| Irinotecan(IRT)(a camptothecin derivative)is a prodrug,which must be converted to its active metabolite 7-ethyl-10-hydroxy camptothecin(SN38)to produce antitumor effect and the metabolic conversion of IRT to SN-38 in vivo is accomplished by carboxylesterases,which exist in plasma and many tissues,especially liver,and even in tumors.Topoisomerase I,Which has high expression in tumor cells,is a ubiquitous enzyme involved in DNA replication and RNA transcription.Tumor cells have more topoisomerase I than normal cells,therefore,SN-38,a topoisomerase I inhibitor,can cause DNA strand breakage and thus induce death or apoptosis of the tumor cells.Therefore,it has inhibitory effects on various tumors such as colorectal cancer,breast cancer,ovarian cancer,small cell lung cancer and etal.The anti-tumor activity of IRT depends on the integrity of the lactone ring in its structure:in an acidic environment with a pH<7,IRT exists mainly in the form of lactone ring and has anti-tumor activity;when in the alkaline environment(pH>7),Some lactone rings will open and lose anti-tumor activity.Currently,the commercially available dosage form is irinotecan hydrochloride injection,which can greatly improve the insolubility of camptothecins,but intravenous drip administration will expose the drug to an alkaline en'vironment with pH 7.4,leading to the lactone ring open,so that the anti-tumor activity is reduced,so a larger dose is required to achieve the desired activity;however,large doses lead to a series of side effects such as severe diarrhea,vomiting,neutropenia and severe inhibition of bone marrow cells.Besides,Camptothecins belong to time-dependent drugs which means their prolonged residence time in-vivo will increase drug efficacy.Therefore,it is of great clinical significance to improve the efficacy of IRT and reduce its toxic and side effects.As a novel nanocarrier drug system,polymer nanoparticles(Polymeric Nanoparticles)have many advantages.Firstly,it can be used as a carrier for many drugs(water-soluble or water-insoluble)to increase their bioavailability;secondly,the application of some special materials(such as PLGA,etc.)can make nano-formulations have a long-lasting,sustained-release effect;lastly,polymer nanoparticles can usually be passively targeted to the tumor site by EPR effect due to proper particle size,thereby improving drug efficacy.Among them,PLGA polymer nanoparticles have received extensive attention and research because of the excellent characteristics of PLGA itself.In this paper,a kind of long-acting,sustained release nanoparticles(IRT-PLGA-Nps)was prepared by emulsification solvent evaporation method using IRT as model drug.The main research contents include:preparation and characterization of IRT-PLGA-Nps;the in vitro release characteristics and preliminary safety evaluation of IRT-PLGA-Nps;pharmacokinetic evaluation of IRT-PLGA-Nps;pharmacodynamic evaluation of IRT-PLGA-Nps.1.Preparation and characterization of IRT-PLGA-NpsFirstly,the in vitro determination method of IRT was established by UV spectrophotometry;secondly,IRT-PLGA-Nps was prepared by emulsification solvent evaporation method,and the factors(including prescription factors and process factors)that may affect the IRT-PLGA-Nps particle size,encapsulation efficiency and drug loading were screened to determine the best prescription;finally,the optimal prescription was reproduced and characterized by a series of characterizations including encapsulation efficiency,drug loading,particle size,Zeta potential,pH,microscopic morphology(TEM),and phase identification(DSC).The results showed that the linear relationship between the UV absorbance and the concentration of IRT DMSO solution was good in the concentration range of 2.55?20.4?g/mL.The method has good accuracy,high precision and strong specificity.Through single-factor screening,the effect of process factors(shear rate and shear time)on the nanoparticle size was investigated,and the optimal process was determined as follows:the shearing rate was 25000 rpm and the shearing time was 3 min;then the prescription factors which effect