Cellular And Systemic Pharmacokinetics Study Of Pegylated Doxorubicin

Most small molecular antitumor drugs have the defects such as poor water solubility,short circulating half life and low bioavailability.Polyethylene glycol modified technology could improve the pharmacodynamic properties of small molecular antitumor drugs,which is one of the hot topics in chemical modification.It also has a good application prospects.Doxorubicin is a commonly used small molecular drug used in cancer chemotherapy.Its adverse reactions include alopecia,myelosuppression,nausea,vomiting,oral mucositis and so on.PEGylated doxorubicin could reduce its adverse reactions.The development of PEGylated drugs requires the establishment of an accurate and robust analytical method capable of analyzing the entire drug and releasing the drug.However,because the molecular weight is not unique,direct analysis of PEGylated drugs is really challenging.Due to the lack of effective methods,pharmacokinetic studies based on intact drug and release drug are difficult to perform.In this paper,a liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was developed for the analysis of PEGylated doxorubicin,free doxorubicin,and free PEG in biological samples and applied to the cellular,tissular and systemic pharmacokinetics study of doxorubicin and PEGylated doxorubicin in rats.This study provides an analytical strategy and theoretical data support for the development and evaluation of PEGylated doxorubicin and other PEGylated medicine.1.Mass spectrometric cleavage of doxorubicin,PEG and PEGylated doxorubicin.Mass spectrometric lysis of doxorubicin,PEG and PEGylated doxorubicin was investigated by high resolution tandem mass spectrometry(Q/Q/TOF)with the Product Ions Scan and MS^all scanning modes.In Product Ions Scan scan mode,fragment ions of doxorubicin were generated by fragmenting precursor ions at the collision energy CE of30 eV,such as m/z 544.0820,526.0735,397.0195,379.0098,361.0028,321.0158,148.0706,130.0634;In MS^all scanning mode,fragment ions related to mPEG subunit with different molecular weights could be obtained under the collision energy CE,such as m/z89.0582,133.0870,177.1152,221.1442 and 265.1738(2,3,4,5,and 6 subunits).Quantitative analysis of different molecular weight mPEGs could be accomplished by these fragment ions related to mPEG subunit.By MS^all scanning,PEGylated doxorubicin undergoes collision-induced dissociation in the quadrupole,PEGylated doxorubicin structurally related fragment ions such as m/z 397.0883,377.0779,361.0673,321.0726associated with the doxorubicin and fragment ions associated with the polyethylene glycol subunit structure m/z 89.0582,133.0836,177.1094,221.1357,265.1619 could be obtained.The experimental results demonstrate that the MS^all scanning mode could be used for the analysis of PEG.It is also a powerful tool for the analysis of polymers with non-uniform molecular weight.2.Pharmacokinetic studies of doxorubicin and mPEG2K-doxorubicin rat.A liquid chromatography tandem mass spectrometric(LC-MS/MS)method for the determination of doxorubicin,mPEG2K-doxorubicin and mPEG2K in rat plasma was developed and applied to the pharmacokinetic studies of doxorubicin and mPEG2K-doxorubicin in rat,the study explains the pharmacokinetic behavior differences between doxorubicin and PEGylated doxorubicin in rats,it also provides a data reference for the development and evaluation of PEGylated drugs.The results show that doxorubicin rapidly distribute from blood to tissues after administration of doxorubicin in rats.After administration of PEGylated doxorubicin,the drug mainly in the plasma is PEGylated doxorubicin,and it gradually release the free drug doxorubicin and PEG.Doxorubicin is the material basis of PEGylated doxorubicin to play the pharmacodynamic effect.PEGylated doxorubicin could reduce the plasma C₀ and C_max concentration of doxorubicin significantly,thereby it could reduce the side effects of doxorubicin;After administration of doxorubicin,the half-life t_1/2 of doxorubicin in rat plasma is 0.498±0.475 h;After administration of PEGylated doxorubicin,the half-life t_1/2/2 of mPEG2K-doxorubicin is 1.41±0.416 h,and the half-life t_1/2 of doxorubicin was 1.38±0.188 h.The PEG modification prolongs the circulation time of doxorubicin in the body.After intravenous administration of 1 mg/kg of doxorubicin to Wistar rat,the AUC_0-t-t of doxorubicin was 97.4±10.4h·?g/L,the AUC_0-?of doxorubicin was 97.7±10.2 h·?g/L;After intravenous administration of 1 mg/kg of mPEG2K-doxorubicin(according to the dose of