Topoisomerase II (inhibitor); DNA intercalator [1][2]
Selective for topoisomerase IIα over topoisomerase IIβ in stabilizing enzyme-DNA covalent complexes [2]

In Vitro: Pixantrone inhibited topoisomerase IIα-mediated kDNA decatenation and induced linear DNA formation in a pBR322 DNA cleavage assay, confirming topoisomerase II poison activity. It produced DNA double-strand breaks in K562 cells as determined by γH2AX assay. [2]
Pixantrone was a stronger DNA intercalator than doxorubicin, as shown by thermal denaturation of DNA (ΔTm slope 3.3-fold larger than doxorubicin) and ethidium bromide displacement assay (Kapp = 1.4 × 10⁷ M⁻¹, 2.6-fold larger than doxorubicin). [2]
Pixantrone (0.01-10 μM) inhibited rat AChR 97-116 peptide-specific T cell proliferation in a dose-dependent manner, with 39.3% inhibition at 0.01 μM. It also suppressed Con A-induced T cell responses. [4]
In etoposide-resistant K562 cells (K/VP.5) with reduced topoisomerase II levels, pixantrone showed 5.7-fold cross-resistance (IC50 0.10 μM in K562 vs 0.85 μM in K/VP.5), confirming topoisomerase II targeting. [2]
In MDCK/MDR cells overexpressing ABCB1 (P-gp), pixantrone showed 77-fold resistance compared to parental MDCK cells, indicating it is a P-gp substrate. [2]
Pixantrone (1 mM) produced a semiquinone free radical in a xanthine oxidase/hypoxanthine enzymatic reducing system under hypoxic conditions (EPR spectroscopy), but did not produce detectable semiquinone in K562 cell suspension. [2]
Pixantrone did not significantly increase DCFH oxidation in K562 cells, indicating low reactive oxygen species production. [2]

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Regardless of cell cycle disruption, pixantrone hydrochloride (0–10 μM, 72 h) causes cell death in a range of cancer cell lines [1]. Pixantrone hydrochloride (25–500 nM, 24 h) can cause significant chromosomal abnormalities, disrupt chromosome segregation, and cause mitotic disaster in PANC1 cells [1]. With an IC50 of 0.10 μM and 0.56 μM, pazantrone (0-100 μM, 72 h) HCl efficiently suppresses the proliferation of human leukemia K562 cells, etoposide-resistant K/VP.5 cells, MDCK, and ABCB1-transfected MDCK/MDR cells. 0.058 μM are, in turn, 4.5 μM and 4.5 μM[2]. Pixantrone (0.01-0.2 μM) hydrochloride acts on topoisomerase IIα to construct linear DNA in a concentration-dependent manner. It also generates semiquinone radicals in enzymatic reduction systems, but not in cellular systems—presumably because cellular absorption is minimal [2]. Pixantrone hydrochloride (0.01–10 μM) exerts strong inhibitory effects on T cell proliferation specific to the rat 97–116 peptide [4].

In Vivo: In a rat model of osteoarthritis, pixantrone (12.5 mg/kg, i.p., every 2 days for 7 days) reversed MIA-induced increases in P2X7R, MMP-13, SP, PGE2, IL-1β, IL-6, and TNF-α in cartilage tissue, and inhibited NF-κB pathway activation. [3]
In a rat model of experimental autoimmune myasthenia gravis (EAMG), pixantrone (16.25 mg/kg, i.v., once weekly for 3 weeks) significantly reduced clinical scores (mean score: 1.1 preventive, 0.9 therapeutic vs 2.4 vehicle), increased muscle AChR content (112.0 and 98.3 fmol/g vs 57.5 fmol/g vehicle), and reduced anti-rat AChR antibody titers (0.46 and 5.81 pmol/ml vs 25.74 vehicle). [4]
In EAMG rats, pixantrone treatment abrogated TAChr-specific LNC proliferation (1.25 μg/ml: 1432 cpm vs 29,382 cpm vehicle) and reduced total LNC counts by 77.3%. [4]
In doxorubicin-pretreated mice, pixantrone (27 mg/kg, i.v., once weekly for 3 weeks) did not worsen pre-existing cardiomyopathy (MTS: 4.6 to 4.7), whereas doxorubicin or mitoxantrone significantly worsened cardiomyopathy (MTS to 6.7 and 8.2, respectively). [1]
In treatment-naïve mice, two cycles of pixantrone (27 mg/kg) induced minimal cardiotoxicity (MTS ≤ 0.5), while doxorubicin or mitoxantrone caused marked or severe degenerative cardiomyopathy (MTS 8.0 and 8.0, respectively). [1]
In Lewis rats, pixantrone (16.25 mg/kg, i.v., once weekly for 3 weeks) reduced splenic cell count by 39.8% and LNC count by 77.3% compared to vehicle-treated EAMG rats. [4]

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Pyridoxantrone hydrochloride (IV, 27 mg/kg 3 times every 7 days) did not worsen pre-existing moderate degenerative cardiomyopathy, generates modest cardiotoxicity in mice following repeated treatment cycles, and In animals pretreated with doxorubicin, mortality was lower than that with mitoxantrone [3]. Pixantrone hydrochloride (16.25 mg/kg intravenously, 3 times weekly) modulates lymph node cell (LNC) responses, affects T-cell subsets in TAChR-immune Lewis rats, and is effective in experimental autoimmune severe disease Myasthenia gravis (EAMG) has also shown preventive and therapeutic effects in rats [4].

