Evaluation Of The Neurotoxicity Of HCPT Sustained-Release Tablets As Brain Implants

BackgroundHydroxycamptothecin(HCPT) is an indole alkaloid extracted from Camptotheca acuminata Decne, which have a wide range of anti-tumor activity, and no cross-resistance to other anticancer drugs. The anticancer of HCPT is different from anti-metabolite and alkylating drugs. As the specific drug for cell periodic, HCPT can effect the cells which in S phase, more significantly than those in G1, G2 phase, and HCPT have no effect on the G0 phase cells. Cell mitosis can be inhibited by HCPT at higher concentrations. In addition, HCPT is the specific inhibitor of topoisomeraseⅠwhich can interfere with DNA replication by selectively inhibiting topoisomerase.Glioma is one of the hardest remedial diseases, which often recurs after surgery, therefore, the chemotherapy after surgery is essential. Most chemotherapeutics are difficult to achieve therapeutic level within brain tissue due to the Blood Brain Barrier(BBB) via vein injection. Serious side effect may be caused by increasing dose. In recent years, interstitial chemotherapy through local embbeding, which wrapped the chemotherapy drugs into biodegradable polymer, has brought widespread attention. However, implant as a new dosage form, it not only bypass the BBB to administer directly into the brain tissue, but also extend the functional time of drug with high concentration result from sustained-release.Using Polylactic acid (PLA) as release material, we have prepared the HCPT sustained-release tablet. Its effect on Glioma have been studied. It showed that HCPT sustained-release tablet can prolong survival time of tumor-bearing rats. The experiment imitate to stduy the toxicity of the HCPT sustained-release tablet which implanted into rat brain. The foundation would be expected to lay for the further development of the preparation.Objective(1)To isolate and to identify the cerebral cortical neurons in vitro methods. (2)To observe the cytotoxicity of HCPT sustained-release tablet through intervening the cultured nerve cells in vitro.(3)To study the damage of HCPT sustained-release tablet on brain organization and function in vivo.(4)To study the damage of HCPT sustained-release tablets on heart, liver, spleen, lung and kidney in vivo.(5)To study the suppression of HCPT sustained-release tablets on the bone marrow in vivo.(6)To study the teratogenic effect of HCPT sustained-release tablets to fetal rat, and evaluate the change of the fetal brain tissue in vivo.Methods(1)The cerebral cortex nerve cells of SD newborn rats were prepared by filtering through a mesh, centrifugation and trypsogen digestion.(2)Neurons were affirmed by immunocytochemical method and morphologies under Light Microscope.(3)The toxicity of HCPT on cerebral cortex nerve cells were evaluated by CCK-8 assay.(4)The cerebral cortex nerve cells'apoptosis were detected by Hoechst33258 staining.(5)Obtain the brain, heart, liver, spleen, lung and kidney tissue of rats after reperfusion at the 30 day and the 120 day from experimental points, then observe the morphological changes in the organization through paraffin and HE staining.(6)The number of Nissl bodies in neurons'cytoplasm were viewed to evaluate the capacity of protein synthesis by Nissl staining at the 30 day and the 120 day from experimental points.(7)The neuronic apoptosis ratio of damaged brain tissue was observated by TUNEL at the 30 day and the 120 day from experimental points.(8)The bone marrow suppression of HCPT sustained-release tablet was observated by bone marrow smear and Wright stain at the 30 day from experimental points.(9)we implant HCPT sustained-release tablets to the brain of pregnant mice, taking Brain of pregnant rats and fetal rats after 1d delivery. Neurons'primary culture in vitro, HE staining to Observation neurons morphological changes. Results(1)The purity of cerebral cortex neurons were more than 95% based on the morphology and immunocytochemistry(NSE) identification in vitro.(2)The results of CCK-8 Test show that:①There was no significantly cytotoxicity of polylactic acid on primary cultured cerebral cortex neurons and astrocytes.②Compared with the control group, significant differences emerged when 40μg/ml HCPT treated cortical neurons(P<0.05); The differences were obvious when the concentration of HCPT achieve to 320μg/ml(P<0.01).③Compared with normal control group, significant differences emerged when 20μg/ml HCPT treated cerebral cortex astrocytes(P<0.05); The differences were obvious when the concentration of HCPT achieve to 80μg/ml(P<0.01).④The cytotoxicity of simulated HCPT sustained-release tablet in vitro was more obvious than HCPT.(3)The results of Hoechst33258 staining showed that:①The apoptosis of cerebral cortex neurons and astrocytes were not obvious induced by polylactic acid;②Compared with the control group, significant variances emerged after 80μg/ml HCPT treated cortical neurons(P<0.05); The differences were obvious when the concentration of HCPT achieved to 160μg/ml(P<0.01);③Compared with normal control group, significant variances emerged after 40μg/ml HCPT treated cerebral astrocytes(P<0.05); the differences were obvious when the concentration of HCPT achieved to 80μg/ml(P<0.01);④The apoptosis ratio of cerebral cortex nerve cells were no significant difference between simulated HCPT sustained-release tablet in vitro than HCPT.(4)After the experiment of rats in vivo, the results of immunohistochemical staining of cerebral cortex showed that:①Polylactic acid have no significant impact in the number and activity of nerve cells;②The cerebral cortex damage of HCPT sustained-release tablets dependented HCPT dose;③Compared with the sham group, no significant differences emerged on the histology of Low-dose tablets of HCPT after implanted into the cerebral cortex;④High dose of HCPT sustained-release had a significant effect on the number and activity of cerebral cortex neurons.(5) After the experiment of rats in vivo, the results of HE staining showed that high doses of HCPT sustained-release tablets had some damage to the liver, and the liver edema emerged. The heart, spleen, lung and kidney did not change significantly. (6) After the experiment of rats in vivo, the results of Wright's stain showed that HCPT sustained-release tablets had no bone marrow suppression.(7) Having a primary culture of the neurons. neurons of Model groups'fetal rats were well-developed in vitro and there were no Significant difference compared with the control groups; Through Experiment of rats in vivo, HE staining demonstrate that there are a certain degree of toxicity whith HCPT release tablets to pregnant rat's brain, while the influence was not obvious for fetal rat's brain tissue.Conclusions(1)A large number of cerebral cortex neurons with high purity had successfully trained used by the serum-free culture methods.(2) The toxic effect of cerebral cortex neurons and astrocytes induced by polylactic acid was not obvious.(3) There was invariably toxicity of cerebral cortex neurons and astrocytes induced by HCPT. The cerebral cortex neurons have more sensitivity to HCPT than astrocytes.(4)The damage of the structure and function of brain tissue was not obvious treated by Polylactic acid in vivo.(5)HCPT sustained-release tablets had some toxicity on the brain, and the higher dose of HCPT, the higher toxicity. Low dose and middle dose of HCPT sustained-release tablets had no clear toxicity on heart, liver, spleen, lung and kidney. In addition to some damage on the liver, high dose of HCPT sustained-release tablets had no significant toxicity to other organs on the histology in vivo.(6)HCPT sustained-release tablets had no bone marrow suppression in vivo.