS could be described by bioexponential model. Compared with the TFu-Sus group, the behaviors of drug release of TFu-PLGA-MS significantly changed. TFu-PLGA-MS could reduce drug elimination, extended residence time of drug in vivo and prevent the fluctuation of drug concentration. Thus TFu-PLGA-MS could reduce the side effects, decrease the administration frequency, increase patient compliance and enhance the therapeutic effect. This study explored a new approach for TFu intra-muscular administration, enriched the research content of intramuscular-injectable delivery system of anticancer drugs and showed a high scientific and technological value and cheerful prospects for clinical application.