Topoisomerase II
DNA topoisomerase II [1][2][3]

Voreloxin Hydrochloride is a novel topoisomerase II poison and inhibitor that causes site-specific DNA DSB, G2 arrest, and apoptosis by intercalating DNA. Voreloxin (0.1–20 µM) causes site-selective DNA DSB in CCRF-CEM cells and inhibits topoisomerase II activity. Voreloxin (0.11, 0.33, 1, 3 µM) partially induces G2 arrest in A549 lung cancer cell line by using topoisomerase II. Voreloxin cytotoxic activity requires DNA intercalation. Nevertheless, Voreloxin (1–9 µM) does not produce a noticeable amount of ROS[1]. In AML cell lines MV4-11 and HL-60, voreloxin exhibits strong cytotoxic activity, with IC50 values of 95 ± 8 nM and 884 ± 114 nM, respectively. In acute leukemia cell lines, voreloxin and cytarabine exhibit additive or synergistic activity[2]. With a mean LD50 of 2.3 μM, voreloxin exhibits activity against primary acute myeloid leukemia (AML). In the myeloid cell lines NB4 and HL-60, voreloxin has an LD50 of 0.59 μM ± 0.25 μM. Voreloxin acts on topoisomerase II and induces cell accumulation in the S and G2 phases of the cell cycle[3].

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In human myeloid leukemia cell lines (HL-60, NB4, U937, K562), Voreloxin (SNS-595) HCl exhibited concentration-dependent antiproliferative activity, with significant inhibition of cell growth observed at nanomolar concentrations [3]
- Voreloxin (SNS-595) HCl induced cell cycle arrest at the G2/M phase in myeloid leukemia cells, accompanied by upregulation of cyclin B1 and downregulation of cdc25C, leading to cell cycle checkpoint activation [3]
- The drug triggered apoptosis in myeloid leukemia cells, as evidenced by increased caspase-3/7 activity, phosphatidylserine externalization, and DNA fragmentation. Apoptosis induction was associated with mitochondrial membrane potential loss and cytochrome c release [3]
- In combination with cytarabine, Voreloxin (SNS-595) HCl showed synergistic antiproliferative and apoptotic effects in myeloid leukemia cell lines, with combination indices (CI) < 1 indicating synergism [2][3]
- Voreloxin (SNS-595) HCl inhibited topoisomerase II-mediated DNA relaxation, stabilizing the enzyme-DNA cleavage complex and preventing DNA strand religation, which contributed to its antitumor activity [1][2][3]

Voreloxin (20 mg/kg, i.v.) administered once every four days, twice (q4d ×2), is the only treatment that reduces the cellularity of CD-1 mice's bone marrow by 80%. In mice, voreloxin at 10 mg/kg combined with cytarabine results in marrow ablation, sinusoidal dilatation, and adipocyte infiltration. In bone marrow and peripheral blood CD-1 mice, voreloxin (20 mg/kg, i.v.) and cytarabine induce a reversible decrease in myeloid and lymphoid cells. When voreloxin (10 mg/kg, q4d ×2) and cytarabine are combined, platelets CD-1 mice experience a more mild reversible neutropenia[2].

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In murine models of myeloid leukemia, intravenous administration of Voreloxin (SNS-595) HCl resulted in significant inhibition of tumor growth, with reduced tumor burden and prolonged survival compared to control groups [3]
- Voreloxin (SNS-595) HCl induced bone marrow aplasia in vivo, characterized by decreased counts of hematopoietic progenitor cells and mature blood cells in treated animals [2]
- Combination treatment with Voreloxin (SNS-595) HCl and cytarabine in leukemia-bearing mice showed enhanced antitumor efficacy compared to either drug alone, without substantial increase in toxicity [2][3]

DNA topoisomerase II activity assay: Purified DNA topoisomerase II was incubated with supercoiled plasmid DNA in reaction buffer. Voreloxin (SNS-595) HCl was added at different concentrations, and the mixture was incubated at 37°C for a specified period. The reaction was terminated by adding a stop solution, and the products were separated by agarose gel electrophoresis. The intensity of supercoiled, relaxed, and cleaved DNA bands was quantified to assess the inhibition of topoisomerase II activity. The results showed that Voreloxin (SNS-595) HCl stabilized the topoisomerase II-DNA cleavage complex, inhibiting DNA religation [1][2][3]

MTS cell proliferation assays are used to conduct in vitro toxicity tests on primary AML mononuclear cells over the course of 48 hours. One can calculate lethal doses (LD50). In a final volume of 90 μL, cells are treated with voreloxin (31.25 nM to 4 μM) and Ara-C (62.5 nM to 8 μM) by serial dilution and incubated for 48 hours. After incubation, the reaction is incubated for an additional 4 hours with the addition of 20 μL of MTS reagent. Spectrophotometry is used to measure the absorbance of the reaction at 490 nm after this point, and the percentage of viable cells is computed in relation to the untreated control cells in the same experiment. Software called Calcusyn is used to calculate IC50 values[3].

