Construction And Evaluation In Vitro And In Vivo Of Lung-targeted Tedizolid Phosphate Loaded Cationic Liposomes

Tedizolid phosphate is a new anti-bacterial drug developed by CUBIST PHARMS.As a prodrug of tedizolid,tedizolid phosphate is transformed into tedizolid through the phosphate esterase after oral or intravenous administration.Tedizolid,the second generation of oxazolidinone antibiotics,is a protein synthesis inhibitor to inhibit bacterial protein synthesis through binding to the bacterial ribosome 50S subunit.Moreover,its antibacterial effect has no cross-resistance with other types of antibiotics.In addition,tedizolid phosphate has a longer half-life compared to the first-generation oxazolidinone antibiotic linezolid.Clinical studies have shown that tedizolid not only has better clinical efficacy than linezolid,and the cycle of treatment is diminished by 40%.However,tedizolid phosphate is unstable at room temperature,easy to hydrolyze,which brings great inconvenience to storage and transportation.Moreover,the lung targeting is not enough high for the treatment of pneumonia.And it will lead to the risk of drug resistance by simply increasing the dose to improve the drug concentration in lung.Liposomes,as a potential sustained release system,can encapsulate both water-soluble drugs and fat-soluble drugs.Especially as an antibiotic drug carrier,liposomes have many advantages:?To improve the pharmacokinetics parameters and biological distribution of drugs;? To improve the antibacterial effect of antibiotics and reduce the risk of antibiotics resistance for pathogenic bacteria;?With local targeted selectivity.However,cationic liposomes can also reduce the changes of particle size during storage of liposomes and maintain the stability of liposomes besides these advantages mentioned above.In addition,the particle size and potential are important factors affecting the passive lung targeting of the carrier.The relatively large particle size and positively charged liposomes have been good for lung targeting.In conclusion,for the antibiotic carrier,cationic liposomes not only have improved stability and lung targeting,but also better biofilm affinity,which has an irreplaceable advantage for the treatment of pneumonia.In this study,with tedizolid phosphate as the model drug,cationic liposomes were prepared by reverse phase evaporation method using soybean lecithin phosphate and cholesterol as the material of liposomes membrane.The aim is to increase the stability and lung targeting of tedizolid phosphate,improve the therapeutic effect and reduce the occurrence rate of antibiotics resistance.The main contents of this research include the preparation and physicochemical properties evaluation of tedizolid phosphate loaded liposomes and tedizolid phosphate loaded cationic liposomes,drug-release behavior in vitro and preliminary safety evaluation,the pharmacokinetics and tissues distribution in mice to evaluate the targeting and sustained release behavior of tedizolid phosphate loaded cationic liposomes in mice.1.Preparation and physicochemical properties of tedizolid phosphate loaded liposomes.The ultraviolet spectrophotometry(UV)was applied to determine the content of tedizolid phosphate.The results showed that the excipients did not interfere with the determination of the drug,and the specificity,precision and recovery rate were all in accordance with the determination requirements.The method of ultrafiltration centrifugation was used to determine the entrapment efficiency.The results showed that this method was energy-saving,having high recovery rate and good reproducibility.Based on the dissolution characteristics of tedizolid phosphate,the tedizolid phosphate loaded liposomes(TDZA-Lips)were prepared by reverse phase evaporation method,and the preparation process was systematically studied with the particle size and entrapment efficiency as the index.On the basis of single factor investigation,the orthogonal experiment was designed to select the optimal prescription and technology.The characteristics of the resulting liposomes according to the optimal prescription and technology were as follows:The liposomes is spherical-like and uniform particles under transmission electron microscopy.The average particle size was(184.9 ± 8.3)nm;the average zeta potential was(-22.8 ±0.15)mV;the encapsulation efficiency was(55.10 ± 1.94)%;the drug loading was(4.51 ± 0.24)%;the average pH value was 6.83 ? 