The Study On Radiosensitization And Its Mechanism And Metabolism Of 629-One Of New Type Bioreductive Drug

As blood vessel has special disposition in tumor, many hypoxic cells are generated in malignancy. These cells are resistant to low LET(lineal energy transfer) radiation,which is the biggest handicap to radiotherapy on tumor. Many methods have been developed to reduce the quantity of hypoxic cells or increase oxygen content in tumor.Using hypoxic cell radiosensitizer is one ideal method to overcome this problem.Bioreductive active drugs are selective cytotoxic prodrug targeting for hypoxic cells in solid tumor. After metabolized by bioreductases in vivo under hypoxic condition, it can be decomposed to cytotoxic metabolites that can enhance therapeutic effiency of radiotherapy and chemotherapy. As a new type of tumor radiosensitizer, bioreductive active drugs are coming to be one important tendency for tumor therapy sensitizer. The key functional factor of bioreductive active drugs is its bioreducases. Studies on these bioreductase are very important to pharmacokinetics and therapeutic effect of bioreductive active drugs, which can provide scientific instruction for applying drugs reasonably in clinic and also supply evidence for new bioreductive drug design. Studies on the interactions between bioreductive drugs and liver drug metabolic enzymes in vitro can predict drug-drug interactions(DDI) occurring in vivo and thus becomes an important approach in the earlier period of drug design and development and reduces the risk of drug elimination since severe DDI.Thus it is very important for new drug development.Objective1. Studies on eytotoxieity and radiosensitization of 629 and MMC: In order to provide the basic experimental and theoretical profiles for 5-(aziridin-l-yl)-3-hydroxy methyl-1-methylindole-4,7-dione(629) can be used in clinic earily, the cytotoxicity and radiosensitization of 629 and its parent compound MMC were studied respectively, as well as the effect of hypoxia and transfection of constitutive androstane receptor (CAR) on these biological effects. At the same time, the effect of 629 and MMC on cell cycle and apoptosis of HepG2 cells and g2car cells was detected by Flow Cytometry(FCM) under different conditions. Furthermore, the possible mechanism of changes was elucidated preliminarily. Finally, cytotoxicity and radiosensitization of 629 were compared with MMC.2. Studies on the mechanism of X-ray induced bystander effect under hypoxic condition:To detect whether X-ray can induce bystander effect on human glioblastoma T98G cells and HepG2 cells under hypoxic condition, then to study the possible mechanism of this effect. Finally, the cytotoxicity and radiosensitization of 629 on HepG2 cells and T98G cells bonding X-ray were investigated.3. Metabolism of 629 In Vitro: In order to provide basic profiles for metabolic interactions and extensive application in clinic, the metabolism of 629 by human liver microsomes was studied in vitro by High Pressure Liquid Chromatography(HPLC). The study on metabolism of 629 include: analysis of enzymatic kinetics and pathway identification are intended and the effect of inducing and inhibiting of 629 on Cytochrome P450.Methods1. The cytotoxicity and radiosensitization of 6291.1 Certify the transfection of constitutive androstane receptor(CAR) gene in HepG2 cells: At first, the previously transfected cells were screened by G418 resistance, then the mRNA expression of CAR and CYP2B6 detected by RT-PCR.1.2 Studies on cytotoxicity of 629 and MMC under different condition: The cytotoxicity of 629 on human primary hepatocyte and the cytotoxicity of 629 and its parent compound--MMC on HepG2 cells and g2car cells were determined by Methyl Thiazolyl Tetrazoliun(MTT) method trader anaerobic or aerobic. The difference cytotoxicity between 629 and MMC was compared to definite that 629 has high performance and low toxicity feature.1.3 Studies on radiosensitization of 629 and MMC under different condition: Based on the cytotoxicity experiment, the radiosensitization of 629 and MMC on HepG2 or g2car cells was evaluated by cell