| Tacrolimus (FK506 or Tac, trade name:Prograf) is a kind of immunosuppressive drug. The functional mechanism of FK506 including:(1)Inhibiting the process of interleukin-2 synthesis; (2)Blocking the calcineurin activation of T cells; (3)Inhibiting the expression of interleukin-2 receptor; (4) Inhibiting the expression of interleukin-2 receptor and interrupting the receptor mediated signal transduction process. FK506 was first discovered by Fujisawa pharmaceutical company (Japan) in 1984 and can be got by total synthesis now. FK506 and cyclosporine A (CsA) are the second generation of immunosuppressant with similar mechanism. Clinical studies showed that immunosuppressive activity of FK506 was stronger than that of CsA. However, large molecular weight and hydrophobic macrocyclic lactone ring limit the absorption of FK506 in gastrointestinal tract to a great extent. FK506-loaded F127-CS/DCA orally macromolecular complex micelles were prepared in order to improve the oral absorption of FK506 in this study. Preparation method, physicochemical properties, in vitro release behavior, intestinal absorption mechanism and in vivo drug absorption kinetics were investigated and evaluated successively. In brief, purpose of this study is to illustrate the improvement of the FK506 gastrointestinal absorption when using F127-CS/DCA micelles as delivery system for oral administration. The main content was detailed into 5 sections as follows:1. Synthesis and characterization of Pluronic F127-chitosan conjugatesPluronic F127 was used as the framework material. Firstly, it reacted with succinic anhydride to obtain single carboxyl modified pluronic F127. Then, ligation reaction occurred on the amine groups of chitosan (CS) and single carboxyl modified pluronic F127. Finally, the structure of F127-CS was identified by infrared spectroscopy (IR) and nuclear magnetic resonance spectrum (1H-NMR). Peaks of CS (nearly 5.0ppm and 3.1ppm) and pluronic F127 (nearly 3.6ppm and 1.1 ppm) on the NMR spectrum indicate the successful synthesis of F127-CS. Moreover, in the infrared spectrum, the peak of carbonyl stretching vibration at 1735.97 cm-1 can prove that CS is successfully connected onto pluronic F127. Result of IR spectrum combined with NMR spectrum could further tell the successful synthesis of pluronic F127-CS.2. The preformulation study of FK506 and preparation of FK506-loaded micellesThis part determined the physicochemical properties of FK506 and FK506-loaded F127-CS/DCA macromolecular complex micelles. The critical micelle concentration (CMC) was investigated using fluorescence probe method. Results indicated that the CMC value of blank F127-CS/DCA micelles was 2.65×10-3mol/L, while that of blank DCA micelles was about 1.8×10-2 mol/L. As a result, the CMC value of blank F127-CS/DCA micelles decreased by one order of magnitude compared with blank DCA micelles. According to CMC determination results, stability of F127-CS/DCA micelles was improved obviously, and F127-CS/DCA micelles could offer better resistance to fluid dilution.FK506 F127-CS/DCA macromolecular complex micelles were prepared utilizing film ultrasonic wave dissolving techniques. Drug loading capacity (DL), particle diameter and zeta-potential were used as indicators to select the optimal prescription. Eventually, the optimal prescription of FK506 F127-CS/DCA complex micelles is as follows:F127-CS/DCA (m/m, X1)=1:1, quantity of FK506 (X2)=8 mg, hydration temperature (X3)=20℃, water volume (X4)=8 ml, hydration time (X5)= 8 min.3. Physicochemical properties and in vitro drug release of FK506-loaded micellesThis section verified the optimal prescription of FK506 F127-CS/DCA macromolecular complex micelles and investigated its physicochemical properties. Results showed that the solution of FK506 F127-CS/DCA FK506 macromolecular micelles is colorless, clear and micelles are in a spherical-like shape under the transmission electron microscope (TEM), whose average particle diameter is about 55.77 nm and average Zeta potential is about-6.38 mV. Simulated gastrointestinal fluid containing 1% Tween-80 (w/v) was used as release medium to investigate the release properties of FK506-loaded micelles in gastrointestinal tract. Through the model fitting of release results, FK506 injections showed a relatively slow release in both kinds of mediums, which was similar to the zero order release. In comparison with FK506 injection, FK506-loaded F127-CS/DCA macromolecular complex micelles exhibited an obvious feature of first order release in both mediums.4. In situ absorption of FK506-loaded macromolecular complex micelles in rat intestinal perfusion modelIn this part, we successfully built in situ rat intestinal perfusion models to investigate the absorption kinetics characteristics and absorption mechanism of FK506-loaded macromolecular complex micelles in rat small intestine. Furthermore, the experiment was carried out to determine which part of rat intestine was the best absorption segment of FK506-loaded micelles. Experiment results showed that,26, 52 and 104 μg/mL FK506 F127-CS/DCA micelles perfusion fluid showed first order kinetics characteristics. Ka values of 26,52 and 104 μg/mL FK506 F127-CS/DCA micelles were 0.2355,0.2326 and 0.221 respectively, which showed no statistical difference (P> 0.05), thus passive diffusion could be thought as the mechanism of FK506 F127-CS/DCA micelles absorption in rat small intestine. The results of different intestine segments absorption experiment were as follows:the absorption percentage in duodenum, jejunum, ileum and colon were (32.51+ 2.31)%, (29.89+2.09)%, (31.45+0.71)% and (13.61+1.70)%, respectively. Drawing a conclusion, duodenum, jejunum and ileum were major absorption sites of FK506 F127-CS/DCA macromolecular complex micelles.5. Pharmacokinetics study of FK506-loaded macromolecular complex micelles in ratsIn this section, we constructed the analysis method of FK.506 in rat serum, and rats FK506 Elisa kit was utilized to detect pharmacokinetics datum of the prepared micelles in rat serum. Compared with FK506 capsule solution, the tmax of FK506-loaded F127-CS/DCA macromolecular complex micelles was shortened to an hour from 3 h, and oral absorption speed was significantly accelerated. Moreover, both of ti/2 and MRT were prolonged. Area under the drug concentration curve (AUC) after oral administration of FK506 capsule solution was 533.79 ng/mL.h. In contrast, AUC was promoted to 742.11 ng/mL.h after oral administration of FK506-loaded F127-CS/DCA micelles. AUC increased by 39.3%, which told us that FK506-loaded F127-CS/DCA macromolecular complex micelles obviously enhanced drug absorption and successfully improved drug bioavailability. |
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