Background
Temozolomide is an oral activity alkylating agent that induces the formation of O6-methylguanine in DNA, which mispairs with thymine during the following cycle of DNA replication, leading to activation of the apoptotic pathways, Temozolomide could crosses the blood-brain barrier and is indicated for malignant gliomas and metastatic melanomas [1]. Temozolomide induced cell cycle arrest at G2/M and to eventually lead to apoptosis [2]. At physiologic pH it is converted to the short-lived active compound, MTIC. MTIC is further hydrolyzed to 5-amino-imidazole-4-carboxamide (AIC) and to methylhydrazine. The cytotoxicity of Temozolomide is mediated by its addition of methyl groups at N7 and O6 sites on guanines and the O3 site on adenines in genomic DNA. Alkylation of the O6 site on guanine leads to the insertion of a thymine instead of a cytosine opposite the methylguanine during subsequent DNA replication, and this can result in cell death [3].
Lymphoma cell counts performed 72 hours after treatment showed that the IC50 of Temozolomide was 44 μM (35-58 μM) when used alone and 16 μM (12-26 μM) when combined with NU1025 [1]. A time-related response in DNA strand-break formation was observed in the U251MG glioblastoma cells treated with Temozolomide (100 μM) alone [4]. Temozolomide was particularly evident when U-118 cells were incubated with Temozolomide concentrations of >100 μM. When U-118 cells were incubated with Temozolomide (250 μM) the proliferation rate was inhibited by 43.2% [5].
Temozolomide consistently demonstrates reproducible linear pharmacokinetics with approximately 100% p.o. bioavailability [6]. In the early stage s.c. implanted SNB-75 astrocytoma model, a 400-mg/kg dose of Temozolomide administered on Day 5 produced 10 of 10 Day 54 tumor-free mice. In later staged s.c. U251 and SF-295 glioblastoma models, a single 600-mg/kg dose produced 9 of 10 Day 86 and 2 of 10 Day 40 tumor-free mice, respectively. In the latter group, a tumor growth delay of >315% was attained [7]. Treatment of CEP 6800 (30 mg/kg) with Temozolomide (17 and 34 mg/kg) resulted in 100% complete regression of U251MG tumors by day 28 versus 60% complete regression caused by Temozolomide alone [4].
替莫唑胺是一种口服活性烷化剂，可诱导 DNA 中 O6-甲基鸟嘌呤的形成，其在随后的 DNA 复制周期中与胸腺嘧啶错配，从而激活细胞凋亡途径，替莫唑胺可穿过血脑屏障并适用于恶性神经胶质瘤和转移性黑色素瘤[1]。替莫唑胺诱导细胞周期停滞在 G2/M 期并最终导致细胞凋亡[2]。在生理 pH 值下，它会转化为短寿命的活性化合物 MTIC。 MTIC 进一步水解为 5-氨基-咪唑-4-甲酰胺 (AIC) 和甲基肼。替莫唑胺的细胞毒性是通过在基因组 DNA 中鸟嘌呤的 N7 和 O6 位点以及腺嘌呤的 O3 位点添加甲基来介导的。鸟嘌呤上 O6 位点的烷基化导致在随后的 DNA 复制过程中插入胸腺嘧啶而不是甲基鸟嘌呤对面的胞嘧啶，这可能导致细胞死亡[3]。
治疗后 72 小时进行的淋巴瘤细胞计数显示，替莫唑胺的 IC50 单独使用时为 44 μM (35-58 μM) 和 16 μM (12-26 μM) 与 NU1025 结合时 [1]。在单独使用替莫唑胺 (100 μM) 处理的 U251MG 胶质母细胞瘤细胞中观察到 DNA 链断裂形成的时间相关反应[4]。当 U-118 细胞与浓度 >100 μM 的替莫唑胺一起孵育时，替莫唑胺特别明显。当 U-118 细胞与替莫唑胺 (250 μM) 一起孵育时，增殖率被抑制了 43.2% [5]。
替莫唑胺始终显示出可重复的线性药代动力学，约为 100 % 口服生物利用度[6]。在早期阶段 s.c.在植入 SNB-75 星形细胞瘤模型后，在第 5 天施用 400 mg/kg 剂量的替莫唑胺产生了 10 只第 54 天无肿瘤小鼠中的 10 只。在后来的 s.c. U251 和 SF-295 胶质母细胞瘤模型，单次 600 mg/kg 剂量分别产生 10 天 86 中的 9 只和 10 天 40 中的 2 只无肿瘤小鼠。在后一组中，肿瘤生长延迟达到 >315%[7]。用替莫唑胺（17 和 34 mg/kg）治疗 CEP 6800（30 mg/kg）导致 U251MG 肿瘤在第 28 天时 100% 完全消退，而单独使用替莫唑胺导致 60% 完全消退[4].
