| Glucagon-like pepide-1(GLP-1) is a peptide that is secreted from L cells in the intestine and nerve cells in the brain. It has many bioactivities. Glucose-dependent insulinotropic properties, suppresses glucagons secretion and appetite, and slowing the gastric emptying. In effect GLP-1 can decrease blood glucose in empty or postprandial stomach. Its effects have been proven in the clinical trails with health volunteers and diabetics. GLP-1 may provide an additional approach for the treatment of type II diabetes. But a major obstacle to the realization of the possible therapeutic use of GLP-1 is its rapid inactivation(half-life is less than 2 min), therefore, GLP-1 must be injected IV continuously or SC repetitiously for therapeutic effect, which may bring inconvenience to patients. This study is designed to use poly(lactic-co glycolic acid) as a biodegradable carrier materials to encapsulate GLP-1, and to control the in vitro release properties of GLP-1 allowing for slow release of GLP-1 from microspheres in a month after single administration.The study was done first with the preparation of PLGA microspheres by double emulsion (w/o/w) technique. Single-factor experiment was designed to investigate the factors influencing microspheres size and encapsulation efficiency. Influential factors include stirring rate when preparing the double emulsion, polyvinyl alcohol(PVA) concentration, PLGA concentration, the viscosity of inner water phase, the osmotic pressure of the outer water phase. It is suggested that stirring rate, PVA concentration and PLGA concentration have significant effects on the appearance and particle size of microspheres and that the appearance and particle size are important factors for control the drug release from microspheres. The smaller of microspheres, the more significant burst effect of microspheres. The encapsulation efficiency can be improved high(more than 80%) through enhancing the viscosity of inner water phase or the osmotic pressure of outer water phase. Other factors did not show significant effects with the exception of PLGA concentration and PVA concentration in our test. PLGA with lower molecular weight has also been found to signally increase the degradation rate of microspheres as well as enhance the accumulated release of GLP-1. When gelatin solution was inner water phase and microspheres prepared PLGA with lower molecular weight, the burst effect small than 20% and accumulated release of GLP-1 exceed 85% and the characteristic of drug release approximately accorded to zero order equation. When the concentration of NaCl in outer water phase was 0.25M, the morphology of microspheres was compact. Though microspheres prepared PLGAwith lower molecular weight, the time lag stage was relatively longe. Added 15%(w/w) PEG6000 to oil phase, the release of GLP-1 can be improved and the time lag can be eliminated, then accumulated release of GLP-1 exceed 80% and the release kinetics fitted the zero order mode.Due to the water/oil interface formed during double emulsion formation, drug may be damaged and inactivated. Therefore, solid in oil in water(s/o/w) and solid in oil in oil(s/o/o) methods were use to prepare GLP-1 loaded PLGA microspheres. The key about those techniques is to micronize peptide powder. To dissolve the GLP-1 and PEG6000 in aqueous solution, frozen and lyophilized, we can gain the microparticles after washed PEG by CH2CI2 In order to decrease the burst effect, zinc ion was added the formulation to form Zinc- GLP-1 complex. Single-factor experiment was designed to investigate those techniques. It was that the encapsulation efficiency of the s/o/w method is low (less than 60%); and stirring rate, PVA concentration, PLGA concentration have significant effect on the appearance and size of microspheres, but not on the encapsulation efficiency. Compared to the s/o/w method, because no aqueous solution in the s/0/0 method, and GLP-1 doesn't dissolve in the outer oil phase, the encapsulation efficiency is higher. In vitro tests suggested the release behavior of microspheres, made by PLGA with molecular weight of 34,000 to consisted of typical three stages: burst release stage, time lag stage and slow release stage. However the time lag stage was relatively longer(15~20days) while accumulated release of the slow release stage shorter. This may be caused by the relatively slow degradation rate of polymeric material. When using PLGA with molecular weight of 14,000, time lag stage was almost nonobservable while the accumulated release of slow release stage increased significantly. Total drug release using this polymer in the slow release stage reached 60%. This suggested PLGA molecular weight was the major influential factor of lag time and slow release stages. Additive such as poloxamer 188(F-68) and ZnCC>3 influence the release behavior of microspheres. When F-68 was added to the oil phase, the burst release effect significantly increased., but there was no significant effect on accumulated release. The burst release effect was completely controlled by adding ZnCCb microparticles to the oil phase in s/0/0 method. After optimization of prescription and manufacture technique of PLGA microspheres, the accumulated in vitro release of GLP-1 reached approximately 70%, burst effect was less than 15%, and the release kinetics fitted the zero order equation.To determine the bioactivity of GLP-1 MS in vivo. SD rats were treated with IP... |
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