clone cultivation method in vitro under anaerobic, aerobic orγ-ray with different doses, and the Sensitization Enhancement Ratio(SER) of 629 and MMC was calculated in different condition, the C1.6 was obtained by linear regression.1.4 The effect of 629 on cell cycle and apoptosis of HepG2 cells and g2car cells under different condition: The effect of 629 on cell cycle and apoptosis of HepG2 and g2car cells under different condition was detected by Flow Cytometry method(FCM), incluing anaerobic, aerobic,γ-ray with different doses, different concentration and different action time of 629. The results were analyzed to show the mechanism of action of CAR.2. X-ray induced bystander effect in hypoxic HepG2 or T98G cells and the radiosensitization of 629 on HepG2 cells and T98G cells under X-ray exposure.2.1 The direct effect of X-ray on HepG2 and T98G cells:HepG2 cells and T98G cells were irradiated with a certain dose of X-ray under hypoxia or normoxia, then the MN yield was detected to definite the radiosensitization of HepG2 and T98G cells2.2 X-ray induced bystander effect in hypoxic HepG2 or T98G cells: Irradiated HepG2 or T98G cells with different doses of X-ray were cultured under the condition of 0.5% oxygen, and cultured the unirradiated HepG2 or T98G cells together with the irradiated cells(co-cultured cells) or with the conditioned medium(conditioned medium transfer), then score the micronuclei(MN) in co-cultured unirradiated HepG2 or T98G cells and conditioned medium transferred unirradiated HepG2 or T98G cells, and the MN yield (Y_MN) was calculated as the ratio of the number of MN to the scored number of binucleated cells.2.3 The effect of free radical in bystander effect of HepG2 and T98G cells: Treated irradiated cells (HepG2 and T98G cells) with the free radical scavenger--1% Dimethyl sulphoxide(DMSO), and the MN yield was detectd in the bystander hypoxic HepG2 cells or T98G cells.2.4 The effect of iNOS in the bystander effect under different condition: HepG2 cells and T98G cells were irradiated by X-rays under hypoxia or normoxia condition, and the conditioned medium was harvested to obtaind oxygenic conditioned medium and anoxic conditioned medium.then treated the unirradiated HepG2 cells or T98G cells under hypoxia or normoxia with oxygenic conditioned medium and anoxic conditioned medium, respectively. (anoxic conditioned medium treated hypoxic cell group(N₂→N₂); anoxic conditioned mediun treated normoxic cells group (N₂→O₂); normoxic conditioned medium treated hypoxic cells group(O₂→N₂); normoxic conditioned medium treated normoxoc cells group(O₂→O₂)). At the same time, the irradiated cells were treated with the specific nitricoxide synthase(iNOS) inhibitor—Aminoguanidine (AG), the MN yield in the bystander cells(HepG2 or T98G cells) was detected to test the mechanism of iNOS and different oxygen content in bystander effect.2.5 The effect of 629 on chromosome alteration of HepG2 and T98G cells with irradiation of X-ray: HepG2 and T98G cells in hypoxia or normoxia were administered with 629 before irradiation, the MN yield was detected and to definite the cytotoxicity and radiosensitization of 629.2.6 The expression of hypoxia inducible factor-1α(HIF-1α) under hypoxia and irradiaton: The expression of HIF-1αinduced by hypoxia or irradiation was detected by Western Blot, then time-effect relationship was identificated.3. The metabolism interaction of 629 In Vitro3.1 Using metabolism model to detect the metabolism stability of 629 in human liver microsomes: Active human liver microsomes were obtained from donor's liver. 