参考文献:
[1]. Tentori L, Leonetti C, Scarsella M, et al. Combined treatment with temozolomide and poly (ADP-ribose) polymerase inhibitor enhances survival of mice bearing hematologic malignancy at the central nervous system site[J]. Blood, The Journal of the American Society of Hematology, 2002, 99(6): 2241-2244.
[2]. Baer J C, Freeman A A, Newlands E S, et al. Depletion of O6-alkylguanine-DNA alkyltransferase correlates with potentiation of temozolomide and CCNU toxicity in human tumour cells[J]. British journal of cancer, 1993, 67(6): 1299-1302.
[3]. Lee S Y. Temozolomide resistance in glioblastoma multiforme[J]. Genes & diseases, 2016, 3(3): 198-210.
[4]. Miknyoczki S J, Jones-Bolin S, Pritchard S, et al. Chemopotentiation of temozolomide, irinotecan, and cisplatin activity by CEP-6800, a poly (ADP-ribose) polymerase inhibitor[J]. Molecular cancer therapeutics, 2003, 2(4): 371-382.
[5]. Carmo A, Carvalheiro H, Crespo I, et al. Effect of temozolomide on the U-118 glioma cell line[J]. Oncology letters, 2011, 2(6): 1165-1170.
[6]. Friedman H S, Kerby T, Calvert H. Temozolomide and treatment of malignant glioma[J]. Clinical cancer research, 2000, 6(7): 2585-2597.
[7]. Plowman J, Waud W R, Koutsoukos A D, et al. Preclinical antitumor activity of temozolomide in mice: efficacy against human brain tumor xenografts and synergism with 1, 3-bis (2-chloroethyl)-1-nitrosourea[J]. Cancer research, 1994, 54(14): 3793-3799.
Protocol
| Cell experiment [1]: |
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Cell lines
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U-118 GBM cell line
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Preparation Method
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Cells were subcultured every 48 h by lifting them up with a cell scrapper. The cells were then centrifuged and resuspended in fresh DMEM. For the experiments, unsynchronized cells were treated with different concentrations of Temozolomide (0, 10, 20, 100, 250 and 500 μM) for 24 and 48 h. Assays were previously performed in the presence of DMSO, which corresponded to each Temozolomide concentration.
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Reaction Conditions
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0, 10, 20, 100, 250 and 500 μM for 24 and 48 h
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Applications
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The effect of Temozolomide was particularly evident when U-118 cells were incubated with Temozolomide concentrations of >100 μM. When U-118 cells were incubated with Temozolomide (250 μM) the proliferation rate was inhibited by 43.2%, as compared to that observed in the control cells.
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| Animal experiment [2]: |
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Animal models
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Male B6D2F1 (C57BL/6 × DBA/2) mice
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Preparation Method
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L5178Y cells (104 in 0.03 mL RPMI-1640) were then injected intracranially, through the center-middle area of the frontal bone to a 2-mm depth, using a 0.1-mL glass microsyringe and a 27-gauge disposable needle. Temozolomide was dissolved in dimethyl-sulfoxide (40 mg/mL), diluted in saline (5 mg/mL), and administered intraperitoneally on day 2 after tumor injection at 100 mg/kg or 200 mg/kg, total dose of 200 mg/kg Temozolomide was divided in 2 doses of 100 mg/kg given on days 2 and 3.
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Dosage form
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Intraperitoneal injection, 100, 200 mg/kg
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Applications
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Intracranial injection of NU1025, immediately before the administration of 100 or 200 mg/kg Temozolomide, significantly increased lifespans with respect to controls or to groups treated with Temozolomide only. When Temozolomide was fractionated, the ILS obtained with this schedule was higher than that observed when NU1025 was combined with a single injection of Temozolomide.
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参考文献:
[1]: Carmo A, Carvalheiro H, Crespo I, et al. Effect of Temozolomide on the U-118 glioma cell line[J]. Oncology letters, 2011, 2(6): 1165-1170.
[2]: Tentori L, Leonetti C, Scarsella M, et al. Combined treatment with Temozolomide and poly (ADP-ribose) polymerase inhibitor enhances survival of mice bearing hematologic malignancy at the central nervous system site[J]. Blood, The Journal of the American Society of Hematology, 2002, 99(6): 2241-2244.
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