Tirapazamine   中文名称: 替拉扎明

DNA-damaging chemotherapeutic agent

体外活性：Trapazamine（也称为 SR-4233；SR259075；Win59075；SR4233）是一种实验辅助药物和 DNA 损伤剂，具有治疗宫颈癌、头颈癌的潜力。当替拉扎明优先在实体瘤的缺氧区域激活其毒性形式时，可以通过减少 HIF-1α 蛋白合成来下调 HIF-1α 表达。当与阿霉素 (DOX) 和 SN-38（伊立替康的活性代谢物）等化疗药物联合使用时，曲扎明可显着抑制 HIF-1α 蛋白的积累，降低 HIF-1α 转录激活，并损害参与相关蛋白的磷酸化。同源重组修复途径，最终导致这两种药物的协同作用。激酶测定：细胞测定：与替拉扎明联合使用，拓扑异构酶 I 抑制剂在几种肝细胞癌细胞系中表现出协同细胞毒性并诱导显着的细胞凋亡。替拉扎明加 SN-38（伊立替康的活性代谢物）诱导的细胞凋亡增强伴随着线粒体去极化和 caspase 通路激活的增加。联合治疗显着抑制了 HIF-1α 蛋白的积累，降低了 HIF-1α 转录激活，并损害了同源重组修复途径中涉及的蛋白的磷酸化，最终导致了这两种药物的协同作用。

在人癌症Bel-7402异种移植小鼠模型中进一步验证了替拉帕扎明与伊立替康联合提高的抗癌功效。 由于替拉帕扎明加SN-38对人肝细胞癌Bel-7402细胞表现出最有效的协同作用，CI值最低（补充表S1），因此选择替拉帕扎明和伊立替康联合治疗的体内疗效，以在裸鼠体内测试Bel-7402异种移植物。如图6A和B以及补充表S2所示，与对照组相比，每2天腹腔注射25mg/kg剂量的替拉帕扎明或每天2.5mg/kg剂量的伊立替康，平均RTV没有显著差异。然而，替拉帕扎明联合伊立替康可引起明显的肿瘤生长抑制（T/C值：36.9%），明显大于替拉帕扎明（T/C值：88.1%）或单独伊立替康治疗（T/C比值：86.4%）。此外，与初始体重相比，用该组合治疗的小鼠在第26天没有明显的体重减轻（图6C）。因此，与单药治疗组相比，替拉帕扎明和伊立替康的组合具有更有效的肿瘤生长抑制作用，但不会导致动物体重减轻。[1]

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大鼠每周一次腹腔注射6次替拉帕扎明，分为5（5TP）和10mg/kg（10TP）两种剂量，而阿霉素的剂量为1.8mg/kg（DOX）。随后两组同时接受两种药物（5TP+DOX和10TP+DOX）。替拉帕扎明降低了心脏脂质过氧化，使阿霉素改变的RyR2蛋白水平恢复正常。DOX组和TP+DOX组的GSH/GSSG比值、总谷胱甘肽、cTnI、AST和SERCA2水平没有显著变化。在10TP和10TP+DOX组中观察到心肌细胞坏死。[2]

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通过尾静脉注射到携带肿瘤的小鼠体内后，TPZ（替拉帕扎明）-Pba-NPs在注射后3小时和24小时的血液浓度分别高出3.17倍，在肿瘤组织中的积聚分别高出4.12倍。在激光照射肿瘤组织后，TPZ-Pba-NPs通过有效的药物递送和体内协同作用成功抑制了肿瘤生长。[3]