the encapsulation efficiency and drug loadingthe of IRT-PLGA-Nps were examined,and the optimal prescription was determined as follows:the ratio of drug to lipid was 1:10,the concentration of PVA in the aqueous phase of the colostrum was 1%,and the concentration of PVA in the dispersed phase was 0.5%,the ratio of oil phase-water phasee was 1:3 and the volume ratio of water phase-disperse phase was 1:5.Finally,a series of characterizations of the best formulation showed that the prepared IRT-PLGA-Nps has an entrapment efficiency of(52.22 ± 2.41)%and a drug loading of(4.75 ± 0.22)%,whats's more,the average particle size was(169.97 ± 6.29)nm and the Zeta potential was(-1.75±0.20)mV,the pH was(6.93 ± 0.04).The reproducibility was good from batch to batch.2.In vitro release characteristics and preliminary safety evaluation of IRT-PLGA-NpsIn order to provide some references for in vivo experiments,we studied the in vitro release characteristics of IRT-PLGA-Nps through a dynamic membrane dialysis method,and IRT-Sol was used as control.The drug release of IRT-Sol and IRT-PLGA-Nps at different time points was determined by UV spectrophotometry.The cumulative release percentage was calculated and the release curve was plotted.Finally,the release results were fitted with a mathematical model to analyze the in vitro release behavior of IRT-PLGA-Nps.In addition,the stability of IRT-PLGA-Nps was investigated by plasma stability experiments,the preliminary safety of IRT-PLGA-Nps was evaluated by hemolysis experiments,which provided a possibility for its in-vivo application.The results showed that there was a good linear relationship between the UV absorbance and concentration of IRT in the release medium at the concentration range of 2.5?20 ?g/mL,and the methodological results showed that the method has high accuracy and good precision.Dynamic membrane dialysis was used for in vitro release experiments.The results showed that compared with the solution group,the nanoparticle group showed a significant sustained-release effect.IRT-Sol released completely at about 6h,and the cumulative release rate reached(93.94 ± 3.98)%.The release rate of IRT-PLGA-Nps was relatively slow,and the cumulative release rate at 12h was(52.06±1.65)%,indicating that the prepared nanoparticles had a certain burst release phenomenon.After 192h,IRT-PLGA-Nps released completely,and the cumulative release percentage exceeds 97%,which indicated that the IRT-PLGA-Nps can initially release a large amount of drug,and then slowly release to maintain a higher therapeutic concentration;a mathematical model fitting of the release curve shows that the in vitro release behaviors of IRT-Sol and IRT-PLGA-NPs occurred in accordance with the Weibull equation and could be expressed as lnln(1/(1-Q/100))=2.18181nt-0.9604(r=0.9996)and Inln(1/(1-Q/100))=1.13931nt-1.8371(r=0.9941)The results of plasma stability test showed that within 24 hours,the average particle size of IRT-PLGA-NPs was relatively stable and could meet the injection requirements.The results of hemolysis experiments showed that the percentage of hemolysis of IRT-PLGA-NPs was less than 5%at the concentration range of 20?100 ?g/mL,which indicated that IRT-PLGA-NPs can be safely used for intravenous injection.Experiments in this part laid a certain foundation for pharmacokinetic experiment and pharmacodynamic experiment in vivo.3.Pharmacokinetic evaluation of IRT-PLGA-NpsIn order to evaluate the in vivo sustained release and pharmacokinetics characteristics of IRT-PLGA-Nps,this section investigated the pharmacokinetics of IRT-PLGA-Nps.The determination method of prodrug(IRT)and its active metabolites(SN38)in plasma was established by high performance liquid chromatography(HPLC);subsequently,ordinary KM mice were used as animal models and IRT-Sol was used as controls,the concentration of IRT and its active metabolite SN38 in mice was determined at the given time point,and drug concentration-time curve was plotted.DAS 2.0 software was used to calculate the pharmacokinetic parameters of prodrug(IRT)and its metabolites(SN38)after IRT-PLGA-Nps and