Preparation And In Vitro & In Vivo Evaluation Of Progesterone Nanocrystal Injection

Objective: Progesterone nanocrystal injection(PNSI)was prepared by using insoluble progesterone(PG)to improve the solubility,dissolution rate of PG,physical stability,efficacy,bioavailability and reduce muscle irritation,side effects.This study is mainly to evaluate the PNSI for the prevention of premature delivery,the feasibility and advantage of tocolysis pregnant women through prescription technology,the research on the physical characterization,crystal stability,in vitro evaluation of nano effect,pharmacokinetics and drug safety assessment compared with PG injection(PI).Content: Study on PG nanocrystal formulation to determine the optimal prescription and the optimum process conditions,with PG nanocrystals particle size,polydispersity,potential as the main evaluation index at the same time;the morphology and particle size of the nanocrystals of progesterone to be determined,microscopic characteristics of potential,structure and crystal stability by transmission electron microscopy(TEM),laser particle size analyzer,X ray diffraction(PXRD),differential scanning infrared spectroscopy calorimetry(DSC)and Fourier transform(FTIR);a method for the determination of progesterone content by HPLC to be established,evaluating PNSI in improving the effect of nanocrystals drug saturated solubility and releasing speed;The PNSI and PI were studied pharmacokinetic evaluation by intramuscular injection of two kinds of drugs in rats;the safety of PNSI were evaluated by hemolysis test and muscle irritation test in rabbits.Methods: PNSI was prepared by using wet grinding mill technology,the particle size,Zeta potential and polydispersity index as index,through the screening of single and compound stabilizer type and dosage,optimization of key process parameters,the optimal injection of progesterone nanocrystals the prescription,grinding process and preservation conditions are determined.Optimization of the key parameters of PNSI,the optimal prescription of grinding process and preservation conditions are determined with the selection of the type,the dosage of single stabilizer and compound stabilizer.By TEM to measure the size and morphology of PG nanocrystals,PNSI were made into nano crystal freeze-dried powder through PXRD analysis of the crystal structure,by DSC investigated the phase transition temperature,analyzed the chemical structure of the FTIR and compared with PG,investigation and analysis of the crystal stability of PG after grinding.Comparison of PNSI and PG saturated solubility and in vitro release by shaking method and dialysis method.The determination of PG content of HPLC was established.Study on pharmacokinetics of the PNSI and PI after intramuscular injection by the establishment of PG in rat plasma HPLC-MS analysis method.The hemolysis rate of the PNSI was qualitatively and quantitatively analyzed by hemolysis test.By comparison with PI,study the irritation of PNSI.Results: Polysorbate 80 as PNSI's sole stabilizer through the type and amount of the single stabilizer and compound stabilizer on the screening of PG.The wet mill was selected as the optimum instrument through the comparison of the instrument and equipment,the optimization of the preparation technology was made by adjusting the rotational speed and the grinding time.According to the requirements of the instrument and the characteristics of the drug,the optimal dilution ratio was 1000 times.In the two months of physical stability experiment.PNSI was white suspension liquid,which was diluted to100?g/mL for morphology analysis by transmission electron microscopy,the results showed that PG nanocrystals was a rectangle,a regular edge was neat,uniform shape and uniform distribution,the particle size was about 300 nm.Analysis by PXRD,PG nanocrystals and PG composed of many amorphous structures and a small amount of amorphous structure,having similar characteristic diffraction peaks in the same diffraction angle,the crystal structure is basically the same.In the DSC analysis,PG nanocrystals and PG peaks at similar temperatures,the transition temperatures were similar.In the analysis of FTIR,PG nanocrystals and PG were on the same wave number peaks,no new absorption peaks and the absorption peak occurred without displacement or disappeared,indicating functional groups,structure was not changed,no new chemical bonds were produced in the preparation.Its chemical structure had not changed.HPLC method for progesterone content determination was established,PG had a good linear relationship within the 2.034-152.52?g/m L(y=60.231x-53.088,r>0.999)concentration range.Comparison of PG nanocrystals and PG,measuring the saturation solubility by table method,PG nanocrystals in water saturated solubility was 8 times of PG,the saturated solubility increased significantly.In the dialysis experiment,PNSI released quickly in PBS solution,the drug released 90% in 2h,while the physical mixture of PG and excipients release was slow,the release amount reached 85% when close to 40 h,and the PG in the preparation of PNSI,the release rate was significantly increased.HPLC-MS method for PG determination of dissolution in rats was established preliminarily,Progesterone had a good linear relationship within the 2ng/mL-5000ng/mL(y=0.887041x+0.028667,r>0.999)concentration range.In order to eliminate the differences in the body,the individual and the endogenous hormones in rats,the male SD rats were selected to carry out the self-control experiment,thigh muscle lateral muscle injection of PNSI and PI respectively.The peak time of two preparations was 8.4±13.5h and 40±6.9h,and the peak concentration was 673.7±889.4?g/m L and 258.3±603.3?g/m L,respectively.The PNSI peak time was significantly less than that PI,Cmax and AUC0-t respectively was 2.6 and 2.7 times of PI,its bioavailability was higher than that PI.In the safety evaluation of PNSI,hemolysis test of them were found two tubes which were added much more PNSI showed a slight hemolysis after 1h,the rest of the tubes had no hemolysis and agglutination phenomenon until 3h.According to the regulations,the third tubes and the back of the tube does not appear hemolysis,partial haemolysis or agglutination occurred in the 1h,the drug can be used for injection.Therefore,mild hemolytic reaction of PNSI was acceptable.Through the quantitative analysis of hemolysis,it was found that the hemolytic rate decreased with the decrease of drug,and all the values were less than 5%.During muscle stimulation test in rabbit,compared with PI,mainly in the muscle tissue of muscle fiber degeneration,inflammatory cell exudate,less bleeding,wide range,belonging to the early inflammatory reaction.The response stimulus was 2,which was mild irritation.While the PI mainly in muscle fiber atrophy,necrosis and fibrous connective tissue hyperplasia,bleeding more widely,accompanied by a large number of vesicles,belonging to the late inflammatory stimulation,3-4 series,which was moderate to severe irritation.So PNSI is less irritating than PI.Conclusion: Preliminary screening materials and the amount of the right through the prescription and dosage of PNSI,the preparation process was optimized by the wet grinding process and the adjustment of the process parameters.Through the laser particle size analyzer,TEM,PXRD,DSC and FTIR analysis,it can be seen that the PNSI was a regular rectangle of about 300 nm,and the distribution was uniform.PNSI saturated solubility and release rate were significantly increased in the shaker and dialysis bag experiment.It had lower hemolysis rate,less irritation and higher safety,and in the pharmacokinetic study compared with PI,its bioavailability was improved significantly.In summary,PNSI technology of wet grinding mechanism was feasible,with uniform particle size and crystal stability.PNSI improved the solubility,dissolution rate,bioavailability and reduced muscle stimulation,PNSI had the clinical value of nanocrystal certain.