Preparation And Evaluation Of Rifapentine Lipid Microsphere Temperature-Sensitive-Gel

Tuberculosis（TB）is a chronic infectious disease caused by mycobacterium Tuberculosis.With the emergence of multi-drug resistant strains of Tuberculosis and the increase of secondary immunodeficiency,the incidence of TB is on the rise globally^[1].Rifapentine（RPT）is a fat-soluble long-acting rifamycin antibiotics.As a semi-synthetic broad-spectrum bactericide,Rifapentine is 2-10 times more effective than rifampicin against mycobacterium tuberculosis in vitro,in particular,the growth of mycobacterium tuberculosis in the stronger bactericidal effect^[2].However,Rifapentine is difficult to dissolve in water,sensitive to light,and its stability is poor.At present,most RPT capsules are taken orally in clinic.The first pass elimination of this dosage form is large and its bioavailability is low,which limits its clinical application to a certain extent（[3]）.In order to overcome these problems,we begin to treat pulmonary tuberculosis by fiberoptic bronchoscopy.However,the new problem is that there is no suitable preparation to achieve better therapeutic effect.Therefore,it is urgent to develop a stable,good retention,self-degradation,high bioavailability Tuberculosis management system.In this study,Rifapentine was prepared into Lipid microsphere Temperature-Sensitive-Gel,and the optimal formulation and preparation technology were selected.The physicochemical properties and safety of the GEL were evaluated.To provide a new dosage form for the treatment of tuberculosis.Part one Pre-prescription studyObjective:To establish a method for determining of the content of Rifapentine,to determine the oil-water partition coefficient of Rifapentine and to determine its equilibrium solubility in different oil phase.Methods:The content of Rifapentine was determined by High-performance liquid chromatography,and its linearity,specificity,precision,stability and recovery rate were investigated.The oil-water partition coefficient of rifapentine was determined by shaking flask method,and the solubility of rifapentine in each oil phase was determined by equilibrium solubility method,and to screen out the suitable oil phase.Results:A HPLC method was established for the determination of rifapentine.Its linearity,specificity,precision,stability and recovery were all in accordance with the requirements of the methodology.The oil-water partition coefficient of rifapentine is 1.98,and its lipophilicity is strong.The oil phase with higher solubility to Rifapentine were selected.Conclusion:The HPLC method for the determination of Rifapentine is suitable for the test and can be used for the determination of Rifapentine.Moreover,the oil-water partition coefficient of Rifapentine was suitable,which laid a foundation for the formulation design and process research in the later period.Part two Preparation and investigation of physical and chemical properties of Rifapentine Lipid Microsphere（RPT-LM）Objective:To determine the optimal formulation and preparation technology of Rifapentine Lipid Microsphere（RPT-LM）,and to investigate its physical and chemical properties.Methods:The appearance and stability of the Lipid microspheres were used as the evaluation indexes,the dosage of each component was selected by single factor investigation.The particle size and the centrifugal stability constant（Ke）were used as the evaluation indexes,the optimum formulation of Lipid microspheres was selected by Orthogonal test,and the optimum preparation technology was also selected.The optimal formulation and preparation technology of RPT-LM were studied.Results:The optimal prescription of RPT-LM was as follows:RPT 50mg,corn oil 1.0 g,soy phospholipid 0.9 g,glycerol 0.25 g,oleic acid 0.05 g,Vitamin E 0.5 g,adding distilled water to 10 ml.The optimum preparation conditions are:preparation temperature 80°C,Shear time 11 min,shear speed11000 r/min.RPT-LM is Orange Opaque,without layer,without oil drop on the surface,with particle size of 482.4 nm,PDI of 0.264,entrapment efficiency of 55.51±0.50%and content of 4.93±0.02 mg/m L.Conclusion:RPT-LM was successfully prepared in this part,which had good stability and improved the solubility of Rifapentine.Part three Preparation and investigation of physical and chemical properties Rifapentine Lipid Microsphere Temperature-Sensitive GelObjective:The preparation of Rifapentine Lipid Microsphere Temperature-Sensitive-Gel（RPT-LM-TSG）was studied.The physical and chemical properties and drug release behavior in vitro were evaluated.Methods:The optimal formulation of RPT-LM-TSG was obtained by using PLGA-PEG-PLGA as the Temperature-Sensitive-Gel Matrix and the Gelation temperature and gelation time were used as evaluation indexes,And Its physical and chemical properties were investigated.The drug release behavior in vitro was investigated by dialysis bag method.Results:The optimal formulation of RPT-LM-TSG was determined as follows:The volume ratio of RPT-LM to PLGA-PEG-PLGA solution with concentration of 20%was 2:1.The particle size of RPT-LM-TSG was 589.4nm,the PDI was 0.142,the gelling temperature was（34.47±0.15）℃,the gelling time was（41.33±1.70）s,and the degradation time was longer in vitro.When the drug was released in vitro for 30 days,the cumulative release rate was more than 80%,and the drug release concentration at each time point was higher than the minimum inhibitory concentration.The release behavior in vitro was in accordance with the Riger-Peppas kinetic model.Conclusion:The RPT-LM-TSG was prepared successfully,the gelling temperature and gelling time were suitable,the release time in vitro was long,it had certain sustained-release effect and accorded with the characteristics of pulmonary administration.Part four Evaluation of cell level and pharmacodynamics of Rifapentine Lipid Microsphere Temperature-Sensitive GelObjective:To evaluate the cytotoxicity and pharmacodynamics of RPT-LM-TSG for pulmonary delivery.Methods:MTT assay was used to determine the survival rate of A549cells,and the safety of A549 cells was evaluated at the cellular level.The uptake of Cou-6-LM was observed by confocal laser scanning microscopy.RPT-LM-TSG was given to SD rats through tracheal intubation to observe the retention of the preparation in the lungs.The bacteriostatic test of lung tissue extract was used to observe the bacteriostatic effect of the preparation and evaluate the pharmacodynamics of the preparation.Results:The blank gel matrix had little cytotoxicity to cells,and the carrier material was safe.In a certain range of concentration and time,the drug uptake of A549 cells increased with the increase of concentration and time.After administration of the drug through a tracheal Tube,orange-colored preparations can be seen trapped in the lungs of rats.Lung tissue containing the drug had some inhibitory effects on Staphylococcus aureus and mycobacterium smegmatis.Conclusion:The PRT-LM-TSG is less toxic,can be ingested by A549cells,and can be retained in the lung after administration through tracheal intubation.