| In the discovery of new drugs,more than 40%of new drug candidates are categorized as practically insoluble.The low solubility leads to a poor absorption,and limits the development of injections,which is not conducive to the development and clinical application of drugs.Increasing the solubility of poorly water-soluble drugs has become one of the important challenges in the development of new drugs.The microparticle delivery system provides a good solution to the problem of insolubility of drugs and has received extensive attention and research.Commonly microparticle delivery systems include:liposomes,solid lipid nanoparticles,microemulsions,polymer micelles,polymer nanoparticles,etc.However,these microparticle delivery systems generally use a large amount of carrier material,which has the limitation of low drug loading.Nanosuspensions are submicron colloidal dispersions which realizes pure drug particles with a small amount of surfactant.as stabilizer.It eliminates the need for a large number of carrier materials and overcomes the above drawbacks of microparticle delivery systems.It is not only suitable for water-insoluble drugs but also for drugs that are insoluble in both aqueous and organic phases.Nanosuspensions have the advantages of good safety,high drug content,simple preparation process and be suitable for large-scale production,and is widely used for the delivery of hydrophobic drugs.On the other hand,intravenous injections of nanosuspensions can also achieve targeted delivery through EPR effects by controlling particle size.Currently,many nanosuspensions of poorly soluble drugs have been successfully marketedTamibarotene(Am80)is a novel synthetic specific retinoic acid receptor(alpha/beta)agonist.It is insoluble in water and has low oral bioavailability.In clinical,it is mainly used for relapse and refractory treatment.In addition,Am80 also has anti-inflammatory and anti-angiogenic activity.It might be key candidate treatment for hepatocellular carcinoma.This project was designed to prepare the insoluble drug Am80 as a nanosuspension with a particle size of<100 nm and a particle size of about 200 nm,and to study its distribution and targeting in mice by intravenous injection,providing a theoretical basis for the clinical application of the new dosage form of Am80.1.Preparation and physicochemical properties of tamibarotene nanosuspensionThe ultraviolet spectrophotometry(UV)method was established to determine the drug content of Am80 nanosuspension and the methodological investigation was conducted.The Am80 nanosuspensions were prepared by nanoprecipitation method.Screening the Am80 nanosuspension with the particle size<100 nm and the smallest particle size by single factor investigation.Then,the prescription was adjusted to prepare Am80 nanosuspensions with a particle size of about 200 nm.And three batches of reproducibility tests and physicochemical properties of Am80 nanosuspensions of two particle sizes were studied.The results showed that the UV method for determining the Am80 content was characterized by high specificity,high precision,good recovery,and met the requirements.The three batches of nanosuspensions of two sizes had good reproducibility.The Am80 nanosuspension particles with particle size<100 nm observed under transmission electron microscope showed good formability and spherical morphology.The particle size was 73.07±10.63 nm,the potential value was-16.1 mV,and the drug content was 4.51%.The particle morphology of Am80 nanosuspension with a particle size of about 200 nm was more rounded,with an average particle size of 206.7 ± 14.3 nm,a potential of-13.5 mV,and a high drug content of 6.89%,which all met the design requirements.2.The in vitro drug release and preliminary safety evaluationFirstly,a UV spectrophotometric method for the determination of Am80 in the release medium was established and the methodology was investigated.Then,the in vitro drug release properties of Am80 nanosuspensions were performed by dynamic membrane dialysis.Finally,the preliminary safety of the two sizes of Am80 nanosuspensions was evaluated by histological examination.The results showed that the UV method for determining the Am80 content in the release medium has a high recovery,the linear relationship of Am80 in the concentration range of 5 μg · mL-1 to 30 μg · mL-1 was good and met the measurement requirements.The in vitro release result displayed that the two sizes nanosuspensions released rapidly in the initial phase,which can reach 58.61%and 56.68%respectively in the first 8 h,and then slowly released.The drug release was complete within 48 h.The in vitro release behaviors of Am80 nanosuspensions were consistent with Weibull Equation equation.Histological examination results showed that intravenous injection of Am80 nanosuspensions of two sizes had good safety,which did not cause inflammation and damage to the heart,liver,spleen,lungs,kidneys,and other tissues and organs of mice.3.The in vivo pharmacokinetics and tissue distribution studiesFirstly,a method for the determination of Am80 in plasma and tissues in mice was established by HPLC,and the methodology was investigated.Then,the mice were randomly divided into three groups,and respectively injected intravenously with Am80 solubilized solution(as a control group),Am80 nanosuspension with a particle size of<100 nm(sample group 1),and Am80 nanosuspension with a particle size of about 200 nm(sample group 2)at a dose of 15 mg·kg-1.At different time points,blood was collected from the mouse eyeball to determine the drug concentration in the plasma.The blood drug concentration-time curve was plotted and the kinetic parameters were obtained via using the DAS 2.0 pharmacokinetic program to study the pharmacokinetics of Am80 nanosuspension in mice.Finally,hearts,liver,spleen,lungs,kidneys and femurs were isolated after sacrificing the mice at each sampling time point to determine drug concentration in each tissue,and targeting parameters were calculated to evaluate the targeting effect of Am80 nanosuspensions of two sizes in bone marrow,liver,spleen and other issues.The results showed that this method is very specific and no endogenous impurities interfere with the determination of the drug.The linearity of Am80 was good in the linear range of 0.2～20 μg·mL-1.The intra-day relative standard deviation(RSD)was less than 5%and daytime RSD was less than 10%.The extraction recovery rate is greater than 80%and RSD<10%.The above results showed that this method meets the requirements of the analysis and determination of biological samples.Compared with the solubilized solution group,the kinetics properties of the nanosuspensions of two particle sizes are similar and there is no obvious sustained release effect.The result of tissue distribution experiment showed that the concentration of Am80 distributed in the liver was very low(both less than 1 μg.g-1)after intravenous injection of Am80 solubilized solution and Am80 nanosuspension of two particle sizes.It may be related to the nature and the in vivo metabolism of Am80.Studies have shown that Am80 can be rapidly metabolized by liver drug enzymes in the liver,and its oral bioavailability is low.In addition,the Am80 nanosuspension with a particle size of<100 nm increased the drug concentration in the bone marrow by approximately 30%.Therefore,the Am80 nanosuspension with a particle size of<100 nm achieved a certain degree of bone marrow targeting(re = 1.3).The Am80 nanosuspension with a particle size of about 200 nm increased the distribution of drugs in spleen(re = 3.16).In conclusion,this project successfully prepared Am80 nanosuspension with particle size<100 nm and particle size about 200 nm,which can be injected intravenously.And it improved the insolubility of Am80 and overcame the shortcomings of conventional injections with large toxic and side effects.In vivo studies results have shown that Am80 nanosuspensions with a particle size of<100 nm increase the distribution of drugs in the bone marrow which had great significance to improve the therapeutic effect of acute promyelocytic leukemia and explored new ideas for clinical treatment of acute promyelocytic leukemia.The drug concentration measured in the liver is very low,and the specific mechanism of action on hepatocellular carcinoma needs further study. |
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