Studies On The Nano-pharmacokinetics Of Doxorubicin Hydrochloride Liposome Injection

When evaluating the pharmacokinetics of nanoparticles,the concentration of released drug should be considered as the foundation,the released drug is the key to connect pharmacokinetics,pharmaceutics and toxicology.With a comprehensive understanding of the distribution,metabolism and excretion of released drug in vivo,the pharmacokinetic parameters obtained are "bioavailable" and could provide a more accurate judgment of the efficacy and toxicity for nanoparticles.A commercial available doxorubicin hydrochloride PEGlyted liposome injection(DOX·HC1-PLI)was selected as the model drug,to evaluate the nano-pharmacokinetics for nanoparticles in rat and tumor-bearing mice.The inhibition of DOX·HC1-PLI to human CYP450 enzymes was also assessed in our study.This paper provided a lot of data for the establishment of nano-pharmacokinetics of DOX·HC1-PLI and a new idea for the evaluation of nanoparticles.1.Pharmacokinetics study of DOX·HCl-PLI in rat plasma.A new sample preparation method for the separation of released doxorubicin(R-DOX)in rat plasma was developed,and a new liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the simultaneous quantitation of DOX and its five metabolites ·has been developed and validated.It has been shown that practically all of the DOX in plasma was in liposome-encapsulated form(>95%).Compared with free drug,the Cmax of R-DOX decreased about 4-fold,and the initial plasma concentration(C0)was decreased about 10-fold,this may reduce the cardiac toxicity of DOX.The area under the plasma concentration-time curve(AUC)of R-DOX was increased about 9.6-fold,and AUC of total DOX(T-DOX)was increased significantly,about 783-fold increase.The volume of distribution of free drug was 72632 mL/kg,of R-DOX was 5376 mL/kg and of T-DOX was only 201 mL/kg.The concentrations of five metabolites in plasma were negligible,indicating that the metabolite rate is slower than that with free DOX.2.Tissue distribution of DOX-HC1-PLI in rat.A new sample preparation method for the separation of encapsulated doxorubicin(E-DOX)in rat tissues was developed and validated.DOX·HC1-PLI could target to v immune system through RES uptaking,the maximum tissue concentration of R-DOX in spleen was increased about 1.5-fold,and of T-DOX was increased 2.1-fold.The cardiac toxicity of DOX is highly correlated with the maximum tissue concentration of DOX in heart,and the maximum tissue concentration in heart of R-DOX was decreased 2.8-fold.The concentration of R-DOX has a dynamic equilibrium in rat tissues,and the R-DOX/T-DOX ratio was between 70-80%in most tissues,and about 60%in lung tissue.The metabolites accumulation in tissues was delayed compared with free drug,the concentrations of five metabolites were almost could not be detected at 1h and 12 h.3.The tissue distribution of DOX·HC1-PLI in tumor-bearing miceDOX·HC1-PLI has enhanced the tissue distribution in tumor,the maximum concentration of R-DOX in tumor has increased about 26%,and T-DOX has increased about 58%.DOX·HCl-PLI could reduce the cardiac toxicity of the drug in mice compared with free DOX;the maximum concentration of DOX was 40-fold higher than R-DOX,and 17-fold higher than T-DOX.The release and elimination rate of R-DOX in tumor,liver,spleen and lung has reached the dynamic equilibrium at 12 h after administration,and at 36,86%of the drug was existed as released form.Ml was the main metabolite in mice tissues,and no M3 was observed.4.Study on bile and urine excretion of DOX·HCl-PLI.A highly sensitive LC-MS/MS method new sample preparation method for the separation of encapsulated doxorubicin(E-DOX)in rat tissues was developed and validated.After a single intravenous injection of 5 mg/kg DOX·HCl,32%of the drug has been excreted through bile within 96 h(DOX,27%;M1,4.4%;M3,0.07%;M4,0.02%and M5,0.02%),and 11.2%of drug has been excreted through urine within 120 h(DOX,5.55%;M3,0.002%;M4,0.01%).After a single intravenous injection of 5 mg/kg DOX·HCl-PLI,6.35%of drug has been excreted by bile within 84 h(DOX,5.55%;M3,0.002%;M4,0.01%),and 3.37%of drug has been excreted by urine within 120 h(DOX,3.105%;M1,0.186%;M2,0.075%).Compared with free DOX,the cumulative excretion amount of drug in bile was increased about 5.0-fold,and the cumulative excretion amount in urine was increased 3.3-fold.M2 was mainly excreted through urine and M3 mainly excreted through bile.5.The inhibition of DOX·HC1-PLI to human CYP450 enzymesA high-throughput LC-MS/MS method based on positive/negative ion exchange technology for the determination of nine metabolites of specific probe substrate drug has been developed and validated.The method could study on the inhibition of CYP1A2,CYP2D6,CYP2E1,CYP2C9,CYP2C19,CYP3A4,CYP2A6 and CYP2B6.The inhibition results indicated that free DOX could inhibit all the CYP450 enzymes,but DOX·HCl-PLI had no or very week inhibition on these CYP450s.After administration of DOX·HCl-PLI,the pharmacokinetics of R-DOX,E-DOX and T-DOX were different.Only the released drug is the "bioactive" compound,so it is inappropriate to evaluate the pharmacokinetics of DOX·HCl-PLI with T-DOX.When evaluating the pharmacokinetics of nanoparticles,the concentration of released drug should be considered as the foundation.