The Study On Metabolic Interactions Of Mitomycin C And Radiosensitization Of Mitomycin C Analog

Incidence and mortality of malignant tumor are increasing year by year, but the effect of radiotherapy, as one of the three most important treatments in clinic, is dissatisfactory, one of the primary reason for this is hypoxic cells exist in solid tumor different extent, these cells are resistant to radiation and chemotherapeutic drugs. Bioreductive drugs are selective cytotoxic prodrug targeting for hypoxic cells in solid tumor, after reduced by bioreductases in vivo under hypoxia, it can produce cytotoxic metabolites which can enhance curative effect of radiotherapy and chemotherapy. As a new type of tumor sensitizer, bioreductive drugs are coming to one of the directions for tumor sensitizer discovery and development in future, the key factor for bioreductive drugs to function well are bioreductases. Studies on bioreductase are very important to pharmacokinetics and curative effect of bioreductive drugs, which can provide academic foundation and instruction for reasonable drug administration in clinic, as well as implications for new bioreductive drug design. While studies on the interactions between bioreductive drugs and liver drug metabolic enzymes IN VITRO or EX VIVO can predict drug-drug interactions (DDI) occurring in vivo in forepart of drug discovery and development, which is important to decrease the risk of elimination for severe DDI, to instruct secure reasonable drug administration in clinic during new drug discovery and development, and to design new agents in future.Objective: (1) Studies on metabolic interactions of mitomycin C (MMC): In order to provide basic profiles for metabolic interactions, metabolism of MMC by human liver microsomes, as well as the MMC interactions in male rat liver, were studied preliminarily IN VITRO and EX VIVO respectively. For study on metabolism of MMC, metabolite identification and analysis of enzymatic kinetics and pathway identification are intended; for study on MMC interactions, the effect (inducing or inhibiting) of MMC on activities of CYP1A, CYP2C and CYP2D is intended IN VITRO and EX VIVO.(2) Studies on radiosensitization of MMC and 629Ac: Thecytotoxicity and radiosensitization 629Ac (5-aziridinyl-3-hydroxy methyl acetate- l-methylindole-4,7- dione, MMC analog) were studied respectively, as well as the effect of hypoxia and transfection of constitutive androstane receptor (CAR) on these biological effects, Furthermore, possible mechanisms of changes were elucidated preliminarily. Finally, cytotoxicity and radiosensitization of 629Ac were compared with MMC.Methods: (1) Studies preliminarily on metabolism of MMC IN VITRO: MMCwas incubated with human liver microsomes. Then metabolites were isolated and detected by High Pressure Liquid Chromatography (HPLC). Types of metabolic enzymes were estimated by the effect of NADPH, Dicumarol (DIC) on metabolism of MMC. Standard, reaction, background control (microsomes was inactivated), negative control (no NADPH), and inhibitor group (adding DIC) were assigned, the results were analyzed by Graphpad Prism 4.0.(2) Studies on the effect of MMC on activities of cytochrome P-450 isoenzymes in liver EX VIVO: Healthy male Sprague-Dauley (SD) rats were administrated with MMC intraperitoneally, dexamethasone (DEX) and P-naphthofiavone (p-NF), as positive control, were selected to induce rat liver CYP2C11 and CYP1A2 respectively, besides blank, control and solvent control. Rats' liver was resected to prepare microsomes and to detect isoenzymes activities after 3 d or 6 d. The results were analyzed with one-way ANOVA. In addition, MMC was preincubated with normal, DEX-induced and P-NF-induced rat liver microsomes, then isoenzymes activities were detected, a-naphthoflavone (a-NF), Quinine (Qui) and Furafylline (Fur) were selected as specific inhibitors of CYP1A2, CYP2D1/2 and CYP2C11 respectively (positive control), besides blank control. The results were analyzed by two-way ANOVA. Activities of CYP2C11, CYP1A2 or CYP2D1/2 in rat liver microsomes were evaluated with reactions of tolbutamide 4-hydroxylation, phenacetin O-deethylation or dextromethorhan O-demethylation respectively. Specific metabolites of three probes themselves were detected by HPLC, so as to calculateactivities of the isoenzymes.(3) Studies on radiosensitization of MMC and 629Ac: Plasmid mCARl/pCR3 was certified by double digestion, PCR and sequencing analysis, then HepG2 cells were transfected with the plasmids mediated by liposome, and vector pCR3 was used as control. Then transfected cells were screened by G418 resistance and limiting dilution. Plasmid mCARl/pCR3 was detected in HepG2 cells, the mock transfectant (HepG2/pCR3 cells, transfected with vector pCR3) and positive clone called g2car cells by PCR, and mRNA expression of mCARl/pCR3 and CYP2B6 by RT-PCR respectively. Cytotoxicity and radiosensitization of MMC and 629Ac in vitro were evaluated in g2car and HepG2 cells by MTT method under anaerobic and aerobic. 