Preparation, In Vitro And In Vivo Evaluation Of Oral Microemulsion Of Insulin-phospholipid Complex

OBJECTIVESAdministration of peptide and protein drugs such as insulin via oral route is always the focus and difficulty of modern pharmaceutics. Microemulsion is thought to be a promising drug delivery system among various formulation approaches. However, as a hydrophilic drug, insulin is always present in the aqueous phase. For O/W microemulsion, insulin can be destoryed in vivo by GI enzymes since insulin is in the external phase, for W/O microemulsion, phase inversion may happen in vivo when the preparation is diluted with a relatively large amount of GI fluid, which will lead to the destroy of insulin still. Therefore, the present study aim to develop insulin microemulsion, in which insulin is loaded in the oil phase by employed phospholipid complex technique preparing insulin-phospholipid complex (IPC) that improving the liposolubility of insulin, to reduce the destroy of insulin in vivo and then improve the oral bioavailability of insulin, which providing basis to the development of oral formulation for peptide and protein drugs such as insulin.METHODS AND RESULTS 1. Preparation and characterization of IPCThe IPC was prepared by a lyophilization method using DMSO containing5%glacial acetic acid as solvent. The phospholipid/insulin quality ratio and oil phase were selected using transmittance as evaluation index. The IR spectra and X-RD analysis were used to verify the formation of the complex. The effect of preparation conditions on the biological activity of insulin was assessed by the blood glucose level after s.c injection. The results showed that, insulin was completely dissolved when the phospholipid/insulin quality ratio was32:1and the oil was Ethyl Oleate. The IR spectra and X-RD analysis confirmed the formation of IPC. The blood glucose level after s.c injection showed that there was no significance effect of preparation conditions on the biological activity of insulin.2. Preparation and characterization of IPC-W/O、IPC-O/W microemulsionThe formulation factors such as surfactant, cosurfactant and Km were screened by pseudo-ternary phase diagrams. The optimal preparations of IPC-W/O、O/W blank microemulsion were as followed:[Lecithin:alcohol:EO:H2O=26.67:26.67:35.55:11.11(m/m/m/m)]、[EL35:1,2-propanediol:EO:H2O=18.18:6.06:6.06:69.70(m/m/m/m)]. The particle sizes of IPC-W/O、IPC-O/W microemulsion were27.3nm and18.6nm, the PDI were0.117and0.141, the concentration of insulin were769.78±0.86μg·mL-1and216.5±0.57μg·mL-1. The drug contents of the preparations decreased at room temperature for30days and were stable at4℃.3. In vitro evaluation of IPC microemulsionEvaluation of IPC microemulsion through enzymatic degradation study and transport experiment with Caco-2cell model in vitro. The enzymatic degradation study showed that IPC microemulsion had significant protective effect against pepsin and trypsin degradation. The protective effect of IPC-W/O microemulsion was significantly higher than that of IPC-O/W microemulsion. The protective effect of the preparations was in the following order:IPC-W/O microemulsion> IPC-O/W microemulsion> IPC. The transport experiment with Caco-2cell model showed that the Papp of IPC, IPC-W/O microemulsion and IPC-O/W microemulsion were significantly higher than that of insulin. The absorption enhancement ratio(R) were1.26,1.48,1.50respectively compared with insulin solution. A higher Papp value was obtained with IPC-W/O microemulsion and IPC-O/W microemulsion in comparison with IPC, but there was no significant difference between IPC-W/O microemulsion and IPC-O/W microemulsion.4. In vivo evaluation of IPC microemulsionEvaluation of IPC microemulsion through pharmacodynamics experiment on STZ-induced diabetic rats in vivo. Rats were divided into five groups:①IPC-W/O microemulsion PO;②IPC-O/W microemulsion PO;③IPC PO;④insulin solution s.c;⑤untreated as control. Doses were50IU-kg-1body and1IU-kg-1body for PO and s.c, respectively. Results showed that IPC-W/O microemulsion, IPC-O/W microemulsion and IPC all had significantly hypoglycemic effect compared with the control group. The hypoglycemic effect of the preparations was in the following order:IPC-W/O microemulsion> IPC-O/W microemulsion> IPC, the lowest glucose level were66.7%,70.8%and82.02%of initial glucose level respectively. The relative pharmacological bioavailability were2.4%,1.8%,1.6%respectively for IPC-W/O microemulsion, IPC-O/W microemulsion and IPC compared with insulin s.c. The hypoglycemic effect of IPC-W/O microemulsion was sustained for a longer time compared to subcutaneous injection. The pharmacodynamic experiment showed that IPC microemulsion especially the IPC-W/O microemulsion had significantly hypoglycemic effect after oral administration, indicated that this drug delivery system can improve oral absorption of insulin, and can be considered as a promising oral delivery system for insulin.CONCLUSIONSThe present study developed IPC-W/O、IPC-O/W microemulsion in which insulin was loaded in the oil phase by employed phospholipid complex technique. This IPC microemulsion especially the IPC-W/O microemulsion can significantly increased the protective effect of insulin against enzymes degradation and enhanced insulin transport across Caco-2cell monolayer in vitro. The pharmacodynamic experiment showed that IPC microemulsion especially the IPC-W/O microemulsion had significantly hypoglycemic effect after oral administration, and the hypoglycemic effect sustained for a longer time compared to subcutaneous injection. It was a promising oral delivery system for insulin. This paper provided experimental basis for the development of oral formulation for peptide and protein drugs such as insulin.