Enzyme Assay: Topoisomerase II decatenation assay: Reaction mixture (20 μl) contained 40 ng kDNA and either 45 ng topoisomerase IIα or 4.5 ng topoisomerase IIβ. Separation was on 1.2% agarose gel containing ethidium bromide. [2]
DNA cleavage assay: Reaction mixture contained 40 ng pBR322 DNA and 60 ng topoisomerase IIα or 6 ng topoisomerase IIβ. Separation on 1.2% agarose gel containing ethidium bromide at 10 V for 18 hours. [2]
γH2AX assay: K562 cells treated with drug for 4 hours, lysed, and 70 μg protein subjected to SDS-PAGE on 14% gel, transferred to PVDF membrane, probed with anti-γH2AX antibody. [2]
ICE (immunodetection of complex of enzyme-to-DNA) assay: K562 cells treated for 1 hour, genomic DNA isolated, 2.5 μg DNA applied to slot-blot apparatus, immunoblotted with anti-topoisomerase IIα or IIβ antibodies. [2]
EPR spectroscopy for semiquinone radical: 15 μl reaction mixture (xanthine oxidase/hypoxanthine or K562 cell suspension) containing drug (1 mM) in gas-permeable Teflon tubing, placed in EPR cavity, hypoxic conditions with nitrogen gas (400 L/h, 37°C). Instrument settings: microwave power 20 mW, modulation frequency 100 kHz, microwave frequency 9.3 GHz, modulation amplitude 2.0 G, scan time 42 s, 1024 data points/scan, magnetic field centered at 3310 G, 50 G scan range. [2]
Thermal denaturation of DNA: Calf thymus DNA (6 μg/ml) in 10 mM Tris-HCl (pH 7.4) with drug, temperature ramp 1°C/min, absorbance at 260 nm, Tm from first derivative maximum. [2]

Cell Assay: K562 human leukemia cells were maintained in minimal essential medium α with 10% fetal calf serum and 20 mM HEPES (pH 7.2). Growth inhibition was measured by MTS assay (72 h) or direct cell counting. [2]
K/VP.5 etoposide-resistant cells (containing decreased topoisomerase IIα and IIβ) were used to assess cross-resistance. [2]
MDCK and MDCK/MDR (ABCB1-overexpressing) cells were used to assess P-gp substrate status. [2]
Rat T cell line specific for AChR 97-116 peptide: Cells were challenged with 5 μg/ml peptide or 2 μg/ml Con A with increasing pixantrone (0.01-10 μM), and proliferation measured by [³H]thymidine incorporation. [4]
Lymph node cells from TAChr-immunized rats: LNCs (2 × 10⁶/ml) challenged with TAChr (0.25 or 1.25 μg/ml), Torpedo 97-116 peptide (10 μg/ml), rat 97-116 peptide (10 μg/ml), or Con A (2 μg/ml) for 3 days, then [³H]thymidine pulsed overnight. [4]
Flow cytometry: Spleen and LNCs stained with FITC-conjugated anti-CD3, PE-conjugated anti-CD4, and FITC-conjugated anti-CD8 antibodies. Data expressed as percentage of positive cells on forward/side scatter-gated population. [4]

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Cell proliferation assay [4]
Cell Types: Lewis rat T cell line
Tested Concentrations: 0.01-10 μM
Incubation Duration:
Experimental Results: 39.3% inhibition of rat 97-116 peptide-specific T cell proliferation at 0.01 μM, complete inhibition of T cells at high concentrations Cell Proliferation.

