Preparation And Study Of CA Smedds

CA was extracted from metabolites of fungus by Fujisawa pharmac- eutical co. ltd in 1982. CA has been applied to prevent and cure of rejection after organ transplantation and its clinical efficacy is better than cyclosporine (10~100 folds). CA has been given a good evaluation since it was used in clinical in 1989. Now, CA has been used as a first-line drug to prevent or treat rejection and autoimmune hepatitis. In the domestic market there are mainly imported CA solid dispersion capsules which are very expensive. Due to its almost insoluble property (2~3μg/ml) and instability in water , the aim of the study is to prepare a formulation of CA to increase its solubility and enhance its stability.Self-microemusifying drug delivery system (SMEDDS) is mainly com- posed of oil, surfactant and co-surfactant. SMEDDS has received great attentions recently for its potential use as the drug delivery system for poorly water soluble drug to improve its bioavailability. After orally administered, the SMEDDS has the ability of forming microemulsion following dilution by aqueous phase in stomach with gentle peristalsis. For combining properties of CA with advantages of SMEDDS in improving solubility of poorly water soluble drugs, this study chose to prepare CA SMEDDS and studied its stability and bioavailability. Yet there are not CA SMEDDS appearing on the market at home and abroad, the research of CA SMEDDS has practical significance.Objective: Select appropriate surfactant, co-surfactant, oil and stabilizer to prepare CA SMEDDS. Vitro and vivo analysis methods of CA SMEDDS were established; Evaluate the quality of CA SMEDDS; Study the stability and bioavailability of CA SMEDDS.Methods:1 Preparation of CA SMEDDS: Firstly, select and identify the mainly components of blank SMEDDS. Then choose stabilizerA which could greatly enhance the stability of CA in SMEDDS as the stabilizer of CA SMEDDS. BHT was chosen as antioxidant of CA SMEDDS. In the optimization process of CA SMEDDS formulation, the content of CA in formulation was identified. In order to greatly improve the stability of CA in SMEDDS, contents of surfactant, co-surfactant, oil and stabilizer in formulation were also optimized. According to the optimized prescription of CA SMEDDS, strong stability samples were made and soft capsules of CA SMEDDS were prepared for dissolution test and animal experiments.2 Quality evaluation of CA SMEDDS: The HPLC methods were established to determine the content and dissolution rate of CA SMEDDS. Study the appearance and viscosity of CA SMEDDS; Study particle size and Zeta potential of CA microemulsion. According to the best prescription, prepare CA SMEDDS. Then carry out high temperature test, strong light test, accelerative test and long-term test to study the stability of CA SMEDDS.3 Pharmacokinetic studies of CA SMEDDS: Use health male dogs as experimental animals. Reference preparation was used as standard to estimate the pharmacokinetics of CA SMEDDS soft capsules. After orally administered single dose of CA SMEDDS soft capsules and reference preparation (0.5mg/kg) to dogs, blood samples were collected from the vein at predetermined time intervals. The content of CA in whole blood was determined by HPLC-MS/MS method. The pharmacokinetics parameters were estimated by 3p87 program. The bioavailability was contrasted between CA SMEDDS and reference preparation.Results:1 Considering the stability, particle size(diluted 200 folds by water), dissolution and bioavailability, the best formulation of CA SMEDDS was: CA 0.5g CremophorRH40 40g 1,2-propylene glycol 15g Micromolecule ester(CP) 44g Stabilizer A 1.0g BHT 0.05g2 The standard curve of HPLC for assay of CA SMEDDS was: A=1.6225×104X-9.9332×103, (r=0.9999, n=7)Select 50% acetonitrile-water solution as solvent in which the conversion ratio of CA isomerⅡwas 4.93%. The average value of relative recovery was 99.89%, intra-day precision were 1.43%, 1.57%, 0.58% (n=6), inter-day precision was 1.32% (n=6), consistent with the regulation of ChP.3 The standard curve of HPLC for determining the dissolution rate of CA SMEDDS soft capsule was: A=7.253×104X-8.996×103, (r=0.9999, n=7)Use the third method of ChP(2005) appendix XC as the method of determination dissolution rate of CA SMEDDS soft capsule. The dissolution test used water with 37±0.5℃as medium and the speed of dissolution tester was 100r/min. The relative recoveries were 99.14%, 99.51%, 99.59%. The intra-day precision of the sample (0.01mg/ml) was 1.43% (n=6), consistent with the regulation of ChP.4 The quality evaluation and stability study of CA SMEDDSAt temperature of 25℃, the CA SMEDDS prepared according to the best prescription was colorless and uniform liquid and its viscosity was 46.0 mPa·s. After diluted 200-folds by double distilled water, the particle size of the CA microemulsion was about 17nm and the Zeta potential was -21.3mV(25℃). High-temperature test indicated that CA SMEDDS had no significant change in appearance, viscosity and particle size at 60±2℃for 10 days, but the content of CA decreased by more than 5%, related substance increased by 0.41%. Strong light test for 10 days showed that the samples were sensitive to light of 4500±500lx, the related substances increased slightly. The accelerated test indicated the three bathes of samples had no significant changes in appearance, viscosity and particle size at 40±2℃, 75±5% relative humidity for 6 months, the content decreased by about 3%, the content of related substance was controlled in 2%. Results of long-term test for 9 months showed that the related substances of the three bathes of samples were 0.29%, 0.27%, 0.24%, the content of CA decreased by less than 1%. Results of long-term test indicated that CA SMEDDS had higher stability at 25±2℃.5 The pharmacokinetics parameters were estimated by 3p87 program. The concentration-time curves could be fitted to a two-compartment model with first order and weight factor=1/c2 for both CA SMEDDS soft capsules and reference preparation. The main pharmacokinetics parameters of CA SMEDDS soft capsules and reference preparation were t1/2(α): 1.37h, 2.39h; t1/2(β): 13.13h, 19.54h; T(peak): 0.79h, 2.92h; C(max): 36.21ng/ml, 11.49 ng/ ml; AUC: 238.29(ng/ml) *h, 174.47 (ng/ml) *h.Conclusion: The CA SMEDDS prepared according to the best formulation had higher physical and chemical stability. At the temperature of 25±2℃for 9 months, the appearance, viscosity, particle size and Zeta of the samples had no significant changes, the content of CA decreased by less than 1%, and the related substance was controlled in 0.3%. CA SMEDDS had higher bioavailability than reference preparation. This study significantly improved the stability of CA in SMEDDS and made a breakthrough in the development of CA SMEDDS.