Temozolomide
(Synonyms: 替莫唑胺; NSC 362856; CCRG 81045; TMZ) 目录号 : GC13667
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。
Cas No.:85622-93-1
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >98.50%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
|
Cell lines |
U-118 GBM cell line |
|
Preparation Method |
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. |
|
Reaction Conditions |
0, 10, 20, 100, 250 and 500 µM for 24 and 48 h |
|
Applications |
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. |
| Animal experiment [2]: | |
|
Animal models |
Male B6D2F1 (C57BL/6 × DBA/2) mice |
|
Preparation Method |
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. |
|
Dosage form |
Intraperitoneal injection, 100, 200 mg/kg |
|
Applications |
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. |
|
References: [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. |
|
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].
References:
[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.
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].
| Cas No. | 85622-93-1 | SDF | |
| 别名 | 替莫唑胺; NSC 362856; CCRG 81045; TMZ | ||
| 化学名 | 3-methyl-4-oxoimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxamide | ||
| Canonical SMILES | CN1C(=O)N2C=NC(=C2N=N1)C(=O)N | ||
| 分子式 | C6H6N6O2 | 分子量 | 194.15 |
| 溶解度 | ≥ 29.61mg/mL in DMSO | 储存条件 | Store at -20°C, protect from light, stored under nitrogen |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
||
| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
 |
1 mg | 5 mg | 10 mg |
| 1 mM | 5.1507 mL | 25.7533 mL | 51.5066 mL |
| 5 mM | 1.0301 mL | 5.1507 mL | 10.3013 mL |
| 10 mM | 0.5151 mL | 2.5753 mL | 5.1507 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Evidence-Based Practice: Temozolomide Beyond Glioblastoma
Purpose of review: Temozolomide is a first-line treatment for newly diagnosed glioblastoma. In this review, we will examine the use of temozolomide in other contexts for treating gliomas, including recurrent glioblastoma, glioblastoma in the elderly, diffuse low- and high-grade gliomas, non-diffuse gliomas, diffuse intrinsic pontine glioma (DIPG), ependymoma, pilocytic astrocytoma, and pleomorphic xanthoastrocytoma. Recent findings: Temozolomide improved survival in older patients with glioblastoma, anaplastic gliomas regardless of 1p/19q deletion status, and progressive ependymomas. Temozolomide afforded less toxicity and comparable efficacy to radiation in high-risk low-grade gliomas and to platinum-based chemotherapy in pediatric high-grade gliomas. The success of temozolomide in promoting survival has expanded beyond glioblastoma to benefit patients with non-glioblastoma tumors. Identifying practical biomarkers for predicting temozolomide susceptibility, and establishing complementary agents for chemosensitizing tumors to temozolomide, will be key next steps for future success.
Potential Strategies Overcoming the Temozolomide Resistance for Glioblastoma
Glioblastoma (GBM) is a highly malignant type of primary brain tumor with a high mortality rate. Although the current standard therapy consists of surgery followed by radiation and temozolomide (TMZ), chemotherapy can extend patient's post-operative survival but most cases eventually demonstrate resistance to TMZ. O6-methylguanine-DNA methyltransferase (MGMT) repairs the main cytotoxic lesion, as O6-methylguanine, generated by TMZ, can be the main mechanism of the drug resistance. In addition, mismatch repair and BER also contribute to TMZ resistance. TMZ treatment can induce self-protective autophagy, a mechanism by which tumor cells resist TMZ treatment. Emerging evidence also demonstrated that a small population of cells expressing stem cell markers, also identified as GBM stem cells (GSCs), contributes to drug resistance and tumor recurrence owing to their ability for self-renewal and invasion into neighboring tissue. Some molecules maintain stem cell properties. Other molecules or signaling pathways regulate stemness and influence MGMT activity, making these GCSs attractive therapeutic targets. Treatments targeting these molecules and pathways result in suppression of GSCs stemness and, in highly resistant cases, a decrease in MGMT activity. Recently, some novel therapeutic strategies, targeted molecules, immunotherapies, and microRNAs have provided new potential treatments for highly resistant GBM cases. In this review, we summarize the current knowledge of different resistance mechanisms, novel strategies for enhancing the effect of TMZ, and emerging therapeutic approaches to eliminate GSCs, all with the aim to produce a successful GBM treatment and discuss future directions for basic and clinical research to achieve this end.
Elucidating the mechanisms of Temozolomide resistance in gliomas and the strategies to overcome the resistance
Temozolomide (TMZ) is a first-choice alkylating agent inducted as a gold standard therapy for glioblastoma multiforme (GBM) and astrocytoma. A majority of patients do not respond to TMZ during the course of their treatment. Activation of DNA repair pathways is the principal mechanism for this phenomenon that detaches TMZ-induced O-6-methylguanine adducts and restores genomic integrity. Current understanding in the domain of oncology adds several other novel mechanisms of resistance such as the involvement of miRNAs, drug efflux transporters, gap junction's activity, the advent of glioma stem cells as well as upregulation of cell survival autophagy. This review describes a multifaceted account of different mechanisms responsible for the intrinsic and acquired TMZ-resistance. Here, we summarize different strategies that intensify the TMZ effect such as MGMT inhibition, development of novel imidazotetrazine analog, and combination therapy; with an aim to incorporate a successful treatment and increased overall survival in GBM patients.
Pericytes augment glioblastoma cell resistance to temozolomide through CCL5-CCR5 paracrine signaling
Glioblastoma (GBM) is a prevalent and highly lethal form of glioma, with rapid tumor progression and frequent recurrence. Excessive outgrowth of pericytes in GBM governs the ecology of the perivascular niche, but their function in mediating chemoresistance has not been fully explored. Herein, we uncovered that pericytes potentiate DNA damage repair (DDR) in GBM cells residing in the perivascular niche, which induces temozolomide (TMZ) chemoresistance. We found that increased pericyte proportion correlates with accelerated tumor recurrence and worse prognosis. Genetic depletion of pericytes in GBM xenografts enhances TMZ-induced cytotoxicity and prolongs survival of tumor-bearing mice. Mechanistically, C-C motif chemokine ligand 5 (CCL5) secreted by pericytes activates C-C motif chemokine receptor 5 (CCR5) on GBM cells to enable DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-mediated DDR upon TMZ treatment. Disrupting CCL5-CCR5 paracrine signaling through the brain-penetrable CCR5 antagonist maraviroc (MVC) potently inhibits pericyte-promoted DDR and effectively improves the chemotherapeutic efficacy of TMZ. GBM patient-derived xenografts with high CCL5 expression benefit from combined treatment with TMZ and MVC. Our study reveals the role of pericytes as an extrinsic stimulator potentiating DDR signaling in GBM cells and suggests that targeting CCL5-CCR5 signaling could be an effective therapeutic strategy to improve chemotherapeutic efficacy against GBM.
TGF-β1 modulates temozolomide resistance in glioblastoma via altered microRNA processing and elevated MGMT
Background: Our previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-β1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-β1 in temozolomide resistance has remained unknown.
Methods: The distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-β1-treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-β1 in temozolomide resistance.
Results: Overexpression of TGF-β1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-β1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-β1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-β1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-β1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-β1 or lncRNA expression benefited from temozolomide therapy.
Conclusions: Our results reveal an underlying mechanism by which TGF-β1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-β inhibitor may serve as an effective therapy for glioblastomas.