| CA is a macrolide immunosuppressant, which was isolated from Streptomyces tssukubaensis, No999, in 1984 from Japan. As a potent new immunosuppressant, CA has a selective inhibition on T cells and mainly through the inhibition of TH cells to release IL-2, IL-3, IFN-y, and inhibition of the expression of IL-2R play its powerful immunosuppressive effects. CA has better immunosuppressive action than cyclosporine A(10-100), and has less rate adverse effect and less adverse action, and can reverse the rejection has occurred, has been used in liver, kidney and other immunosuppressive therapy for organ transplants.Because of its poorly solubility in water and its solubility is only 2-3μg/mL, orally administration of CA often can not be completely absorbed, which limits its therapeutic application. Nanosuspensions technology is a nano-loading system recently developed, which can improve the solubility of insoluble drug and the dissolution rate of poorly soluble drugs to improve bioavailability; In addition, Nanosuspensions technology is a simple process and high drug loading characteristics compared to other nanoparticulate drug delivery systems. The study introduced nano-suspension technology for preparation of the CA, which has a reference value on the other formulations of insoluble drugs.Objective:Appropriate prescription and preparation processes were selected to prepare CA nanosuspensions and freeze-drying. The microscopic morphology of CA nanosuspensions was charactered. Analysis method of CA in vivo and vitro were builded to study the stability, physical and chemical properties and bioavailability of CA nanosuspensions.Methods: 1 Preparation of CA nanosuspensions:On the basis of preliminary experiment, the preparation method of CA nanosuspensions was determined. Adopting the single factor tests were practiced for the identification of factors affecting CA nanosuspensions. Taking nanoparticle size, size distribution and Zeta potential as indexes to select the types of surfactants, suspending agent amount, concentration of CA and parameters of high pressure homogenization process. Four factors and three levels orthogonal experiment was used to determine the best prescription and process. In the study, it was found that aggregation and settlement of nanoparticles would occur in the process of long-term storage of CA nanosuspensions. The study improved the formulation stability of nanoparticles through freeze drying technology.2 Evaluation of CA nanosuspensions:Using transmission electron microscopy to observe the shape and surface morphology of the nanosuspensions. The particle size distribution was measured by particle size analyzer. Zeta potential was measured by the Zeta potential analyzer. The content and related substances were determined by HPLC.3 The stability test of CA nanosuspensions:Prepare CA nanosuspensions by the best prescription. The chemical and physical stability of CA nanosuspensions were investigated through high temperature test, high humidity test, strong light test, accelerated test and long-term stability test.4 Pharmacokinetics study in vivo:Four healthy male dogs were selected as experimental animals. With single dose oral administration and crossover design, the pharmacokinetics study of CA nanosuspensions was performed and compared with the reference preparation. Drug content in whole blood was determined by LC-MS/MS method, The pharmacokinetics parameters were estimated by 3p97 program. Comparing the bioavailability between CA nanosuspensions and reference preparation.Results:1 On the basis of preliminary experiment, CA nanosuspensions were prepared by precipitation and high pressure homogenization combined with technology. The optimal prescription and process were defined by orthogonal experiment. The optimal formula was as follow:the 200mg Poloxmer188 and 2g HPMC were dissolved in 100mL water, to prepare water phase; CA were solved in alcohol, to prepare organic phase, the concentrations of CA,2mg/ml. The CA of organic phase was added to the water phase, under stirring at room temperature; after then placed the crude suspension to high pressure homogenization machine, under the pressure of the 1300bar,16 times, CA nanosuspensions were prepared. By adding appropriate freeze-drying cryoprotectant to the nanosuspensions, the CA nanoparticles powders was obtained by freeze-drying technology.2 CA nanosuspensions obtained from precipitation and high pressure homogenization combined with technology were spherical or near-spherical in shape, the average particle size was about 254nm, with narrow size distribution(PI=0.016), Zeta potential was -27.0mv. Because of its small particle size in the range of nanometer, the dissolution of CA nanosuspensions was significantly improved than the original drug. High-temperaure test indicated that the appearance, particle size and content of CA nanosuspensions had no significant changes at 60℃for 10 days. The weight did not change significantly; the appearance, particle size and content had no significant changes in the 92.5% relative humidity. The strong light test for 10 days found that with strong light 4500±500 lx the appearance and particle size had no significant changes, but the content of CA decreased. The results of accelerative test (40℃)and long-term test (25℃)for 6 months indicated that the appearance, particle size and content of CA nanosuspensions had no significant changes. The results revealed that the CA nanosuspensions had higher stability.3 Pharmacokinetics study in vivo:the main pharmacokineticsparmeters of CA nanosuspensions and reference preparation were C(max):32.46 ng/mL, 26.94ng/mL, T(max):1.44h,1.68h, AUC:290.72 h*ng/mL,224.16 h*ng/mL. The pharmacokinetics study indicated that the relative bioavailability increased to 129.67% compared with reference preparation.Conclusion:CA nanosuspensions were prepared by precipitation and high pressure homogenization combined with technology. The nanoparticles were homogeneous with small particle size and narrow size distribution, the nanoparticles had higher physical stability and chemical stability. Experimental results showed that CA nanosuspensions had higher bioavailability than the reference preparation. This technology has advantages of simple technological process and high drug loading capacity. Introduced nanosuspensions to CA preparation, might provide experimental basis for the study of formulations of poorly soluble drugs.
|
PDF Full Text Request