拓扑异构酶I抑制剂是一类具有广泛临床活性的抗癌药物。然而，它们对癌症的疗效有限。在这里，我们提出了体外和体内证据，表明肝细胞癌中缺氧诱导因子-1α（HIF-1α）的极高水平与拓扑异构酶I抑制剂的耐药性密切相关。在我们小组之前进行的一项研究中，我们发现替拉帕扎明可以通过减少HIF-1α蛋白合成来下调HIF-1α的表达。因此，我们假设替拉帕扎明与拓扑异构酶I抑制剂联合使用可能会克服化疗耐药性。在这项研究中，我们研究了拓扑异构酶I抑制剂与替拉帕扎明联合使用时，在几种肝细胞癌细胞系中表现出协同细胞毒性并诱导显著凋亡。替拉帕扎明加SN-38（伊立替康的活性代谢产物）诱导的细胞凋亡增强伴随着线粒体去极化和胱天蛋白酶途径激活的增加。联合治疗显著抑制了HIF-1α蛋白的积累，降低了HIF-1β的转录激活，并损害了参与同源重组修复途径的蛋白质的磷酸化，最终导致这两种药物的协同作用[1]。

替拉帕明/Tirapazamine 和拓扑异构酶 I 抑制剂一起表现出协同细胞毒性，并显着减少几种肝细胞癌细胞系中的细胞数量。线粒体去极化和 caspase 通路激活的增加与替拉扎明加 SN-38（伊立替康的活性代谢物）诱导的细胞凋亡增强相关。这两种药物通过联合治疗协同作用，显着减少 HIF-1α 蛋白积累，减少 HIF-1α 转录激活，并阻碍同源重组修复途径中涉及的蛋白磷酸化。

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凋亡和线粒体膜电位（ΔΨm）的流式细胞术分析[1]
细胞在常氧或缺氧条件下用SN-38、Tirapazamine 或其组合处理12小时。在收获并用冷PBS缓冲液洗涤两次后，使用Annexin V-FITC/PI凋亡检测试剂盒分析凋亡细胞。PI染色后亚G1期的分析也用于评估细胞凋亡。对于PI染色，收获处理过的细胞，在-20°C下用70%乙醇固定，然后用RNaseA孵育，然后在黑暗中进行PI染色30分钟。为了测定线粒体电位，将细胞重新悬浮在含有0.1μmol/L JC-1的PBS中，并在37°C的黑暗中孵育15分钟。所有样本均使用FACS Calibur细胞仪进行分析。

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克隆形成试验[1]
用替拉帕扎明或SN-38/TPT/HCPT/MONCPT或其组合处理的细胞在软琼脂上以三份的形式铺在60毫米的培养皿中。一旦设置好，将培养皿覆盖2.5 mL培养基，在37°C的缺氧条件下孵育10天，然后对菌落进行评分和拍照。

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免疫荧光[1]
将细胞铺在玻璃培养玻片上，并与SN-38/替拉帕扎明或SN-38+Tirapazamine /替拉帕扎明或载体[0.1%DMSO（v/v）]一起孵育不同时间。然后用4%多聚甲醛固定细胞，并用含有0.1%Triton X-100的PBS渗透。用5%牛血清白蛋白阻断30分钟后，将细胞与HIF-1α或γ-H2AX原代抗体（1:100稀释）孵育1小时，用PBS洗涤三次，然后分别在黑暗中与Alexa Fluor 488偶联抗体和罗丹明二抗孵育。通过DAPI（4′6-二脒基-2-苯基吲哚）染色观察细胞核。使用Olympus Fluorview 1000共聚焦显微镜分析荧光信号。

Six times a week, rats received intraperitoneal injections of tirapazamine (5 mg/kg (5TP) and 10 mg/kg (10TP)) and doxorubicin (1.8 mg/kg, DOX) intraperitoneally. The next two groups (5TP+DOX and 10TP+DOX) were given both medications at the same time. Tirapazamine normalised the level of RyR2 protein that had been affected by doxorubicin and decreased heart lipid peroxidation.

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Measurement of in vivo activity [1]
Tumors were established by injection of Bel-7402 cells (5 × 106 cells per animal, subcutaneously into the armpit) into 5- to 6-week-old BALB/c male athymic mice. Treatments were initiated when tumors reached a mean group size of about 100 mm3. Tumor volume (mm3) was measured with calipers and calculated as (W2×L)/2, where W is the width and L is the length. Athymic mice were intraperitoneally injected with CPT-11 (2.5 mg/kg) dissolved in physiologic saline once daily and Tirapazamine  (25 mg/kg) dissolved in a cremophor:ethanol:0.9% sterile sodium chloride solution (1:1:8, volume) every 2 days. Mouse weight and tumor volumes were recorded every 2 days until the animals were sacrificed. Animal care was in accordance with institutional guidelines.

