Study On Probucol Self-micro Emulsifying Drug Delivery System

Probucol has been widely used in clinical practice to reduce serum cholesterol and prevent the progression of atherosclerosis. However, probucol is slightly absorbed in the gastrointestinal tract due to its poor solubility in water. The main aims of this research were to study the intestinal absorption mechanisms of probucol, and investigate the factors that affect the absorption of probucol for preparing a novel oral drug delivery system. Self-microemulsifying drug delivery system (SMEDDS) of probucol was developed and characterized. The stability of probucol SMEDDS was investigated. Finally, the studies on pharmacokinetics and mechanism of absorption of different formulations of probucol were determined.Detection of probucol was accomplished by reverse-phase HPLC.The Caco-2 cell model was used to study the uptake and transport of probucol. The results indicted:(1)Probucol is actively absorbed primarily. The uptake of probucol was positively correlated to concerntrations of donor solution. When the concerntration increased to 10μg/ml, uptake of probucol increased non-linearly. And saturation was observed at a high donor concerntration. The uptake of probucol was positively correlated to temperature. The P-glycoprotein inhibitor, cyclosporine A, significantly enhanced the uptake amount of probucol, especially with a low donor concerntration. But the p-gp inhibitor, verapamil, had no significant effect on the uptake of probucol. (2) The absorptive transport of probucol was pH and temperature dependent and the transport was enhanced at pH7.4 and 37℃on the apical side. (3) The transport of probucol was time and concerntration dependent. But saturation was observed when donor concerntration was 20μg/ml. (4) Transport of probucol across Caco-2 cell monolayers was directional. Papp of Basolateral to Apical was much more than that of Apical to Basolateral (1.1-2.1 folds). In the presence of CsA, inhibitors of P-glycoprotein, the Papp BL-AP of low concerntration donor solution decreased, while Papp AP-BL increased, respectively. But no difference was observed when donor concerntration was high. Verapamil had no significant effect on the transport of probucol.Probucol may be substrate of other efflux proteins rather than P-glycoprotein. The effect of CsA on transport of probucol when donor concerntration was low may due to competitive combination of two agents with micelle in donor solution. (5) Indometacin, the MRP1 inhibitor, did not have significant effect on transport of probucol in two directions. (6) Sodium azide, the ATP inhibitor, decreased transport of probucol from two directions, which indicates transport of prbucol is energy dependent.In the study of preformulation, the equilibrium solubility of probucol in different oil, surfactants and cosurfactants was investigated and pseudo-ternary phase diagrams were constructed to evaluate the formulation effect. An optimized formulation consisted of Lauroglycol FCC:Olive oil (2:1) as oil phase, Cremopher EL:Tween 80 (1:1) as surfactants,and PEG-400 as co-surfactants. With the amount of olive oil in oil phase, the amount of oil in formulation and Km as the independent variables, the optimized formulation was determined by the central composite design-response surface methodology. The response variables were particle size, Zeta potential, solubility, and the cumulative percentage of probucol in 5 min. The optimized formulation of probucol was quickly and conveniently obtained by the central composite design-response surface methodology, i.e. oil:surfactants:co-surfactants =50%:33%:17%.The effect of dilution on the particle size of probucol in different media was investigated. The results indicated that dilution had hardly effect on the particle size of microemulsion. When the SMEDDS was diluted with water in the ratio of 1:250, the size of microemulsions was(92.7±47.7) nm with a Gaussian distribution. The SMEDDS was stable.Influence test of probucol SMEDDS indicated that high temperature and humidity can affect the drug content and particle size, and probucol SMEDDS should keep in the shade and cool. Accelerating test indicates that probucol SMEDDS were stable.The pharmacokinetic parameters of probucol suspension, oil solution, and SMEDDS were investigated after oral administration to rats at a single dose of 6mg/kg and 60mg/kg, respectively. With a 6mg/kg dose, none plasma concerntration was detected, and results of oil solution and SMEDDS were as follows:Cmax were (0.40±0.07)、(0.58±0.12)μg·ml-1,respectively;AUC were(7.60±3.11)、(12.91±5.81)μg·ml-1·h,respectively. The relative bioavailability of probucol SMEDDS to oil solution was 170%.With a 60mg/kg dose, results of suspension, oil solution and SMEDDS were as follows:Cmax were (0.16±0.07)、(1.80±0.43)、(3.36±0.84)μg·ml-1, respectively;AUC were (7.20±1.63)、(34.16±10.65、(73.59±31.21)μg·ml-1·h, respectively. The relative bioavailability of probucol oil solution and SMEDDS were 474.4%、1022.1%.MTT results showed the SMEDDS in lower dilution presented certain cytotoxicity. Study of toxicity of SMEDDS and excipients on gastrointestine tract in mice showed that SMEDDS and excipients had no significant toxicity on gastrointestine tract in mice.Rats were treated with intraperitoneal injection of cycloheximide, a chylomicron flow blocking substance to investigate the lymphatic transport of drugs. Then the animals were given the oral gavage of three probucol formulations (60mg/kg), and the pharmacokinetic experiment was conducted. Results showed that, Cmax of three formulation were (0.16±0.05)、(0.69±0.21)、(1.72±0.30)μg·ml-1, respectively; AUC were (2.90±1.37)、(13.17±6.96)、(22.91±13.34)μg·ml-1·h, respectively.59.72%、61.45%、68.87% of drug were absorded via lymphatic system, respectively. Probucol had a strong ability to associate with chylomicron in vitro.It indicated that probucol has a good tendency to be absorbed through lymphatic system, and excipients in oil solution and SMEDDS can stimulate the secretion of chylomicron to promote lymphatic absorption of drugs.