doxorubicin)to Wistar rat,the AUC_0-t-t of doxorubicin was 177±56.2 h·?g/L,the AUC_0-?of doxorubicin was 184±55.3 h·?g/L.After pegylation,the AUC and bioavailability of the free drug doxorubicin could be increased and the anti-tumor effect of doxorubicin could be improved.After administration of doxorubicin,the apparent volume of distribution of doxorubicin Vz was 7.52±7.37 L/kg;after administration of mPEG2K-doxorubicin,the apparent volume of distribution of doxorubicin Vz was 11.7±4.02 L/kg.As the total volume of rat is 0.667 L/kg,the results indicate that doxorubicin has a wide range of tissue distribution in rats.After pegylation,the apparent volume of doxorubicin has been increased,which means that doxorubicin may have a specific distribution in some tissues.3.The tissue distribution of doxorubicin and mPEG2K-doxorubicin in tumor-bearing miceA LC-MS/MS method was developed for the analysis of doxorubicin and PEGylated doxorubicin in tumor-bearing mice tissues such as brain,heart,liver,genitalia,spleen,lung,kidney and tumor,comparing the distribution differences of doxorubicin and PEGylated doxorubicin in tumor-bearing mice tissues.The results showed that PEGylation could enhance the accumulation and distribution of PEGylated doxorubicin in the tumor tissue,it has a better anti-tumor effect and longer time to maintain efficacy.However,due to the lower concentration of free drug,the efficacy may not be improved significantly in short-term;PEGylated doxorubicin reduces the distribution of doxorubicin in the heart,kidney,lung,spleen,genitalia and brain,it reduces the toxicity of doxorubicin to normal tissues.The safety of PEGylated doxorubicin is better.4.Study on urine and feces excretion of doxorubicin and PEGylated doxorubicin in rats.A LC-MS/MS method for the determination of doxorubicin,mPEG2K-doxorubicin and mPEG2K in rat urine and feces was developed and applied to the study on urine and feces excretion of doxorubicin,mPEG2K-doxorubicin and mPEG2K in rats after administration of doxorubicin or mPEG2K-doxorubicin,respectively.The results showed that doxorubicin could be excreted by urine and feces,the accumulated excretion of doxorubicin in 120h was 6.895%,indicating that a wide range of metabolism occurred in the body in addition to the prototype.PEGylated doxorubicin can be metabolized to doxorubicin and free PEG in the body and excreted in the form of doxorubicin,free PEG and PEGylated doxorubicin.Renal excretion is the main excretion route of free PEG and PEGylated doxorubicin.After pegylation,there was a significant decrease in urine and feces excretion of doxorubicin,while PEG and PEGylated doxorubicin had a larger amount of renal excretion.It should pay more attention on whether PEG and PEGylated doxorubicin have some toxicity to the kidneys or not.5.Cellular pharmacokinetics study of doxorubicin and pegylated doxorubicinA LC-MS/MS method was developed to analyze doxorubicin and PEGylated doxorubicin in cells.The distribution difference of doxorubicin and PEGylated doxorubicin in A549 cells was determined.PEGylated doxorubicin gradually released doxorubicin after it entered the A549 cells.After the A549 cells were administered at 1 h,the doxorubicin amount in the cells accounted for 11.15%of the PEGylated doxorubicin and 10.0%of the total dose(doxorubicin and PEGylated doxorubicin);As time went on,intracellular doxorubicin ratio of the total dose increased slowly to 26.4%at 4 h,the ratio was remained steady at around 20%from 8 h to 72 h,indicating that the slow release could be achieved after pegylation.The amount of doxorubicin in A549 cells after administration of doxorubicin 1-48 h was far greater than the amount of doxorubicin released after the A549 cells cells were administrated with PEGylated doxorubicin.Therefore,during this time,the toxicity and efficacy of doxorubicin should be much larger,indicating that doxorubicin take effect faster than PEGylated doxorubicin.However,due to the high concentration of doxorubicin,the toxicity was greater.Cell culture alone does not have the EPR effect,the amount of PEGylated doxorubicin and doxorubicin in the cells can not directly represent the true situation in vivo.However,the experimental data could explain how PEGylated doxorubicin release doxorubicin in tumor cells and the dynamic ratio changes.The amount of doxorubicin in A549 cells after administration of PEGylated doxorubicin at 72h is 109.85%compared with administration of doxorubicin,indicating that PEGylated doxorubicin has a longer circulation in cells and it could take effect for a long time.