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The study was conducted on sexually mature male albino rats of Wistar CRL: (WI)WUBR strain, obtained from a commercial breeder. Animals with the initial body weight of 160–195 g were maintained in stable conditions at 22°C with a 12 h light/dark cycle and given standardized granulated fodder LSM. The rats were intraperitoneally (i.p.) exposed to doxorubicin and/or Tirapazamine  [2].
The animals were randomly divided into six groups (n = 7): DOX: doxorubicin 1.8 mg/kg; 5TP: Tirapazamine  5 mg/kg; 10TP: tirapazamine 10 mg/kg; 5TP+DOX: 1.8 mg/kg doxorubicin and 5 mg/kg tirapazamine; 10TP+DOX: 1.8 mg/kg doxorubicin and 10 mg/kg tirapazamine; control was given i.p. 0.9% NaCl solution. Doxorubicin (1.8 mg/kg) and tirapazamine in both doses were intraperitoneally injected once a week for six weeks in all study groups. A week after administration of both compounds the study was terminated. The animals were sacrificed and the blood and heart samples were collected during autopsy [2].

135413511 Rat Intraperitoneal LD50 59390 ug/kg Archives of Toxicology, 66(100), 1992 [PMID:1605723]
135413511 Rat Intravenous LD >36 mg/kg Kidneys, ureters and bladder: renal tubular changes (including acute renal failure, acute tubular necrosis); endocrine: other changes; blood: bone marrow changes not included in the above. The Toxicologist, 12(154), 1992
135413511 Mouse Intraperitoneal LD50 89 mg/kg International Journal of Radiation Oncology, Biology and Physics, 16(977), 1989 [PMID:2703405]
135413511 Intravenous LD50 in mice: 101 mg/kg. Behaviors: lethargy (reduced overall activity); skin and appendages (skin); hair; others. British Journal of Cancer Supplement, 20(84), 1993

[1]. Tirapazamine sensitizes hepatocellular carcinoma cells to topoisomerase I inhibitors via cooperative modulation of hypoxia-inducible factor-1α. Mol Cancer Ther. 2014 Mar;13(3):630-42.

[2]. Tirapazamine-doxorubicin interaction referring to heart oxidative stress and Ca2? balance protein levels. Oxid Med Cell Longev. 2012;2012:890826.

[3]. Optimized Combination of Photodynamic Therapy and Chemotherapy Using Gelatin Nanoparticles Containing Tirapazamine and Pheophorbide a. ACS applied materials & interfaces vol. 13,9 (2021): 10812-10821.

[4]. Electronic structure and reactivity of tirapazamine as a radiosensitizer. Journal of molecular modeling vol. 27,6 177. 22 May. 2021.

