| Gliomas are the highest incidence tumors in the central nervous system at the presenttime, both at home and abroad. The principle of treatment of malignant glioma is toemphasize the surgical resection, and combined with reasonable radiation, chemotherapyand other comprehensive treatment after surgery, but the curative effect is not satisfiedwith. The chemotherapy drugs in gliomas has made some progress in recent years.Temozolomide (TMZ) is a new drug which emerge in recent years for the therapy ofgliomas, which aroused widespread concern just because of its wider antitumor spectrum,the easy way to take, the easy way to through the blood brain barrier, and also because ithave no superposition toxicity with other drugs, can be used for the patients who have drugresistant to nitrosourea.Since the clinical application of TMZ, it have made some survival benefit than theformer chemotherapy drugs and reduced the drug side effects. The method of oraladministration of TMZ is simple, patients were easy to accept, but it’s gastrointestinalreaction is obvious, although be aided by the conventional clinical drugs that suit thegastrointestinal reaction, it can not be used to the glioma patients who suffer fromgastrointestinal diseases at the same time. In view of this situation, some scholars makeTMZ and it’s active metabolites TMZA as the leading compounds and designed twotemozolomide water-soluble derivatives2T-P400,2T-P600. Then we observe the curativeeffect of2T-P400and2T-P600against human gliomas through experiments both in vitroand in vivo. Part Ⅰ Observation of tumor inhibitory effect of temozolomidederivatives (2T-P400、2T-P600) on human glioma cell line SHG-44in vitroObjective: To observe the inhibitory effect of temozolomide derivatives on humanglioma cell line SHG-44in vitro.Methods: SHG-44cells were cultured in DMEM culture medium containing10%fetal calf serum, take tumor cells in good condition and digest them with0.25%trypsin,then dilute the cells to cell suspension (1×104/ml) with culture medium. Then cellssuspension was transfered into96-well plates (100μl/well). After cells growed for24hours,divide the cells into6groups, including2T-P400group,2T-P600group, TMZ group,PEG400group, PEG600group and blank control group. Every group have5concentrationgradients, among them, the final concentration of TMZ, PEG400, PEG600were200umol/L,100umol/L,50umol/L,25umol/L and10umol/L, on the other hand, the finalconcentration of2T-P400and2T-P600were100umol/L,50umol/L,25umol/L,12.5umol/L and5umol/L. Then, put the96-well plates into the incubator and continue tocultivate it with the blank control group adding equal volume of DMEM medium. ODvalues at570nm of all groups were measureed96hours after drug administration, and thecorresponding IC50value were calculated. Make a Comparison between the inhibitionrates against glioma cell SHG-44of all drug groups relative to the the blank control group.Results: The experimental results showed that the inhibition rates of2T-P400,2T-P600and TMZ against glioma cell SHG-44gradually go up with the rising of drugconcentration. Experimental data confirmed that the inhibition effect of2T-P400and2T-P600is close to that of TMZ and have no statistical difference (P>0.05), but havesignificant difference with that of two negative control groups PEG400,PEG600(P<0.01).The inhibition effect of PEG400, PEG600have no statistical difference with that of theblank control group (P>0.05). The IC50values of TMZ,2T-P400,2T-P600respectively is9.73±0.24ug/ml,12.9±0.34ug/ml,15.7±0.36ug/ml, and the IC50values of two negativecontrol groups PEG400, PEG600respectively is150.8±3.36ug/ml,171.6±4.11ug/ml. Conclusion: Experiments in vitro confirmed that2T-P400,2T-P600retained theinhibiting activity against tumor of TMZ.2T-P400and2T-P600provided new drugs andnew way of drug administration for patients who suffer from gliomas.Part Ⅱ Experimental studies on anti-glioma effect of temozolomidederivatives (2T-P400、2T-P600) in subcutaneous xenograft modelObjective: To observe the anti-glioma effect of2T-P400,2T-P600in SHG-44subcutaneous xenograft model.Method: SHG-44glioma cells (1×107) were implanted subcutaneous position of rightarmpit of Balb/c nude mice.After subcutaneous tumor formed, resect it and cut into pieces,and inoculated subcutaneously (2mm3/mouse). When tumor growd to about100mm3,tumor-bearing