Design Of Drug Delivery System For Oral Natural Progesterone

As the female steroidal hormones,progesterone has been found to metabolize quickly and extensively after orally administrated,which leads to its poor and variable bioavailability,so progesterone is normally administrated by intramuscular route, however,local pain at the injection site is not insignificant and becomes an increasingly important issue as progesterone administration is prolonged.Liposome is one of the fastest growing drug delivery systems,for the delivery of sensitive molecules/compounds such as proteins and hormone,liposome could protect the incorporated agents from degradation,increase the absorption and bioavailability of drugs.In our article,a new kind of progesterone proliposome was made.After orally administrated or mixed with water,the liposome could be formed and the properties of progesterone liposome for improving absorption and protection against metabolism were determined.At first,progesterone liposome was made by a new kind of proliposome method invented in our laboratories,and the prescription of liposome was optimized,and then the properties including morphology,particle size,protective effect in gastric and intestinal contents,acute toxicity,the absorption in vitro,the bioavailability in vivo and the first-pass effect were determined.There was not such report about progesterone liposome in china.To optimize the prescription of progesterone proliposome,the single factor experiment and ternary phase diagram were used,and the ratio of drug and lecithin was determined. After proliposome was made,the acute toxicity was determined and the result demonstrated that progesterone liposome was safe with a low toxicity to mice and the LD50 was less than micronized progesterone soft capsule.The stability of liposome was observed through the stress testing and accelerated testing,the results showed that liposome was susceptive to temperature and light,so the liposome should be stored in shady and cool place.During the determination of entrapment efficiency,High performance liquid chromatography was used to determine the contents of progesterone.The transmission electron microphotography and scanning electron microphotography was used to observe the morphology of progesterone liposome and blank liposome,the results showed the liposome was round with multilayer.The particle sizes and zeta potential of liposome were determined by using the nano-particle sizer,the results demonstrated that the average particle size of liposome was 103.7nm and zeta potential was -88.9mv.The gel filtration chromatography method was established to determine the entrapment efficiency of liposome,and then the influencing factors of entrapment efficiency were studied.The results showed that the entrapment efficiency of liposome was 54.95±9.98%and the ratio of drug and lecithin,diluted times might take an effect on the entrapment efficiency.The leakage rate of liposome at room temperature and 4^C were also studied through the determination of entrapment efficiency,and a low leakage rate in 4℃was observed. Furthermore,progesterone liposome was compared with progesterone liposome made by film evaporation method.The results indicated that the morphology of two kinds of liposome was same,but the entrapment efficiency of liposome made by our method was higher than the other,and the particle size of the former was lower than the latter,which the liposome made by our method was better than that made by film evaporation method. All the results indicated that progesterone liposome could be formed with our method.To study the protective of liposome against degradation,a method in vitro was established and the residual contents of progesterone liposome versus time after mixed respectively with the artificial gastric juice,artificial intestinal juice or the grastric and intestinal contents of rats were determined.The results indicated that liposome could protect effectively progesterone against the trypsin when it was mixed with the duodenum and colon contents of rats.However,the results were not obvious when liposome was mixed with the other contents as above,because the conditions of intestinal contents was more like the true conditions than the artificial intestinal juice,it could be believed that the liposome could protect effectively progesterone against degradation at intestinal contents when taken orally.A good absorption model in vitro,Caco-2 cell model,was established in our laboratory, and then the transport of liposome was determined after Caco-2 cell grew for up to twenty-one days and TEER was above 300Ω·cm².The results indicated progesterone belonged toâ…¡type of drugs in BCS and its transport was active transport.The P_app at basolateral to apical direction was higher than that at apical to basolateral direction,and the P-gp inhibitor could improve the accumulative transport flux,which indicated there was P-glycoprotein at basolateral to apical direction.P-glycoprotein could act as a "drug pump" which could pump the drug out of the cells when the drug was absorped.On the other hand,progesterone liposome could improve the drug absorption through facilitating the transcellular transport and opening of tight junction to allow the paracellular pathway. Lastly,the cell toxicity of progesterone and progesterone liposome was determined through MTT experiments,the results showed that progesterone liposome had a little cell toxicity when diluted into 250 times with water.The pharmacokinetics of progesterone liposome in rats removed ovaries was studied and compared with micronized progesterone soft capsule,and the relative bioavailability was calculated.The radioimmunoassay was used to determine the drug contents during the experiment.Furthermore,cross-over design was used in order to avoid the individual difference.The results indicated that the plasma concentration-time curves for progesterone liposome and micronized progesterone soft capsule fit two compartment open model after intragastric administration to the rats,there was significant difference in AUC between liposome and micronized progesterone,which suggested that liposome had higher bioavailability than micronized progesterone,the relative bioavailability is 289.19%.It was also studied about the first-pass effect in rats of liposome through three administration route:intragastric administration,caudal injection and hepatic portal injection.The results demonstrated that in liposome group there were 49.49%drugs metabolized by liver and 26.00%drugs of total drugs metabolized by gastric and intestine. When administrated through the caudal injection,the bioavailability of liposome is higher than micronized progesterone soft capsule and the clearance of the former is less than the latter.All those results indicated progesterone liposome could improve the bioavailability of progesterone through improving the absorption and protecting progesterone against metabolism.Moreover,in order to protect ulteriorly against metabolism in vivo and improve ulteriorly the bioavailability,liposome was tried to modify with chitosan,and the properties of chitosan modified liposome was determined and compared with liposome. The results indicated that the entrapment efficiency of chitosan modified liposome was lower than the liposome,the zeta potential of chitosan modified liposome was positive. All that results indicated that the chitosan modified liposome had been formed.In our article,the feasibility of progesterone liposome was evaluated through the determination of liposome properties,and the absorption mechanism,the pharmaco- kinetics in vivo,and then the first-pass effect was studied.The results would lay a foundation for the development of progesterone proliposome.