Prelinimary Studies On Temozolomide Delayed Release Microspheres Against Glioma

Gliomas are the most common primary tumors in the central nervous system. However,the treatment of malignant gliomas,namely surgical resection combined with radiotherapy and chemotherapy,can not achieve satisfactory therapeutic effect.In recent years,great progresses have been made on chemotherapy of gliomas.Temozolomide(TMZ) is a new alkylating agent for chemotherapy with high efficiency(median survival time extended one month compared with the conventional chemotherapy),low toxicity,easy to use,etc.,which re-enhanced our enthusiasm on glioma chemotherapy,and provided new hope for treatment of malignant gliomas.As previously reported in the literature,BCNU sustained release tablets implanted in the intraoperative tumor residual cavity after surgical resection of glioma can help to improve therapeutic efficacy,and delay tumor recurrence.Based on the same idea,TMZ delayed release microspheres(P-TMZ) were designed,and the anti-tumor effects against human gliomas were studied both in vitro and in vivo.PartⅠObservation of tumor inhibitory effect of temozolomide delayed release microspheres on human glioma cell line SHG-44 in vitroObjective:To observe the inhibitory effect of temozolomide delayed release microspheres on human glioma cell line SHG-44 in vitro.Methods:SHG-44 cells were cultured in DMEM culture medium containing 10%fetal calf serum,then cell suspension(1×10~4/ml) was transfered into 96-well plates (100μl/well).After cell growth for 24 hours,P-TMZ,TMZ,BCNU,and P-0 were added at a concentration gradient(the final concentration were 1μg/ml,5μg/ml,10μg/ml,20μg/ml, 40μg/ml,respectively.) with blank control(adding equal volume of DMEM medium).The inhibitory effects of temozolomide delayed release microspheres against glioma cells were detected with MTT method.OD values at 570nm of all groups were obtained 96 hours after drug administration,and the corresponding IC50 value were calculated.Results:MTT data showed that P-TMZ,TMZ,and BCNU have significant anti-tumor effect against SHG-44 glioma cells in vitro,their IC50 value was 6.63±0.26μg/ml, 7.98±0.24μg/ml and 8.41±0.26μg/ml,respectively,which were all less than 10μg/ml, when compared with P-0(P＜0.01).Conclusion:P-TMZ retained anti-tumor activity of TMZ,which has made the foundation for further studing the anti-tumor effect in vivo. PartⅡExperimental studies on anti-glioma effect of temozolomide delayed release microspheres in subcutaneous xenograft modelObjective:To observe the anti-glioma effect of P-TMZ in SHG-44 subcutaneous xenograft model.Method:SHG-44 glioma cells(1×10~7) were implanted subcutaneously into Balb/c nude mice,then tumor was resected and cut into pieces,and inoculated subcutaneously (2mm~3/mouse).When tumor growth reached to about 65mm~3,tumor-bearing mice were divided randomly into 6 groups(7 mice/group),namely P-TMZ group,TMZ group, P-TMZ plus TMZ group,BCNU group,blank microspheres(P-0) group and normal saline (NS) group.P-TMZ(500mg/kg),P-0(500mg/kg)were suspended with DMEM to the final volume of 150μl and injected slowly into the tumor tissues.TMZ(50mg/kg) was administered with gavage once daily for 5 days.BCNU(40mg/kg) was systemically applied through tail vein injection once,NS was injected directly(150μl/mouse) into tumor tissue.Body weight and subcutaneous tumor size were measured every 4 days till 28 days after treatment.Results:Tumor inhibition ratio of P-TMZ,P-TMZ plus TMZ,and BCNU were higer than P-0(P＜0.01);P-TMZ,P-TMZ plus TMZ,and BCNU were more effective than TMZ (P＜0.05).TMZ showed definite anti-tumor effect when compared with P-0,NS(P＜0.05).Conclusion:P-TMZ retained anti-tumor activity of TMZ and had better therapeutic effect against glioma when administered locally.PartⅢExperimental studies on temozolomide delayed release microspheres against human glioma in intraeranial xenograft modelObjective:To evaluate the therapitic effect of P-TMZ against human glioma in SHG-44 intracranial xenograft model.Method:2.0mm~3 SHG-44 tumor tissue was transplanted intracranially into the right caudate nucleus of each Balb/c nude mice.Then tumor-bearing mice were sacrificed(three mice each time) in the different time points(0d,5d,10d,15d,20d,25d) after inoculation. The whole brains were taken out,and continuous brain axial frozen sections were proceeded.After HE staining,the shortest tumor diameter(a) and the longest diameter(b) in the biggest tumor slice were measured microscopically.Tumor volume was calculated according to the formula V=a~2×b/2.After standard tumor growth curve was completed, the SHG-44 intracranial xenograft model was established.With the same method, tumor-bearing mice were randomly divided into 7 groups after 10 days of tumor inoculation,namely P-TMZ group,TMZ group(gavage),P-TMZ plus TMZ(gavage) group, TMZ group(intracranial injection),BCNU(tail vein injection) group,blank microspheres (P-0) group and normal saline(NS) group.P-TMZ(100mg/kg),TMZ(50mg/kg) and P-0 (100mg/kg) were suspended with DMEM to the final volume of 20μl and injected slowly into the corresponding intracranial tumor tissues;TMZ(50mg/kg) was administered throuph gavage once daily,total of five days.BCNU(40mg/kg) were applied systemically via tail vein injection once.28 days after treatment,nude mice were sacrificed.Frozen section,and HE staining of the brain were performed,diameters of tumor were measured microscopically.Results:The intracranial tumor size of P-0 and NS group were significantly larger than other groups(P＜0.01).The tumor inhibition rate of P-TMZ group,P-TMZ plus TMZ group,BCNU group showed no obvious difference(P＞0.05),but were all higer than TMZ(gavage) group(P＜0.01 ),and higher than TMZ(intracranial injection) group,too (P＜0.05),TMZ(gavage) grou and TMZ(intracranial injection) group had no obvious difference(P＞0.05).Conclusion:Experimental data showed that P-TMZ not only retained the tumor inhibitory activity of TMZ,and had better therapeutic effect than TMZ when administered directly in the intracranial tumor during observation phase of nealy one month.