Tirapazamine belongs to the benzotriazine class of compounds, with the structure 1,2,4-benzotriazine, containing an amino substituent at the 3-position and two oxygen substituents at the 1 and 4-positions. It possesses antitumor, apoptosis-inducing, and antibacterial activities. Tirapazamine is an N-oxide, belonging to the benzotriazine class of compounds, and is also an aromatic amine. It is functionally related to 1,2,4-benzotriazine. Tirapazamine, also known as SR-4233, is an experimental anticancer drug activated under hypoxic conditions. This activation mechanism is highly useful because hypoxia is common in human solid tumors, a phenomenon known as tumor hypoxia. Therefore, Tirapazamine is activated only in hypoxic regions of solid tumors. Notably, cells in these hypoxic regions are typically resistant to radiotherapy and most anticancer drugs. For all these reasons, it is strongly recommended to use Tirapazamine in combination with other anticancer therapies. Tirapazamine entered Phase III clinical trials in 2006 for the treatment of head and neck cancer, gynecological tumors, and other types of solid tumors. Tirapazamine is a benzotriazine dioxide with potential antitumor activity. It can be selectively activated by various reductases, forming free radicals in hypoxic cells, thereby inducing DNA single- and double-strand breaks, base damage, and cell death. The drug also enhances the sensitivity of hypoxic cells to ionizing radiation and inhibits radiation-induced DNA strand break repair by inhibiting topoisomerase II. (NCI04)
A triazine derivative that causes DNA strand breaks in hypoxic cells, thus making tumor cells more sensitive to the cytotoxic activity of other drugs and radiation.
Indications
For the treatment of head and neck cancer.
Mechanism of Action
Numerous preclinical trials have demonstrated its selective toxicity to hypoxic cells through a single-electron reduction reaction of the parent molecule to generate free radicals, which interact with DNA, producing single- and double-strand breaks and fatal chromosomal aberrations. The drug also shows activity when used in combination with fractionated radiotherapy and certain chemotherapeutic agents, particularly cisplatin and carboplatin.

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Pharmacodynamics
Terapamycin is an anticancer drug that is inactive in well-oxygenated normal tissues but active in the hypoxic environment of solid tumors. Therefore, this drug can kill these hypoxic or hypoxic cells while limiting toxicity to normal tissues. Since these hypoxic cells are often resistant to radiation and commonly used anticancer drugs, terapamycin may be highly effective when used in combination with standard anticancer therapies. Topoisomerase I inhibitors are a class of anticancer drugs with broad clinical activity. However, their efficacy against hepatocellular carcinoma is limited. In this article, we present in vitro and in vivo evidence showing that extremely high levels of hypoxia-inducible factor-1α (HIF-1α) in hepatocellular carcinoma are closely associated with resistance to topoisomerase I inhibitors. In previous studies in our group, we found that terapamycin can downregulate HIF-1α expression by reducing HIF-1α protein synthesis. Therefore, we hypothesize that the combination of terapamycin with a topoisomerase I inhibitor may overcome chemotherapy resistance. This study found that the combination of topoisomerase I inhibitors and telapamine produced synergistic cytotoxicity and significantly induced apoptosis in various hepatocellular carcinoma lines. The enhanced apoptosis induced by telapamine combined with SN-38 (the active metabolite of irinotecan) was accompanied by mitochondrial depolarization and caspase pathway activation. The combination therapy significantly inhibited HIF-1α protein accumulation, reduced HIF-1α transcriptional activation, and weakened phosphorylation of proteins related to the homologous recombination repair pathway, ultimately leading to the synergistic effect of the two drugs. Furthermore, the enhanced anticancer efficacy of telapamine combined with irinotecan was further validated in a human hepatocellular carcinoma Bel-7402 xenograft mouse model. In summary, our data demonstrate for the first time that HIF-1α is closely associated with resistance to topoisomerase I inhibitors in hepatocellular carcinoma. These results suggest that HIF-1α is a promising target and provide a theoretical basis for conducting clinical trials to investigate the efficacy of topoisomerase I inhibitors combined with telapamine in the treatment of hepatocellular carcinoma. [1]

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Doxorubicin (DOX) can cause chronic cardiomyopathy, the etiology of which is related to oxidative stress and systolic dysfunction. Tirapazamine (TP) is an experimental adjuvant drug with the same redox conversion process as DOX. This study aimed to evaluate the effects of Tirapazamine on oxidative stress, contractile protein levels, and cardiomyocyte necrosis in rats treated with doxorubicin. Rats were intraperitoneally injected with Tirapazamine once a week for a total of six times at doses of 5 mg/kg (5TP) and 10 mg/kg (10TP), while simultaneously receiving doxorubicin at a dose of 1.8 mg/kg (DOX). Subsequently, both groups of rats were treated with both drugs simultaneously (5TP+DOX and 10TP+DOX). Tirapazamine reduced cardiac lipid peroxidation levels and restored doxorubicin-induced changes in RyR2 protein levels to normal. There were no significant changes in the GSH/GSSG ratio, total glutathione, cTnI, AST, and SERCA2 levels between the DOX and TP+DOX groups. Cardiomyocyte necrosis was observed in both the 10TP group and the 10TP+DOX group. [2]