7 doses for MMC within 0-50 μg/mL, 8 doses for 629Ac from 0 to 2000 ng/mL were assigned in cytotoxic experiments, the results were used to calculate IC50 of MMC or 629Ac in different conditions first, then analyzed by t-test. Based on cytotoxic experiments, 3 doses for MMC and 4 doses for 629Ac under 20% IC₅₀of themselves were selected to study their radiosensitization, 7 doses for γ-ray within 0⁴0 Gy were assigned, the results were used to calculate sensitization enhancement ratio (SER) in different condition, then C_1.6 were got by linear regression.Results: (1) Studies preliminarily on metabolism of MMC IN VITRO: 3NADPH-dependent metabolites were isolated by HPLC after MMC were incubated with human liver microsomes, their retention time were 10.0 min, 14.0 min, 14.8 min (named as Ml, M2, M3) respectively, their formation kept to enzymatic kinetics (Michaelis-Menten equation), their Km were 0.42 mmol/L (95% CI, 0.31-0.56), 0.81 mmol/L (95% CI, 0.59-1.10), 0.54 mmol/L (95% CI, 0.41-0.71) respectively. The data indicated that the three peaks isolated by HPLC were metabolites of MMC. DIC can inhibit formation of M2, its IC₅₀ was 0.074 mmol/L (95% CI, 0.057-0.096), which indicated DT-diaphorase could take part in formation of M2.(2) Studies on the effect of MMC on activities of cytochrome P-450isoenzymes in liver EX VIVO:â‘  Administration of saline to SD rats intraperitoneally for 3 d or 6 d showed no effect statistically on activities of CYP1A2, CYP2C11, CYP2D1/2 in liver, nor did corn oil—β-NF solvent for 3 d on CYP1A2. While DEX administrated intraperitoneally increased the activities of CYP1A2, CYP2D1/2 and CYP2C11 by 1.3 fold (PO.05), 1 fold (P<0.01), and 2.3 fold (P<0.01) respectively. p-NF was a strong inducer of CYP1A2, increasing the activity of CYP1A2 by 13.7 fold (PO.01). Rats also were treated intraperitoneally with 1 mg/kg.d MMC for 3 d or 6 d, and there were no significant effect on the activities of CYP2D1/2, CYP2C11 and CYP1A2 in rat liver.â‘¡ 50 μmol/L MMC can inhibit CYP1A2 activity in normal rat liver microsomes, and inhibited the activities of CYP2D1/2, CYP2C11, and CYP1A2 in dexamethasone-induced rat liver microsomes by 17.94% (P<0.05), 82.20% (P<0.01) and 38.46% (P<0.05) respectively, and the activity of CYP1A2 was decreased by 59.31% (P<0.01) in p-naphthoflavone-induced rat liver microsomes.(3) Studies on radiosensitization of MMC and 629Ac:â‘  mRNA expression of CAR and CYP2B6 can not be detected in HepG2 cells and HepG2/pCR3 cells but can in g2car cells. Neither under anaerobic and aerobic, there were significant difference between the cytotoxicity of MMC to HepG2 cells and to HepG2/pCR3 cells, nor did 629Ac (P>0.05). This meant Plasmid mCARl/pCR3 was transfected into g2car cells successfully and target gene CYP2B6 was transactivated by CAR, and transfection of vector pCR3 showed no effect on cytotoxicity of MMC and 629Ac.(2) The results of cytotoxicity indicated that hypoxia, transfection of CAR can affect cytotoxicity of MMC and 629Ac.a. Anaerobic VS aerobic: Under anaerobic, IC50 of MMC to HepG2 cells and g2car cells were 21.23 umol/L (95% CI, 19.06-23.37), 12.01μmol/L (95% CI, 9.87-12.80) respectively; under aerobic,ICjoof MMC to HepG2 cells and g2car cells were 5.87 umol/L (95% CI, 3.73-8.28), 4.61 umol/L (95% CI, 2.47-6.75) respectively. Under anaerobic, ICsoof 629Ac to HepG2 cells was 0.21 umol/L (95% CI, 0.02-0.40), decreased by 0.57 umol/L (anaerobic: aerobic=l:3.71 , P<0.05) compared with under aerobic, indicating cytotoxicity of 629Ac increased; Under aerobic and anaerobic, IC50 of 629Ac to g2car cells were 1.77 umol/L (95% CI, 1.58-1.96), 1.79 nmol/L (95% CI, 1.59-1.98) respectively. These data indicated that the changes of cytotoxicity of MMC or 629Ac to g2car cells between anaerobic and aerobic were less than that of to HepG2 cells, it' means g2car cells were insensitive to hypoxia.b.. HepG2 cells VS g2car cells: There was no significant difference between IC50 of MMC lo g2car cells and HepG2 cells under aerobic (P>0.05), but IC50 of MMC to HepG2 cells was 9.22 umol/L higher than to g2car cells (HepG2:g2car=1.77:l, P<0.05), indicating that g2car cells was more sensitive to MMC. IC50 of 629Ac to g2car cells under aerobic and anaerobic were 0.99 umol/L (HepG2:g2car=l:2.7, P<0.05), 1.58 umol/L (HepG2:g2car =1:8.52, /J<0.05) higher than to HepG2 cells, which meant that g2car cells was less sensitive to 629Ac than HepG2 cells whatever under aerobic or hypoxia.c. MMC VS 629Ac: Under aerobic, lC50of MMC to g2car cells and HepG2 cells was higher than that of 629Ac 2.84 umol/L ( MMC:629Ac=2.60:l, PO.05), 5.09 umol/L (MMC:629Ac= 7.53:1, PO.05) respectively; while under hypoxia, ICsoof MMC to g2car cells and HepG2 cells was higher than that of 629Ac 10.22 umol/L ( MMC:629Ac=6.71:l, P<0.05), 21.02 umol/L (MMC:629Ac= 101.09:1, P<0.05) respectively, which indicated that the cytotoxicity of 629Ac to g2car cells and HepG2 cells was stronger than that of MMC whatever under aerobic or hypoxia.Radiosensitization experiments indicated. For parent HepG2 cells, hypoxia selective radiosensitization of MMC was observed, it's C1.6...