| Irinotecan(CPT-11)is a semi-synthetic derivative of cytotoxic antitumor drugs and belongs to the anticancer drugs of camptothecin.CPT-11 has been used to treat a variety of solid tumors in clinical application,such as gynecologic tumors,colorectal cancer,gastric cancer,lung cancer,liver cancer and so on.However,the chemical structure of CPT-11 is easily transformed into carboxylate form under the pH condition of human bodyafter intravenous infusion,and the effect would be reduced.Currently,the research of ultrasound guided intratumoral injection for tumor has become a hot topic.This technology can achieve high local drug concentration in the tumor without obvious adverse reactions.CPT-11 lactone structurein the tumor acidic environment to maintain activity in form to realize better efficacyafter intratumoral injection.After local injection of CPT-11,CPT-11 can maximize the effective structure,and improve the amount of drugs converted into SN-38.Microspheres drug delivery system is a trend of anticancer drug research.As a kind of slow controlled release preparation,microspheres can release drugs slowly,achieve long-term effect,improve the efficacy of drugs,reduce the toxic and side effects of drugs.Irinotecan loaded PLGA microspheres(CPT-11-PLGA-MS)were prepared by emulsion-solvent evaporation method using CPT-11 as a model drug and biodegradable material poly lactic acid glycolic acid(PLGA)as the carrier.It is expected to find new treatments and new ideas for the treatment of cancer by intratumoral injection.The main research contents of this subject include:thepreparationand the physicochemical properties of CPT-11-PLGA-MS,in vitro release of CPT-11-PLGA-MS,drug leakage and retention of tumor tissue in vivo,CPT-11-PLGA-MS for treatment of H22 solid tumor research after intratumor injection.1.Study on the preparation and physicochemical properties of CPT-11-PLGA-MSCPT-11-PLGA-MS prepared by emulsion solvent evaporation method,the UV method for determination of content and entrapment efficiency of CPT-11-PLGA-MS spectrophotometric method.The encapsulation efficiency and drug loading as evaluation index,through the single factor test and orthogonal test to optimize the prescription and preparation process,to reproduce the optimal formulation of 3 batches of CPT-11-PLGA-MS were prepared in 1%mannitol as protective agentby using freeze-drying method.Verified the formation of CPT-11-PLGA-MS by DSC method.The results showed that the determination method can determine the encapsulation efficiency and drug loading of CPT-11-PLGA-MS accurately.The absorbance and concentration showed a good linear relationship,high recovery rate,good precisionin 4.9～24.5 μg/ml concentration range.The surface of the microspheres was round by optimization of prescription and process,the average particle size was(9.29 ± 0.02)μm,the encapsulation efficiency was(77.97 ± 1.26)%,and the drug loading was(7.08 ± 0.11)%.The dried microspheres were prepared by low temperature freeze-drying,the average particle size of the lyophilized microspheres after redissolution was(9.51 ± 0.28)μm.DSC verified the formation of CPT-11-PLGA-MS.2.Study on in vitro drug release of CPT-11-PLGA-MSIn vitrorelease contrastive study of CPT-11-PLGA-MS and CPT-11-Sol were studied by dialysis method in pH6.0 PBS(0.1%NaN3).A method for determining the content of CPT-11 in vitro release medium was established.The cumulative release percentages of CPT-11-PLGA-MS and CPT-11-Sol at different time points were measured,and drawing the curve of re lease.The methodological study results showed that the recovery rate,the stability is high,have good linear relationship in the concentration range of 3.00-18.00 μg/ml,could be used for accurate determination of CPT-11 and CPT-11-PLGA-MS in the content of CPT-11 in vitro release.In vitro drug release test showed that cumulative release amount is(47.93 ±0.23)%of CPT-11-Sol in 1h,the cumulative release percentage reached(93.13 ± 0.13)%in 6 h however,the cumulative release amount is(21.00 ± 0.37)%of CPT-11-PLGA-MS in 1 h,reached(96.33 ±0.16)%in 20d.Two group comparison shows that the release of CPT-11-PLGA-MS.3.Study on drug leakage and tumor tissue retention in vivo afterintratumoral injection of CPT-11-PLGA-MS.This partestablished the model of tumor-bearing mice firstly,and established a method for determination of drug concentration in plasma and tumor tissue in mice by HPLC method.The mice were randomly divided into two