629 with different concentrations was incubated with the human liver microsomes, and its metabolic products were isolated by HPLC.3.2 Identifieate the metabolic pathway of 629 in human liver microsomes in Vitro: 629 with different concentrations was incubated with haman liver microsomes, and the specific inhibitor of CYP1A2, CYP2B6, CYP2A6, CYP2C19, CYP2E1, CYP3A4, CYP2D6 and CYP2C9 was added respectively, the residual 629 was deteted by HPLC. At the same time, suitable negative control(NC) and posstive control (PC) were assigned, and the experimental groups were compared with NC or PC in order to definite the metabolic enzyme of 629.3.3 The inhibition effect of 629 onCYP2A6, CYP2D6, CYP3A4 and CYP2B6 In Vitro: 629 was incubated with human liver microsomes together with a specific substrate including of CYP1A2, CYP2A6, CYP2D6 and CYP3A4. The metabolizing products of above enzyme were detected by HPLC. IC₅₀ of 629 was analyzed by using the software Graphpad Prism 4.0.3.4 The induction effect of 629 on CYP1A2 and CYP3A4 in Vitro: 629 was incubated with human primary hepatocyte 2 days, the specific inhibitor of CYP1A2 and CYP3A4 was added into each reaction system, respectively. The specific metabolizing products of CYP1A2 and CYP3A4 were detected by HPLC. The experimental group was compared with NC or PC, then definite the effect of 629 on the activity of CYP1A2 and CYP3A4 was definatedResults1. The eytotoxieity and radiosensitization of 6291.1 Certify the transfeetion of constitutive androstane receptor(CAR) gene in HepG2 cells: After screening by G418 resistance and RT-PCR, mRNA expression of CAR and CYP2B6 was not deteted in HepG2 cells, but detected in g2car cells.1.2 Studies on cytotoxicity of 629 and MMC under different condition: 629 had no cytotoxicity effect on human primary hepatocyte. But the cytotoxicity effect of 629 and MMC on HepG2 and g2car cells under hypoxia was higher than that under normoxia(P＜0.05), transfection of CAR increased the cytotoxicity effect of MMC on HepG2 cells (P＜0.05) but not of 629 (P＞0.05). Hence CAR could adjust the metabolite o transcriptional level and increase drug toxicity, otherwise, CYP2B6 might be the primary metabolic enzyme on MMC, but not on 629. In addition, the toxicity of 629 was higher than it parent compound—MMC(P＜0.05).1.3 Studies on radiosensitization of 629 and MMC under different condition: The radiosensitization of 629 was stronger than MMC, in addition, the radiosensitization of g2car cells toγ-ray was higher than HepG2 cells. These data indicated that CAR cought affect the radiosensitization of 629 and MMC on HepG2 cells.1.4 The effect of 629 on cell cycle and apoptosis of HepG2 cells and g2car cells under different condition: The effect of 629 on cell cycle and apoptosis of HepG2 and g2car cells under different condition was detected by Flow Cytometry method(FCM). Different phases of cell cycle of HepG2 and g2car cells were changed under different treatment, but the change was not regularly. The percentage apoptic HepG2 cells and g2car cells increased with drug treatment time, concentration and irradiation doses. All these data indicated 629 could affect the hypoxic cytotoxicity and radiosensitization by changing cell cycle progress. On the other hand, CAR affected the biological effect of 629. 2. X-ray induced bystander effect in hypoxic HepG2.2.1 The direct effect of X-ray on HepG2 and T98G cells: The MN ratio of two kinds cells(HepG2 and T98G cells) was obtained in normoxia higher than in hypoxia. The MN ratio of T98G cells was higher than HepG2 cells, the OER(oxygen enhancement ratio) of HepG2 cells and T98G cells was 1.55 and 2.39, The data indicated that T98G cell line was more sensitive than HepG2 cell line to X-rays irradiation;2.2 X-ray induced bystander effect in hypoxic HepG2 or T98G cells: The micronuclei(MN) yield of both bystander cells of HepG2 cells or T98G cells, either co-cultured with irradiated cells or treated with conditioned medium, was higher than the control, which gave a direct evidence of irradiation induced bystander response under hypoxia.2.3 The effect of free radical in bystander effect of HepG2 and T98G cells: Free radical savanger 1% DMSO decreased the MN yield of above bystander HepG2 and g2car cells, indicating that ROS played a key role in this bystander effect under hypoxia.2.4 The effect of iNOS in the bystander effect under different condition: The MN ratio of the normoxic condition medium treat hypoxic cells group(O₂→N₂) on HepG2 cells or T98G cells was higher than other three groups(normoxic condition medium treat normoxic cells group(O₂→O₂), hypoxic condition medium treated hypoxic cells group(N₂→N₂) and hypoxic condition medium treat normoxic cells group(N₂→O₂))(P＜0.05), when cells treated with the specific inhibitor of iNOS before irradiation, the results show: When T98G cells were irradiated with 5Gy X-ray, the MN ratio of all groups was reduced(P＜0. 