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 LD50 intraperitoneal 59390 ug/kg Archives of Toxicology., 66(100), 1992 [PMID:1605723]
135413511 rat LD intravenous >36 mg/kg KIDNEY, URETER, AND BLADDER: CHANGES IN TUBULES (INCLUDING ACUTE RENAL FAILURE, ACUTE TUBULAR NECROSIS); ENDOCRINE: OTHER CHANGES; BLOOD: CHANGES IN BONE MARROW NOT INCLUDED ABOVE Toxicologist., 12(154), 1992
135413511 mouse LD50 intraperitoneal 89 mg/kg International Journal of Radiation Oncology, Biology, Physics., 16(977), 1989 [PMID:2703405]
135413511 mouse LD50 intravenous 101 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); SKIN AND APPENDAGES (SKIN): HAIR: OTHER 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 is a member of the class of benzotriazines that is 1,2,4-benzotriazine carrying an amino substituent at position 3 and two oxido substituents at positions 1 and 4. It has a role as an antineoplastic agent, an apoptosis inducer and an antibacterial agent. It is a N-oxide, a member of benzotriazines and an aromatic amine. It is functionally related to a 1,2,4-benzotriazine.
Tirapazamine, also known as SR-4233, is an experimental anticancer drug that is activated in hypoxic conditions. This activation is very useful as this hypoxic state is found in human solid tumors in a common phenomenon known as tumor hypoxia. Hence, tirapazamine is solely activated in those hypoxic areas of solid tumors. It is important to take into consideration that normally, the cells in these hypoxic regions are resistant to radiotherapy and most anticancer drugs. For all these reasons, the combination of tirapazamine with other anticancer treatments is highly recommended. Tirapazamine entered phase III testing in 2006 for patients with head and neck cancer and gynecological cancer, as well as for other solid tumor cancer types.
Tirapazamine is a benzotriazine di-N-oxide with potential antineoplastic activity. Tirapazamine is selectively activated by multiple reductases to form free radicals in hypoxic cells, thereby inducing single-and double-strand breaks in DNA, base damage, and cell death. This agent also sensitizes hypoxic cells to ionizing radiation and inhibits the repair of radiation-induced DNA strand breaks via inhibition of topoisomerase II. (NCI04)
A triazine derivative that introduces breaks into DNA strands in hypoxic cells, sensitizing tumor cells to the cytotoxic activity of other drugs and radiation.

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Drug Indication
For the treatment of head and neck cancer.

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Mechanism of Action
Extensive preclinical testing has established that the mechanism for the selective toxicity towards hypoxic cells is the result of a one-electron reduction of the parent molecule to a free radical species that interacts with DNA to produce single- and double-strand breaks and lethal chromosome aberrations. It has also shown activity when combined with fractionated irradiation and when combined with some chemotherapy agents, particularly cisplatin and carboplatin.

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Pharmacodynamics
Tirapazamine is a anticancer drug that is inactive in normal tissues that are well oxygenated, but becomes active at the low oxygen levels found in solid tumors. As a result, the drug kills these poorly oxygenated or hypoxic cells while limiting toxicity in normal tissue. Tirapazamine may prove highly effective when used in combination with standard anticancer therapy, as these hypoxic cells are characteristically resistant to radiation and common anticancer agents.

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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. Moreover, the increased anticancer efficacy of tirapazamine combined with irinotecan was further validated in a human liver cancer Bel-7402 xenograft mouse model. Taken together, our data show for the first time that HIF-1α is strongly correlated with resistance to topoisomerase I inhibitors in hepatocellular carcinoma. These results suggest that HIF-1α is a promising target and provide a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and tirapazamine in hepatocellular cancers. [1]

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Doxorubicin (DOX) causes long-term cardiomyopathy that is dependent on oxidative stress and contractility disorders. Tirapazamine (TP), an experimental adjuvant drug, passes the same red-ox transformation as DOX. The aim of the study was to evaluate an effect of tirapazamine on oxidative stress, contractile protein level, and cardiomyocyte necrosis in rats administered doxorubicin. 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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In combination therapy, synergetic effects of drugs and their efficient delivery are essential. Herein, we screened 12 anticancer drugs for combination with photodynamic therapy (PDT) using pheophorbide a (Pba). On the basis of combination index (CI) values in cell viability tests, we selected tirapazamine (TPZ) and developed self-assembled gelatin nanoparticles (NPs) containing both Pba and TPZ. The resulting TPZ-Pba-NPs showed a synergetic effect to kill tumor cells because TPZ was activated under the hypoxic conditions that originated from the PDT with Pba and laser irradiation. After they were injected into tumor-bearing mice via the tail vein, TPZ-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. These overall results suggest that in vitro screening of drugs based on CI values, mechanism studies in hypoxia, and real-time in vivo imaging are promising strategies in developing NPs for optimized combination therapy.[3]

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Tirapazamine (TP) has been shown to enhance the cytotoxic effects of ionizing radiation in hypoxic cells, thus making it a candidate for a radiosensitizer. This selective behavior is often directly linked to the abundance of O2. In this paper, we study the electronic properties of TP in vacuum, micro-hydrated from one up to three molecules of water and embedded in a continuum of water. We discuss electron affinities, charge distribution, and bond dissociation energies of TP, and find that these properties do not change significantly upon hydration. In agreement with its large electron affinity, and bond breaking triggered by electron attachment requires energies higher than 2.5 eV, ruling out the direct formation of bioactive TP radicals. Our results suggest, therefore, that the selective behavior of TP cannot be explained by a one-electron reduction from a neighboring O2 molecule. Alternatively, we propose that TP's hypoxic selectivity could be a consequence of O2 scavenging hydrogen radicals.[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.)