IRT-Sol were injected into the mice,so as to evaluate the kinetic characteristics of IRT-PLGA-Nps and IRT-Sol.The methodological results show that the linear relationships between the plasma concentration and peak area of both IRT and SN38 are good at the range of 0.04?6.4 ?g/mL,and the method has high specificity,good accuracy,and high precision.The results of pharmacokinetics experiments showed that the retention time of IRT and SN38 in the IRT-PLGA-Nps group was significantly larger than that of the IRT-Sol group after tail vein injection.Firstly,from the results of the determination of the concentration of the prodrug IRT,we can concluded that the t1/2?,MRT,and AUC values of the IRT-PLGA-Nps were 6.89,3.61,and 2.93 times higher than those of the IRT-Sol group,respectively,which meaned the nanoparticles group can significantly prolong the retention time of IRT in plasma and increase the bioavailability of IRT.Secondly,the pharmacokinetic parameters of metabolite SN38 were analyzed.The t1/2?,MRT,and AUC values of the IRT-PLGA-NPs group were 2.55,4.43 and 2.31 times those of the IRT-Sol group.Similarly,it can be concluded that the IRT-PLGA-NPs group can prolong the residence time of SN38 in plasma,which is expected to improve the anti-tumor efficacy of IRT.Overall,IRT-PLGA-NPs can increase the retention time of IRT and SN38 in vivo,which is crucial for improving the drug efficacy of time-dependent drugs.4.Pharmacodynamic evaluation of IRT-PLGA-NpsIn order to evaluate the efficacy of IRT-PLGA-Nps systematically,pharmacodynamics experiments were performed by using KM mice bearing H22 tumors as animal models,IRT-Sol and physiological saline(negative control)were used as controls.Tumor Growth Inhibition(TGI),tumor Specific Grow Rate(SGR)and tumor Doubling Time(DT)were calculated by the measurement of tumor volume at given time points in each group to assess the in vivo anti-tumor effect of three groups quantitatively.At the same time,the body weight of mice in each group was recorded at different time points during the experimental period,the body weight change was used for evaluating the toxicity of each group.Next,tumor tissues and main organs(heart,liver,spleen,lung,kidney)of tumor-bearing mice were removed for HE pathological sections,the anti-tumor effect of each group was evaluated by observing the sections of the tumor,and the in vivo safety of each group was evaluated by observing sections of major organs.The results showed that during the experimental period,the tumor volume of the saline group showed a rapid growth trend.The TGI of the IRT-PLGA-Nps group was 1.3 times that of the IRT-Sol group,the SGR was 0.47 times that of the IRT-Sol group,and the DT was 2.13 times of IRT-Sol group.Which indicateed that the IRT-PLGA-Nps group could inhibit tumor growth better than the IRT-Sol group.At the same time,statistical results(t-Test)showed that compared with the negative control group(NS),IRT-PLGA-Nps had no significant difference on the body weight of mice(p>0.05),which indicated that the nanoparticles prepared in this experiment had fewer side effects.HE staining of tumor tissue of mice showed that IRT-PLGA-Nps had significant anti-tumor effect compared with the other groups;the HE sections of major organs(heart,liver,spleen,lung,kidney)of IRT-PLGA-Nps group showed no significant difference with the control group,which indicated that IRT-PLGA-Nps had no significant toxic and side effects on the main organs of mice,so that had a certain potential for clinical application.Conclusion:In this study,an IRT-PLGA-Nps with high encapsulation efficiency,high drug loading,and moderate particle size was prepared by emulsified solvent evaporation method.The in vitro-release of IRT-PLGA-Nps was slow,the safety and stability is good.Pharmacokinetics and pharmacodynamics experiments showed that IRT-PLGA-Nps can prolong the retention time of IRT and its metabolite SN38 in vivo,so that the anti-tumor effect was significantly enhanced,and the toxic and side effects were reduced,which laid a certain theoretical basis for clinical application. |
PDF Full Text Request