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In combination therapy, the synergistic effect of drugs and their effective delivery are crucial. This study screened 12 anticancer drugs for combined use with pheophoric acid a (Pba) photodynamic therapy (PDT). Based on the combination index (CI) value in the cell viability test, we screened telapamine (TPZ) and prepared self-assembled gelatin nanoparticles (NPs) containing Pba and TPZ. The obtained TPZ-Pba-NPs showed a synergistic killing effect on tumor cells because TPZ was activated under hypoxic conditions generated by Pba photodynamic therapy (PDT) and laser irradiation. After TPZ-Pba-NPs were injected into tumor-bearing mice via the tail vein, the blood drug concentration and tumor tissue accumulation increased by 3.17 times and 4.12 times, respectively, at 3 hours and 24 hours after injection. After laser irradiation of tumor tissue, TPZ-Pba-NPs successfully inhibited tumor growth through efficient drug delivery and in vivo synergistic effect. These overall results suggest that in vitro drug screening based on CI values, mechanism studies under hypoxic conditions, and real-time in vivo imaging are effective strategies for developing nanoparticles for optimizing combination therapies. [3]

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Terapramine (TP) has been shown to enhance the cytotoxic effects of ionizing radiation in hypoxic cells and is therefore a candidate for radiosensitizers. This selective behavior is usually directly related to the abundance of oxygen (O2). This paper investigates the electronic properties of TP in vacuum, microhydration (containing 1 to 3 water molecules), and embedded in continuous water bodies. We discuss the electron affinity, charge distribution, and bond dissociation energy of TP and find that these properties do not change significantly after hydration. Consistent with its high electron affinity, bond breaking induced by electron attachment requires energy above 2.5 eV, which excludes the direct formation of bioactive TP radicals. Therefore, our results suggest that the selective behavior of TP cannot be explained by the single-electron reduction of neighboring O2 molecules. We propose that the hypoxia selectivity of TP may be due to the scavenging of hydrogen radicals by O2. [4]

C7H6N4O2

178.05

178.049

C, 47.19; H, 3.39; N, 31.45; O, 17.96

27314-97-2

135413511

Orange to dark orange-red solid powder

1.7±0.1 g/cm3

493.6±28.0 °C at 760 mmHg

220ºC

252.3±24.0 °C

0.0±1.3 mmHg at 25°C

1.777

-0.31

89.83

1

4

0

13

191

0

[O-][N+]1=C(N([H])[H])N=[N+](C2=C([H])C([H])=C([H])C([H])=C12)[O-]

ORYDPOVDJJZGHQ-UHFFFAOYSA-N

InChI=1S/C7H6N4O2/c8-7-9-11(13)6-4-2-1-3-5(6)10(7)12/h1-4H,(H2,8,9)

1,4-dioxido-1,2,4-benzotriazine-1,4-diium-3-amine

SR 4233; SR-4233; TIRAPAZAMINE; 27314-97-2; 3-Aminobenzo[e][1,2,4]triazine 1,4-dioxide; 1,2,4-Benzotriazin-3-amine, 1,4-dioxide; 3-Amino-1,2,4-benzotriazine 1,4-dioxide; Tirazone; Win-59075; WIN 59075; SR4233; SR259075; SR-259075; SR 259075; WIN 59075; WIN-59075; WIN59075; NSC130181; NSC-130181; NSC 130181; Tirazone; TP; Tirapazamine; 3-Aminobenzo[e][1,2,4]triazine 1,4-dioxide; 3-Amino-1,2,4-benzotriazine 1,4-dioxide; 1,2,4-Benzotriazin-3-amine, 1,4-dioxide; Tirazone; Win-59,075; SR-4233;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: 2.5 mg/mL (14.03 mM) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (11.68 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (11.68 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: 10 mg/mL (56.13 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。