DNA-damaging chemotherapeutic agent

Moreover, the increased anticancer efficacy of tirapazamine combined with irinotecan was further validated in a human liver cancer Bel-7402 xenograft mouse model. The combination of tirapazamine and irinotecan arrests tumor growth Because the tirapazamine plus SN-38 exhibited the most effective synergistic effect with the lowest CI values on human hepatocellular carcinoma Bel-7402 cells (Supplementary Table S1), the in vivo efficacy of the tirapazamine and irinotecan combination therapy was chosen to test against Bel-7402 xenografts in nude mice. As shown in Fig. 6A and B and Supplementary Table S2, the intraperitoneal administration of tirapazamine at a dose of 25 mg/kg every 2 days or irinotecan at a dose of 2.5 mg/kg every day produced no significant difference in mean RTV compared with that of the control group. However, tirapazamine plus irinotecan caused marked tumor growth inhibition (T/C value: 36.9%) that was significantly greater than that caused by tirapazamine (T/C value: 88.1%) or irinotecan treatment alone (T/C value: 86.4%). Furthermore, compared with the initial body weights, the mice treated with the combination showed no significant body weight loss on day 26 (Fig. 6C). Thus, the combination of tirapazamine and irinotecan exerted more potent tumor growth inhibitory effects compared with the monotreatment groups, but caused no extended bodyweight loss to the animals.[1]

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Rats were intraperitoneally injected six times once a week with tirapazamine in two doses, 5 (5TP) and 10 mg/kg (10TP), while doxorubicin was administered in dose 1.8 mg/kg (DOX). Subsequent two groups received both drugs simultaneously (5TP+DOX and 10TP+DOX). Tirapazamine reduced heart lipid peroxidation and normalised RyR2 protein level altered by doxorubicin. There were no significant changes in GSH/GSSG ratio, total glutathione, cTnI, AST, and SERCA2 level between DOX and TP+DOX groups. Cardiomyocyte necrosis was observed in groups 10TP and 10TP+DOX.[2]

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After they were injected into tumor-bearing mice via the tail vein, TPZ (tirapazamine)-Pba-NPs showed 3.17-fold higher blood concentration and 4.12-fold higher accumulation in tumor tissue 3 and 24 h postinjection, respectively. Upon laser irradiation to tumor tissue, TPZ-Pba-NPs successfully suppressed tumor growth by efficient drug delivery and synergetic effects in vivo. [3]

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Topoisomerase I inhibitors are a class of anticancer drugs with a broad spectrum of clinical activity. However, they have limited efficacy in hepatocellular cancer. Here, we present in vitro and in vivo evidence that the extremely high level of hypoxia-inducible factor-1α (HIF-1α) in hepatocellular carcinoma is intimately correlated with resistance to topoisomerase I inhibitors. In a previous study conducted by our group, we found that tirapazamine could downregulate HIF-1α expression by decreasing HIF-1α protein synthesis. Therefore, we hypothesized that combining tirapazamine with topoisomerase I inhibitors may overcome the chemoresistance. In this study, we investigated that in combination with tirapazamine, topoisomerase I inhibitors exhibited synergistic cytotoxicity and induced significant apoptosis in several hepatocellular carcinoma cell lines. The enhanced apoptosis induced by tirapazamine plus SN-38 (the active metabolite of irinotecan) was accompanied by increased mitochondrial depolarization and caspase pathway activation. The combination treatment dramatically inhibited the accumulation of HIF-1α protein, decreased the HIF-1α transcriptional activation, and impaired the phosphorylation of proteins involved in the homologous recombination repair pathway, ultimately resulting in the synergism of these two drugs[1].

Tirapazamine  and topoisomerase I inhibitors together demonstrated synergistic cytotoxicity and significantly reduced the number of cells in several hepatocellular carcinoma cell lines. Increased mitochondrial depolarization and caspase pathway activation correlated with the enhanced apoptosis induced by tirapazamine plus SN-38, the active metabolite of irinotecan. These two drugs work in concert because of the combination treatment, which significantly reduced HIF-1α protein accumulation, reduced HIF-1α transcriptional activation, and hampered the phosphorylation of proteins involved in the homologous recombination repair pathway.

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Flow cytometric analysis of apoptosis and mitochondrial membrane potential (ΔΨm) [1]
Cells were treated with SN-38, Tirapazamine , or their combination under normoxia or hypoxia for 12 hours. After harvesting and washing twice with cold PBS buffer, the Annexin V-FITC/PI apoptosis Detection Kit was used to analyze apoptotic cells. Analysis of the sub-G1 phase after PI staining was also used to assess apoptosis. For PI staining, treated cells were harvested and fixed with 70% ethanol at −20°C, then incubated with RNaseA followed by PI in the dark for 30 minutes. For determination of mitochondrial potential, cells were resuspended in PBS containing 0.1 μmol/L JC-1 and were incubated at 37°C for 15 minutes in the dark. All samples were analyzed using a FACS-Calibur cytometer.

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Clonogenic assays [1]
Cells treated with Tirapazamine  or SN-38/TPT/HCPT/MONCPT or their combination were plated in 60 mm dishes in triplicate on soft agar. Once set, the dishes were overlaid with 2.5 mL of medium and incubated at 37°C for 10 days in hypoxic conditions, at which time the colonies were scored and photographed.

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Immunofluorescence [1]
Cells were plated onto glass culture slides and incubated with SN-38/Tirapazamine  or SN-38 + tirapazamine or vehicle [0.1% DMSO (v/v)] for different time periods. Cells were then fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.1% Triton X-100. After blocking with 5% bovine serum albumin for 30 minutes, cells were incubated with primary HIF-1α or γ-H2AX antibodies (1:100 dilution) for 1 hour, washed three times with PBS and then incubated with Alexa Fluor 488–conjugated and rhodamine secondary antibodies, respectively, in the dark. Nuclei were visualized by staining with DAPI (4′6-diamidino-2-phenylindole). Fluorescence signals were analyzed using an Olympus Fluorview 1000 confocal microscope.

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

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)

Solubility in Formulation 1: 2.5 mg/mL (14.03 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (11.68 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (11.68 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: 10 mg/mL (56.13 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)