| Reverse micelle(RM) can solubilize hydrophilic drugs in oil with the help of amphiphilic molecules.This technology can not only resolve the stability problems of hydrophilic drugs,but also have the potential to improve the oral absorption of drugs. Due to the simplicity of its preparation method,variety and safety of formulations,it is promising in formulation development and drug delivery of both small molecular weight drugs and proteins/peptides.In this work,we took insulin as the model drug, and focused on formulation development and the mechanism of RM formation.In addition,we also deeply studied one of the important mechanisms of bile salt as absorption enhancer of insulin,the suitability of preparation method-freeze-drying tert-butanol(TBA)/water co-solvent solutions in protein formulation.The whole work is divided into four parts as following:The first part includes confirmation and physical characterization of phase region consisting of TBA,bile salt solution and phosphilipid,the compatibility of ingredients of formulation,lyo/cryo-protectant and oil.During the studies of three phase system,we developed a new method to prepare three phase diagram-double phase titration method.Through the physical characterization of two phase system,we revealed the mechanism of solubilization of phosphilipid into TBA/bile salt solution. The result showed that at the concentrations of TBA higher than 9.7%,v/v,bile salt did not display any notable surface activity due to the competitive absorption of TBA on the surface,but still could form micelle with the help of hydrophobic interaction. When the concentration of bile salt was above 28.0mg/ml,the area of clear solution region significantly extended because of the solubilization of phosphilipid by micelle. At the concentrations of bile salt up to 70.0mg/ml,phosphilipid could be solubilized by bile salt in the absence of TBA.In the study of compatibility,we found that the lyophilized powder consisting of bile salt and phosphilipid with a ratio of 7:3 could be solubilized in medium chain mono-glyceride or di-glyceride.And its dispersion in other oil,such as short chain,medium chain and long chain tri-glyceride,can be realized with the help of medium chain mono-glyceride or di-glyceride.In addition, the lyophilized formulations including lyo/cryo-protectant could not be dispersed evenly in all oil abovementioned.The second part is about the important mechanisms of bile salt as absorption enhancer of insulin,the effect of bile salt on the dispersion states,structure and bioactivity of insulin.As one of the absorption-enhanced mechanisms of bile salt,the exact dissociation process of insulin and the driven force induced by bile salt were experimentally proved.The results were as following:(1) in pH 10 buffer,the solution behaviour of insulin was a complex and dynamic equilibrium of monomers,dimers, tetramers and hexamers depending on the concentration and pH;and the transform from monomer,dimer to tetramer,hexamer happened over the concentration range of 0.055-0.11 mg/ml;(2) at the high concentrations of insulin,the hydrophobic interaction between bile salt and insulin oligomers indirectly shifted the equilibrium of insulin multi-components towards insulin monomers,whereas electrostatic repulsion was proposed to directly contribute to the dissociation of insulin oligomers; (3) at molar ratios of bile salt to insulin above 20:1,all insulin oligomer was dissociated into insulin monomer;and these insulin monomers together with bile salt or bile salt micelle further form a complex.During these processes,no notable structural change happened.The third part is to study the interactions of between co-solvent/bile salt and insulin to reveal the self-assembly mechanism of RM.We found that TBA,over the concentrations of 0-15%,v/v,had a similar effect on the dissociation of insulin oligomer as bile salt.However,the entire preservation of insulin structure was due to the molecular segregation happened between insulin and TBA.When the concentration of TBA was high 20%,v/v,the co-solvent could not only dissociate all insulin oligomers(1.56mg/ml) into monomers,but also induced a series of structural changes:the content of a helix increased from 59.3%to 79.5%;at the same time,the contents ofβsheet and random coil decreased from 12.8%and 22.7%to 3.7%and 11.3%,respectively,and the content ofβturn did not have any notable change.In the case of the presence of TBA and bile salt,the stability of insulin decreased.Based on the effect of co-solvent system on insulin structure and bioactivity in the presence of bile salt,we further confirmed the phase region which could be used to prepare RM-the upper limit 10%(v/v) of TBA in co-solvent system.Finally,we revealed the self-assembly mechanism of bile salt and insulin in co-solvent system,which probably contributed to the formation of ARM originally:at the low concentrations of TBA,bile salt and insulin with a molar ratio of 98:1 could assembly into insulin/bile salt complex without any notable change in insulin structure.The fourth part is to reveal both the effect of RM preparation process on structure and bioactivity of insulin and the relative mechanism.The results showed that the factors exerting effect on insulin structure included:condensation induced by freezing,interface denaturation and dehydration resulting from drying,and that the composition of co-solvent was the major factor responsible for the structural change of insulin during lyophilization.And the eutectic system with a volume percent of TBA close to 25%was more destructive compared with other solvent system.Under these conditions,lyophilized insulin had a maximum hypoglycemic effect on STZ-induced diabetic mice at 0.5 hour after injection subcutaneously.However,the formulation with molar ratios of bile salt to insulin of 50:1 and 100:1 could protect insulin through minimizing the denaturation of insulin in phase interface during lyophilization.These formulations displayed a maximum hypoglycemic effect on STZ-induced diabetic mice at 1.0 hour after injection subcutaneously.In addition,the ingredients of formulation,such as trehalose and bile salt,could inhibit the formation of intermolecularβsheet of insulin through physical dilution during lyophilization. The results of spectroscopic studies after the hydration of formulation showed that,in the absence of any ingredient,the formulation with a volume percent of TBA 50% was irreversible in the secondary structure of insulin.However,all other formulations displayed entire recovery in insulin structure.The followed biological activity analysis showed that these formulations with a great recovery in insulin structure still had different hypoglycemic effect on STZ-induced diabetic mice.The different hypoglycemic effect agreed with the degree of structural change during preparation process very well.We proposed that the subtle but decisive incomplete rearrangement or mistake in insulin receptor binding site should be responsible for these differences. Thus,avoiding great structural disruption during the protein formulation process is probably more important than preserving the final native-like structure,although we cannot neglect the directive function of the native structure with regard to the physical and chemical stability of the protein.The results and mechanisms revealed in this study are very important and can be used for reference of other protein and formulation development in lyophilization and studies of protein/peptide drug delivery. |
PDF Full Text Request