groups,the drug(CPT-11 and SN-38)concentrationswas determined by HPLC in plasma and tumor tissues after intratumoral injection of CPT-11-PLGA-MS and CPT-11-Sol,to prove the leakage rateby comparing to the contents of CPT-11 and SN-38 at different time points in plasma after administration.To demonstrate the expected treatment effectby comparing to the content of CPT-11 and SN-38 at different time points after the treatment of tumor tissues.To prove the release characteristics of microspheres that the kinetic parameters was calculated by the drug dynamics program.The method results showed that the method have good specificity andintraday precision,repeatability and recovery rate of RSD was less than 2%,to meet the requirements of determination of biological samples.The retention time of the drug(CPT-11 and SN-38)in the tumor tissue was 72 h after intratumoral injection of CPT-11-Sol,while the concentration of drug in tumor tissue was always higher than that in solution group,and the retention time of drug(CPT-11 and SN-38)was as long as 15 d after intratumoral injection of CPT-11-PLGA-MS.At the same time,compared with CPT-11-PLGA-MS group,the CPT-11 content of blood leakage into the tumor group was relatively high after intratumoral injection of CPT-11-Sol,and no drug was detected in plasma after 1 CPT-11-PLGA-MS intratumoral injection.When determining the content of SN-38 in plasma,it is found that the content of CPT-11 and SN-38 is relatively small and is accompanied by absorption and transformation.The concentration of SN-38 transformation is very small,and its concentration is not yet determined.Pharmacokinetics mathematical model parameters showed that clearanceof CPT-11 and SN-38 in microspheres group is 0.087 times and 0.167 times than the solution group,respectively;the AUC is 11.963 times and 6.160 times than the solution group;the mean retention time is 4.943 times and 5.502 times of the solution group.It indicates that microspheres can release CPT-11 slowly in tumor tissues for a long time,reduce the leakage of drugs into the body circulation,reduce the side effects of drugs,and improve the anti-tumor effect.4.The pharmacodynamic study afterintratumoral injection CPT-11-PLGA-MSfor the treatment of H22 solid tumorsThis part firstly established the model of tumor bearing mice,and normal saline as control,intratumoral injection of CPT-11-PLGA-MS using CPT-11 solution as control group,observation and measurement of tumor bearing mice tumor volume,tumor volume in each group is obtained through TGI calculation,SGR and DT,to evaluate the inhibitory effect of each agent on the tumor.Determination of different time of mice body weight of the animal,the indirect evaluation of the toxicity of pharmaceutical preparations on the body.The tumor tissues obtained at different time points were stained with HE and observed by pathology.Through the necrotic and repair changes of tumor cells in tissue sections,it was proved that CPT-11-PLGA-MS had certain sustained release effect,and the onset time was longer,and the anticancer effect was better than that of CPT-11-Sol group.The results showed that solution group and microspheres group on tumor bearing mice tumor tissue have different degrees of inhibition,and the inhibitory effect of CPT-11 microspheres group in tumor tissues was significantly higher in CPT-11 solution group,that is 2.64 times the solution group,the inhibition rate is 2.13 times the solution group.Tumor specific growth rate is 0.38 times of the solution.Body weight of mice in saline group decreased almost linearly,while the CPT-11 group and the preparation of microspheres group were higher than those in the saline group,and the upward trend in the later period of the test mice weight of CPT-11 microspheres group,can indirectly determine less toxic release microspheres in mice.HE staining of tumor tissue in mice showed that CPT-11 microspheres had a long-acting anti-tumor effect.CPT-11 solution quickly spread into the blood circulation in tumor tissues and had more toxic and side effects on the body.The effect of the microspheres on the sustained release and the inhibitory effect of the tumor tissue is evaluated as a whole better than the solution group.In this paper,the intratumoral injection of CPT-11-PLGA-MS was prepared and evaluated in vitro and in vivo,which provided a new way of thinking and ways for the treatment of cancer by CPT-11. |
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