0.06,which accorded with the requirement of injection.The results of liposomal reproducibility test of three batches showed that the prescription was reasonable and the preparation process was feasible.2.Preparation and physicochemical properties of tedizolid phosphate loaded cationic liposomes.Firstly,chitosan-coated tedizolid phosphate loaded liposomes(c-TDZA-Lips)and stearylamine-modified tedizolid phosphate liposomes(SA-TDZA-Lips)were prepared,respectively.The optimal prescription of c-TDZA-Lips was ensured by single factor investigation for molecular weight of chitosan,concentration of chitosan,potential and other factors.The optimal prescription of SA-TDZA-Lips was determined by single factor study of potential.The c-TDZA-Lips and SA-TDZA-Lips prepared before were observed under transmission electron microscopy.The results indicated that the size distribution was uniform and the particle size of chitosan-encapsulated liposomes increased slightly.The average particle size of c-TDZA-Lips and SA-TDZA-Lips were(214.3 ± 4.2)nm and(194.9 ± 2.93)nm,respectively.The Zeta potential of the two tedizolid phosphate loaded cationic liposomes are all converted from negative charge to positive charge.The average zeta potential of c-TDZA-Lips and SA-TDZA-Lips were(19.5 ± 2.10)mV and(20.4 ?1.10)mV,respectively.The encapsulation rates of c-TDZA-Lips and SA-TDZA-Lips were(55.38 ± 2.18)%And(53.52 ± 2.18)%,respectively.The average pH values of c-TDZA-Lips and SA-TDZA-Lips were 4.30 and 8.86,respectively.The reproducibility test of the three batches of c-TDZA-Lips and SA-TDZA-Lips all showed that the reproducibility of the prescriptions were good and the preparation process was feasible.3.Evaluation of in vitro release behavior and preliminary safety of three kinds of tedizolid phosphate loaded liposomes.The in vitro release properties of TDZA-Lips,c-TDZA-Lips and SA-TDZA-Lips were compared by dynamic membrane dialysis method.Compared with the injection of tedizolid phosphate,the in vitro drug release curves of the three kinds of tedizolid phosphate were similar,and the preparations all had a certain sustained-release effect.Moreover,the release curves were all conforming to the weibull equation.The c-TDZA-Lips had a relatively slower release followed by SA-TDZA-Lips.In addition,The hemolysis test in vitro were applied to evaluate liposomal preliminary safety using erythrocyte from healthy rabbits in this part.The hemolysis and agglutination did not occur on TDZA-Lips and SA-TDZA-Lips by naked eye observation,indicating that the two kinds of liposomes preparations were safe and reliable and could be applied to intravenous injection.However,the hemolysis and agglutination with different degree occured on c-TDZA-Lips except for lowest drug-concentration.Therefore,the c-TDZA-Lips with poor safety were not suitable for intravenous injection.4.Pharmacokinetics and tissues distribution in mices.The HPLC method was established to determine the concentration of tedizolid phosphate in mice plasma and tissues.The results showed that the method could meet the determination requirement of biological sample.The plasma pharmacokinetics of TDZA-Lips and SA-TDZA-Lips were studied after intravenous injection.The pharmacokinetic results showed that compared with TDZA-Inj group,the MRT0-6 of TDZA-Lips group and SA-TDZA-Lips group was prolonged to 2.39 times and 2.80 times,the CL decreased by 0.64 times and 0.71 times,the t1/2? was extended to 1.15 and 1.51 times,the AUC0-? increased by 1.76 times and 2.40 times,respectively.All the changes were helpful to improving the antibacterial effect of tedizolid phosphate.In addition,the drug distributions in diferent tissues like heart,liver,spleen,lung,kidney and brain were also determined.Then the target parameters of TDZA-Lips and SA-TDZA-Lips were calculated in diferent tissues.The results showed that the AUC of the SA-TDZA-Lips was increased by 0.72 times in the lung.This was due to the positively charged stearylamine which contributed to the increased affinity to the lung cells and cell intake.The relative uptake rate of the SA-TDZA-Lips in the lung was 1.527>1,which indicated the lung targeting.These changes indicated that the modification of stearylamine increased the distribution and targeting behavior of liposomes in the lungs,which was in favor of the treatment of pneumonia.In summary,the in vitro evaluation,pharmacokinetics and tissues distribution of SA-TDZA-Lips have basically reached the purpose expected before.Based on the fact that the application of antibiotics is indispensable to the basic principles of treatment,the stearylamine-modified tedizolid phosphate loaded liposomal delivery system constructed in this study are expected to be developed for clinical use as a new carrier for the long term treatment of chronic pneumonia.