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Myeloid leukemia cell antiproliferative assay: Leukemia cell lines (HL-60, NB4, U937, K562) were seeded in 96-well plates at a density of 5×10³ cells/well. Cells were treated with serial concentrations of Voreloxin (SNS-595) HCl alone or in combination with cytarabine. After 72 hours of incubation at 37°C with 5% CO₂, cell viability was measured using a colorimetric assay. The IC50 values for antiproliferative activity were calculated based on dose-response curves [2][3]
- Cell cycle analysis: Leukemia cells were treated with Voreloxin (SNS-595) HCl at the IC50 concentration for 24-48 hours. Cells were harvested, fixed with ethanol, stained with propidium iodide, and analyzed by flow cytometry. The distribution of cells in G0/G1, S, and G2/M phases was quantified to determine cell cycle arrest [3]
- Apoptosis assay: Treated leukemia cells were stained with annexin V-FITC and propidium iodide. Flow cytometric analysis was performed to detect apoptotic cells (annexin V-positive/propidium iodide-negative for early apoptosis; annexin V-positive/propidium iodide-positive for late apoptosis). Caspase-3/7 activity was measured using a luminescent assay kit to confirm apoptotic signaling [3]
- Synergy assay: Cells were treated with various combinations of Voreloxin (SNS-595) HCl and cytarabine. The combination index (CI) was calculated using the Chou-Talalay method to determine synergism (CI < 1), additive effect (CI = 1), or antagonism (CI > 1) [2][3]

Before beginning therapy, the animals are weighed, randomized according to their body weight, and placed into study groups. On day zero and day four (q4d ×2), voreloxin is given intravenously (IV) at a dose of 10 or 20 mg/kg each time. Day zero and day four (tid q4d ×2) are given cytarabine subcutaneously (SC) at 20 or 60 mg/kg every 8 hours. Three or fewer animals per treatment group, but no more than ten, have their tissues and blood sampled on days six, eight, and twelve. Femurs are immersed for 24 to 48 hours in either the Streck Tissue Fixative solution or the 10% formalin solution, and then they are dehydrated to 70% (ethanol, isopropanol, or methanol). At Biopathology Labs, femurs undergo decalcification, paraffin embedding, and sectioning. Hematoxylin-eosin is used to stain the four micron sections (H&E). Femurs stained with H&E are inspected, and the bone marrow's percentage cellularity is calculated. Using Image-Pro Plus v6.1 software, digital photos of representative femur sections are captured on a Leica DM2000 microscope[2].

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Leukemia xenograft model: Immunocompromised mice were intravenously injected with human myeloid leukemia cells to establish systemic leukemia. When leukemia engraftment was confirmed by flow cytometric detection of human CD45-positive cells in peripheral blood, mice were randomly divided into treatment groups. Voreloxin (SNS-595) HCl was dissolved in a sterile aqueous solvent and administered intravenously at doses ranging from 5 to 20 mg/kg, with dosing intervals of 3-4 days for a total of 3-4 doses. Cytarabine was administered intraperitoneally at a dose of 50 mg/kg daily for 5 days in combination groups. Mice were monitored for body weight, survival time, and peripheral blood cell counts. At the end of the experiment, bone marrow, spleen, and liver tissues were collected for pathological and flow cytometric analysis [2][3]
- Bone marrow toxicity evaluation: Immunocompetent mice were administered Voreloxin (SNS-595) HCl intravenously at doses of 10-30 mg/kg. Bone marrow samples were collected 7 days after treatment, and hematopoietic progenitor cells were cultured in semisolid medium. Colony-forming units (CFU) were counted to assess the effect on hematopoietic function [2]

Blood toxicity: Voeloxin (SNS-595) HCl can induce dose-dependent bone marrow suppression in vivo, manifested as a decrease in bone marrow hematopoietic progenitor cells, leukopenia, thrombocytopenia and anemia [2][3] - Bone marrow aplasia: High doses of Voeloxin (SNS-595) HCl can cause severe bone marrow aplasia in mice, manifested as a decrease in cell number and impaired recovery of hematopoietic function [2]

[1]. Vosaroxin is a novel topoisomerase-II inhibitor with efficacy in relapsed and refractory acute myeloid leukaemia. Expert Opin Pharmacother. 2015 Jun;16(9):1395-402.

[2]. Voreloxin, a first-in-class anticancer quinolone derivative, acts synergistically with cytarabine in vitro and induces bone marrow aplasia in vivo. Cancer Chemother Pharmacol. 2010 Oct;66(5):881-8.

[3]. The topoisomerase II inhibitor voreloxin causes cell cycle arrest and apoptosis in myeloid leukemia cells and acts in synergy with cytarabine. Haematologica. 2011 Mar;96(3):393-9.

Voreloxin (SNS-595) HCl is a first-in-class anticancer quinolone derivative with a mechanism of action that is quite different from that of traditional quinolone antibiotics.[2][3] - Mechanism of action: This drug exerts its anticancer effect by binding to DNA and inhibiting DNA topoisomerase II, stabilizing the enzyme-DNA breakage complex, inducing DNA double-strand breaks, and ultimately leading to cell cycle arrest and apoptosis.[1][2][3] - Clinical application: This drug has been proven effective in treating relapsed and refractory acute myeloid leukemia (AML), and clinical trials have shown that it has significant efficacy in patients with advanced AML.[1] - Synergistic effect: Voeloxin (SNS-595) HCl has a synergistic effect with cytarabine, a standard chemotherapy drug for AML, supporting its use in combination therapy.[2][3]

C18H20CLN5O4S

437.9

437.092

175519-16-1

175414-77-4

10343042

White to gray solid powder

2.221

3

10

5

29

662

2

CN[C@H]1CN(C[C@@H]1OC)C2=NC3=C(C=C2)C(=O)C(=CN3C4=NC=CS4)C(=O)O.Cl

JJZCCQHWCOXGCL-QNTKWALQSA-N

InChI=1S/C18H19N5O4S.ClH/c1-19-12-8-22(9-13(12)27-2)14-4-3-10-15(24)11(17(25)26)7-23(16(10)21-14)18-20-5-6-28-18;/h3-7,12-13,19H,8-9H2,1-2H3,(H,25,26);1H/t12-,13-;/m0./s1

7-[(3S,4S)-3-methoxy-4-(methylamino)pyrrolidin-1-yl]-4-oxo-1-(1,3-thiazol-2-yl)-1,8-naphthyridine-3-carboxylic acid;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 mg/mL (4.57 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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