Development And In Vitro/In Vivo Release Evaluation Of Control Released Delivery Systems For Thymopentin

In recent years,rapid progress in the field of biotechnology resulted in discovery, isolation and production of therapeutic peptides and proteins.However,clinical applications of these agents have been limited for many years due to several problems associated with physical and chemical properties of these compounds.High molecular weight and instability in the GI environment do not allow for conventional formulation of peptides and proteins and preclude the oral route of administration from being effective.As a result,peptide drugs are normally administered parenterally and require frequent administrations due to short biological half-lives caused by rapid chemical and enzymatic degradation in body fluids and to complicated mechanisms of action requiring continuous delivery of a small amount of the drug over extended periods of time.This frequent dosing regiment is responsible for poor long-term patient compliance and lowering of life quality of thousands of people undergoing chronic treatment.Thymopentin(TP5),the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr,as an immunoregulatory therapeutic agent is being used extensively in clinical treatment of immunologically mediated disease.The product available is mainly sterile freeze-dry powder and is administered once per day for about 1～24 months.Therefore, development an extended release formulation of TP5 can improve the patients' compliance and decrease the costs of patients.Objective:To find a fast,dependable and adjustable in vitro release method to evaluation sustained released depot of thymopentin.To develop a set of technology and formulations that is suitable for middle scale-up produce.Methods and Results:A HPLC method is established to characterization TP5 content in microspheres and release medium.The methodology validation results shows that this method is accuracy and sensitive for assay of TP5 concentration in the study.Utilize morphous,particle diameter,span,yield,encapsulation efficiencies,drug loading,burst release and release curve as the main index to evaluate prepared microspheres.Mannitol microspheres was prepared and the single factor test results shows that the particle diameter is decreased as the decrease of PLGA concentration in dichloromethane,O/W ratio and the increase of agitating speed and time.The drug stability test results shows that the drug is instable at 50℃in phosphate buffer saline for 30h,and it is stable -20℃in phosphate buffer saline or under sonification for 12min.In vitro release test of TP5 microspheres was Carried out in different release conditions.Effect of the sorts of release medium and its characters(ion strength,pH, surface active agent) and instrument's parameter(agitating speed,temperature) on TP5 release from preparation was investigated.The release rate in different medium was ethanol solution> acetate phosphate> buffer saline;ion strength suppress release and the affection Tween80 concentration was minute;drug releasing in acid pH environment was faster than in neutral medium;affection of agitating speed on release was small and the release rate constant with temperature was fitted with Arrhenius equation.In vivo release test of TP5 microspheres was carried out in mice by assaying residual agent in subcutaneous' microspheres.Based on in vivo results,a new in vitro release method was established chose 20%ethanol solution as the medium and with a step by step temperature rising program.Choose one of PLGA produced by different companies to prepare microspheres and through DSC analysis found there may be some interactions between the TP5 and PLGA polymers.This finding may be able to explain why different microspheres PLGA has different release curve.Based on orthogonal experiment,obtained the optimized formulation and produced three batch microspheres repeatedly.The microspheres were spherical and range in diameter from 15～50μm when observerd under microscope.The drug loaded in microspheres showed a smoothly curve with minimal burst release(＜10%) and high encapsulation efficiencies(＞90%).The in vitro release curve fulfill the Ritger-Peppas equation.The influences of primary emulsion' sonification time and its inject speed, solidation temperature,water content were investigated.The in vitro release rate increased with the inject time,solidation temperature and water cotent increase,but decreased with the sonification time.According to the scale-up principles and study experiences,amplified the formulation step by step from 2,6 to 18 times.Conclusion:The normal distribution of particles range in diameter from 15～50μm,high encapsulation(＞85%),minute initial release(＜5%) and extended release for one month microspheres are achieved by the optimization of the formulation and production process.