05). The decreasing degree in O₂→N₂ group was noticeable(P＜0.01). No change in OGy group; For HepG2 cells, the MN ratio in O₂→N₂ group was significant decreased (P＜0.05) and other three groups((O₂→O₂ group , N₂→N₂ group and N₂→O₂ group) did not significant change (P＞0.05) after treated with AG.2.5 The effect of 629 on chromosome alteration of HepG2 and T98G cells with irradiation of X-ray: Treating HepG2 cells and T98G cells with 629, the MN ratio was 1.28 folds and 1.25 folds under hypoxia to normoxia, respectively. Otherwise, when HepG2 cells and T98G cells were irradiated by 2Gy X-ray under hypoxia, the MN ratio was 1.78 folds and 2.05 folds with 629 to without 629, respectively. The results shown that 629 had function of radiosensitization and cytotoxicity on HepG2 cells and T98G cells under hypoxia.2.6 The expression of hypoxia inducible factor-1α(HIF-1α) under hypoxia and irradiaton: The expression of HIF-1αprotein was detected in HepG2 cells and T98G cells under hypoxia and irradiation. To T98G cells, the expression of HIF-1αprotein achieved to the highest level at 48h under hypoxia, and no expression under normoxia. To HepG2 cells, HIF-1αexpression achieved to the highest at 24h under hypoxia, and the expression cought also been seen slightly under normoxia.3. The metabolism evaluation of 629 In Vitro3.1 Using metabolism model to detect the metabolism stability of 629 in human liver microsomes: The protein concentration in microsome was 1mg/ml. 629 could be metabolized stabile in human liver, the Km value was 78μm/ml, 95% confidence interval was 55-111μg/ml.3.2 Identificate the metabolic pathway of 629 in human liver microsomes in Vitro: The metabolism of 629 with 8 kinds of CYP 450: CYP1A2, CYP2B6, CYP2A6, CYP2C19, CYP2E1, CYP3A4, CYP2D6 and CYP2C9 were detected by HPLC. NC: without NADPH, could not be metabolized; PC: with NADPH and without the specific inhibitor of these metabolic enzyme, so the subatrate can be metabolized. Results showed that 629 was metabolized by CYP1A2, CYP2B6 and CYP2A6 significantly(P＜0.05), but not by CYP2C19, CYP2E1, CYP3A4, CYP2D6 and CYP2C9(P＞0.05).3.3 The inhibition of 629 onCYP2A6, CYP2D6, CYP3A4 and CYP2B6 in Vitro: Results shown that 629 cought inhibite the activity of CYP2A6, CYP2D6 and CYP3A4, the half-lethal dose(IC₅₀) was: 938.4ng/ml, 779.2ng/m and 692.8ng/ml respectively, but no on CYP2B6.3.4 The induction of 629 on CYP1A2 and CYP3A4 in Vitro: Results showen that 629 cought not induce the activity of CYP1A2 and CYP3A4 in its concentration extent(P＞0.05).Conclusion1. 629 had no hypoxic cytotoxicity in human primary hepatocyte, but was toxicin to HepG2 cells, g2car cells and T98G cells, and its cytotoxicity was stronger than its parent compound—MMC. In addition, 629 had radiosensitization effect not only to γ-ray but also to X-ray.2. Transfection of CAR increased expression of CYP2B6 mRNA in HepG2 cells, and enhanced cytotoxicity and radiosensitization of 629 and MMC under hypoxia.3. 629 changed the cell cycle and induced apoptosis in HepG2 and g2car cells, and thus had effects of hypoxic cytotoxicity and radio-radiosensition.4. Bystander effect was observed in HepG2 and T98G cells under hypoxix condition. Free radicals of ROS and NO played key role in the bystander effect. Moreover, Otherwise, HIF-1αrotein was induced by hypoxia and irradiation treatment. It supposed that the HIF-1αgene may play a part in bystander effect, It cought provide the basic profile of theoretical and experimental on the mechanism of the antiradiation of hypoxic cells.5. 629 cought be metabolized in human liver microsom by CYP1A2, CYP2B6 and CYP2A6 significantly. On the microsomal level, 629 cought inhibit the activity of CYP2A6, CYP2D6 and CYP3A4, but not on CYP2B6; 629 had no induction on CYP1A2 and CYP3A4 at the test concentrations in incubation with human primary hepatocytes.