mice were divided randomly into5groups (10mice/group), named2T-P400group,2T-P600group, TMZ group, PEG group and blank control group (NS group).Among them, TMZ group was positive control group, PEG group was negative controlgroup and NS group was blank control group. The dosage and administration wasrespectively that TMZ (50mg/kg/d) was administered with gavage once daily for5days,2T-P400(98mg/kg/d) and2T-P600(123mg/kg/d) was applied through tail vein injectiononce daily for5days, PEG (103mg/kg/d) was also applied through tail vein injection oncedaily for5days, NS (0.2ml/mouse) was applied through tail vein injection once daily for5days. Body weight and subcutaneous tumor size were measured every4days till28daysafter the treatment was began to do. The tumor size was calculated according to theformula, V=a2×b/2,“a” refers to the short diameter and “b” refers to the long diameter. Thelast measure is on the28th day after the treatment was began to do, then calculate therelative tumor proliferation rate according to the experimental data.Results: Experimental data showed that tumor proliferation rates of2T-P400group,2T-P600group and TMZ group were close to each other (P>0.05), have no statisticaldifference, but were significantly lower than that of PEG group and NS group (P<0.01). There is no significance difference between PEG group and NS group (P>0.05).Conclusion: The experiment confirmed that2T-P400and2T-P600retained anti-tumor activity of TMZ and had similar therapeutic effect against glioma whenadministered through tail vein injection.2T-P400and2T-P600have high solubility andstability in water, so they make it possible to avoid gastrointestinal reaction, increase thedrug dosage and keep stable blood drug concentration.Part Ⅲ Experimental studies on temozolomide derivatives (2T-P400、2T-P600) against human glioma in intracranial xenograft modelObjective: To evaluate the therapitic effect of2T-P400and2T-P600against humanglioma in SHG-44intracranial xenograft model.Method:1.0mm3SHG-44tumor tissue was transplanted intracranially into the rightcaudate nucleus of Balb/c nude mice. Then tumor-bearing mice were sacrificed (three miceeach time) in the different time points (0d,5d,10d,15d,20d,25d) after inoculation. Thewhole brains were taken out, and put them in10%Formalin fixed fluid,24hours later,embedded in paraffin, then sliced. After HE staining, the shortest tumor diameter (a) andthe longest diameter (b) in the biggest tumor slice were measured microscopically. Tumorvolume was calculated according to the formula V=a2×b/2. After standard tumor growthcurve was completed, the intracranial xenograft tumor model used for drug testing wasestablished in the same way. Tumor-bearing mice were randomly divided into5groupsafter10days of tumor inoculation, named2T-P400group,2T-P600group, TMZ group,PEG group and NS group,10mice each group. Among them, TMZ group was positivecontrol group, PEG group was negative control group and NS group was blank controlgroup. The dosage and administration was respectively that TMZ (50mg/kg/d) wasadministered with gavage once daily for5days,2T-P400(98mg/kg/d) and2T-P600(123mg/kg/d) was applied through tail vein injection once daily for5days, PEG(103mg/kg/d) was also applied through tail vein injection once daily for5days, NS (0.2ml/mouse) was applied through tail vein injection once daily for5days. On the28thday after the beginning of treatment, nude mice were sacrificed and the whole brains weretaken out, then put them in10%Formalin fixed fluid in time,24hours later, embedded inparaffin, then sliced. After HE staining of the brain were performed, measure and calculatethe intracranial tumor size.Results: The experiment confirmed that the intracranial tumor size of2T-P400group,2T-P600group and TMZ group were significantly smaller than that of PEG group and NSgroup (P＜0.01). There is no statistical difference among2T-P400group,2T-P600groupand TMZ group (P>0.05). The difference between PEG group and NS group have nostatistical significance (P>0.05).Conclusion: The experimental data showed that the inhibition effect of2T-P400and2T-P600against human glioma in intracranial xenograft model is similar to that of TMZ.The experiment also confirmed that2T-P400and2T-P600can go through the blood brainbarrier to inhibit glioma. |
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