| A prodrug is a pharmacological substance which is administered in an inactive or significantly less active form, and can be metabolized into its active form in vivo by enzymes or non-enzymatic activation, accordingly alter the therapeutic index by the improved efficacy and stability as well as the reduced toxicity. The design of prodrugs has drawn more and more attention benifited from its low risk and high return. N3-O-toluyl-Fluorouracil (TFu) was synthesized as one of the newest effective prodrugs of fluorouracil (5-Fu). It was expected to exhibit tumor-target therapeutic effect by selectively releasing5-Fu in tumor tissues.However, the clinical application of TFu might be limited due to its poor aqueous solubility. Hence, a new formulation is highly desirable to improve the solubility, bioavailability and therapeutic index of TFu.Nanosuspensions have revealed their potential to tackle the problems associated with the delivery of poorly water-soluble drugs. Nanosuspensions are submicron colloidal dispersions of pure drug particles in an outer liquid.In the present study, TFu-loaded nanosuspensions (TFu-Ns) were prepared by high-pressure homogenization technology. The TFu-Ns were evaluated in terms of pharmaceutical characteristics and bio-characteristics, respectively. This study provided experiment and theoretical basis for utilizing nanosuspensions in the delivery of poorly water-soluble drugs. The main methods and results were as follows:1. Determination and in vitro stability studies of TFuThe drug concentration of TFu and5-Fu were determined by high-performance liquid chromatography (HPLC) method. The in vitro stability of TFu were evaluated after incubating with phosphate buffered saline (pH=7.4), simulated gastric fluid, simulated intestinal fluid and the blank pasma of Wistar rats, respectively.The results showed that HPLC method was simple and correct. TFu was relatively stable in phosphate buffered saline (pH=7.4) and simulated gastric fluid, while it had a40%degradation in simulated intestinal fluid and almost entirely converted to an equimolar amout of5-Fu in blank pasma of Wistar rats within24hours.2. Preparation and in vitro evaluation of TFu-loaded nanosuspensionsTFu-Ns were obtained by high-pressure homogenization technology. The particle size and stability were taken as the index to evaluate the formulations and preparation process. Then, lyophilization was carried out to improve the stability of TFu-Ns. A series of in vitro evaluation were conducted, including the morphology, particle size and size distribution, zeta potential, in vitro release behaviors, preliminary stability as well as the cytotoxicity.Soya lecithin was selected as the stabilizer. The optimized formulation was as follows:drug/soya lecithin (1:15, w/w), drug concentration (3.5mg/mL); and the optimized preparation process was as follows:high speed shearing (3-5min), high pressure homogenization (200bar×5cycles,500bar×10cycles,800bar×10cycles,1000bar×10cycles). The obtained lyophilized nanosuspensions appeared as white loose powder. The lyophilized nanosuspensions showed good redispersibility and the reconstituted nanosuspensions showed light blue opalesce. TFu-Ns had a mean particle size of (43.85±1.34) nm and a zeta potentional of (-7.17±1.21) mV, while the lyophilized TFu-Ns had a mean particle size of (180.03±3.11) nm and a zeta potentional of (-8.02±1.43) mV. Three batches of samples showed good reproducibility.The in vitro release behavior of TFu from TFu solution (acetonitrile: water=40:60) could be described by Higuchi equations and expressed by the following equations:Q=83.49t1/2-24.88(r=0.9920); while the TFu release behavior from TFu-Ns in vitro was in accord with double phase kinetics model and expressed by the following equations:100-Q=4.655e-0.764t+2.161e-0.093t (rα=0.9975, rβ=0.9905). More than90%of TFu can be released from both of the TFu solution and TFu-Ns within4hours. The preliminary assessment of stability showed that the lyophilized TFu-Ns should be stored in low temperature, in sealed condition, and away from light. The lyophilized TFu-Ns were almost intact at4℃for3months. Compared with5-Fu Injection, TFu-Ns revealed more cytotoxicity against HepG2cells in the presence of liver microsomal enzymes, while blank nanosuspensions almost showed no cytoxicity, which illustrated TFu-Ns were safe and effective.3. Pharmacodynamics, pharmacokinetics and tissue distribution of TFu-loaded nanosuspensionsThe in vivo antitumor efficacy of TFu-Ns was evaluated in Kunming mice bearing H22after intraperitoneal administration, while an equimolar amout of5-Fu injection was chosen as a control formulation. The pharmacokinetic properties of TFu-Ns were investigated in Wistar rats after administration comparing to5-Fu injection. The tissue distribution of TFu-Ns was investigated in Kunming mice bearing H22after intravenous administration. The levels of TFu and5-Fu were both measured by the HPLC method in the pharmacokinetics sdudy as well as the tissue distribution study.The inhibitory rate of tumor weight after administrated with TFu-Ns and5-Fu injection were78.57%and75.00%, respectively, while the inhibitory rate of tumor volume were66.67%and60.00%, respectively, indicating that the antitumor efficacy of TFu-Ns is consistent with that of5-Fu injection. The results of the body weight variations of mice showed the blank nanosuspensions almost had no toxicity compared with normal saline during the therapeutic procedure. The results also suggested the mean body weight of TFu-Ns group was slightly heavier than that of5-Fu injection group, which might be explained by the sustained release of5-Fu from TFu-Ns.The pharmacokinetics study in Wistar rats showed that5-Fu would be released rationally from TFu-Ns, and the AUC of the released5-Fu were1.4fold of that of the injected equal mole5-Fu injection.The tissue distribution study showed that5-Fu can be detected in each organ after TFu-Ns was administrated intravenously to mice. In the group of5-Fu injection,5-Fu was inclined to accumulating in the heart, spleen and kidney rapidly, and eliminated quickly. The release of5-Fu from TFu-Ns was relatively slow, and even a higher concentration can be obtained at45min after administration, which increased the retention time of5-Fu. It was also demonstrated that TFu-Ns decreased the5-Fu concentration in the heart and kindey, which would be expected to reduce the side effects of the drug. The tumor accumulation of5-Fu in the two groups was almost the same, and verified the results provided by the pharmacodynamics study.In this study, TFu-Ns were successfully prepared, which significantly improved the solubility of TFu and showed good biocompatibility. In addation, no organic solvents were added during the preparation process. High-pressure homogenization technology provides a relatively easy scale up opportunity and is better suited for industry. Above all, TFu-Ns might be a promising drug delivery system for cancer therapy. |
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