Animal Protocol: Mouse cardiotoxicity model (doxorubicin-pretreated): CD1 female mice received doxorubicin (7.5 mg/kg, i.v., once weekly for 3 weeks). Six weeks later, mice received vehicle, doxorubicin (7.5 mg/kg), pixantrone (27 mg/kg), or mitoxantrone (3 mg/kg) once weekly for 3 weeks. Hearts collected at week 8 and 16 for histopathology. Cardiotoxicity scored by severity (1-2) × extension (0-5) = total score (0-10). [1]
Mouse single-agent cardiotoxicity model: CD1 female mice received 1 or 2 cycles of vehicle, doxorubicin (7.5 mg/kg), pixantrone (27 mg/kg), or mitoxantrone (3 mg/kg) once weekly for 3 weeks. Hearts collected at weeks 8, 14, 16, and 22. [1]
Rat osteoarthritis model: Male Wistar rats received intra-articular MIA (5 mg/kg) in left hind knee. Pixantrone (12.5 mg/kg, i.p.) injected every 2 days for 7 days starting 2 weeks after MIA. Behavioral tests (paw withdrawal threshold, weight-bearing asymmetry, knee edema) evaluated at days 3,7,10,14,15,17,19,21. Rats sacrificed on days 7,14,21 for tissue collection. [3]
Rat EAMG model: Female Lewis rats immunized with TAChr (50 μg) in CFA in hind footpads. Pixantrone (16.25 mg/kg, i.v., once weekly for 3 weeks) administered preventively (day 4 post-immunization) or therapeutically (week 4 post-immunization). Clinical score (0-4) and body weight recorded twice weekly. At endpoint (day 64), muscle AChR content measured by [¹²⁵I]α-BTX binding, anti-rat AChR antibodies by RIA, LNC proliferation by [³H]thymidine. [4]

Pixantrone showed reduced cellular uptake compared to doxorubicin and mitoxantrone in K562 cells (1.5 nmol vs 6.5 and 8.1 nmol after 1 hour at 10 μM). The low uptake correlated with its highly negative log D at pH 7.4 (-3.2) and log P (0.0). [2]
Maximum pixantrone plasma concentration of 1.2 μM is seen after a 37.5 mg/m² infusion in humans. [2]

In neonatal rat cardiac myocytes, pixantrone was 10- to 12-fold less damaging than doxorubicin or mitoxantrone as measured by LDH release (equipotent at 10 μM vs 0.8-1.0 μM for doxorubicin/mitoxantrone). [2]
Pixantrone did not bind Fe³⁺ (no spectral changes), unlike doxorubicin and mitoxantrone, which may contribute to its reduced cardiotoxicity. [2]
Pixantrone formed a weak complex with Cu²⁺ (Cu²⁺:pixantrone ratio ~1.33:1), but this is unlikely to be pharmacologically significant. [2]
In mouse cardiotoxicity studies, pixantrone (27 mg/kg, 2 cycles) induced minimal cardiotoxicity (MTS ≤ 0.5) with no mortality, while doxorubicin and mitoxantrone caused marked cardiomyopathy (MTS 8.0) with 40-68% mortality. [1]
In doxorubicin-pretreated mice, pixantrone did not worsen pre-existing cardiomyopathy (MTS 4.6 to 4.7), whereas further doxorubicin or mitoxantrone caused significant worsening (MTS to 6.7 and 8.2). [1]
Pixantrone did not produce detectable semiquinone free radical in K562 cell suspension and did not significantly increase reactive oxygen species production. [2]
No signs of cardiotoxic damage were observed in hearts of pixantrone-treated rats in EAMG studies. [4]

[1]. Pixantrone induces cell death through mitotic perturbations and subsequent aberrant cell divisions. Cancer Biol Ther. 2015;16(9):1397-406.

[2]. Mechanisms of Action and Reduced Cardiotoxicity of Pixantrone; a Topoisomerase II Targeting Agent with Cellular Selectivity for the Topoisomerase IIα Isoform. J Pharmacol Exp Ther. 2016 Feb;356(2):397-409.

[3]. Pixantrone (BBR 2778) has reduced cardiotoxic potential in mice pretreated with doxorubicin: comparative studies against doxorubicin and mitoxantrone. Invest New Drugs. 2007 Jun;25(3):187-95.

[4]. Pixantrone (BBR2778) reduces the severity of experimental autoimmune myasthenia gravis in Lewis rats. J Immunol. 2008 Feb 15;180(4):2696-703.

Pixantrone is an aza-anthracenedione structurally related to mitoxantrone, designed to reduce cardiotoxicity while retaining efficacy. It was approved in the European Union for treatment of adult patients with multiply relapsed or refractory aggressive B cell non-Hodgkin lymphoma. Pixantrone selectively targets topoisomerase IIα over topoisomerase IIβ, which may contribute to its reduced cardiotoxicity as topoisomerase IIβ predominates in postmitotic cardiomyocytes. The drug has also shown efficacy in experimental autoimmune myasthenia gravis and may have potential for treating autoimmune diseases. [1][2][4]

C17H19N5O2.HCL

361.826

325.154

175989-38-5

Pixantrone;144675-97-8;Pixantrone free base;144510-96-3

45262985

Typically exists as solid at room temperature

2.144

5

7

6

25

472

0

C1=CC(=C2C(=C1NCCN)C(=O)C3=CC=NC=C3C2=O)NCCN.Cl

NXSHYUUREPUCRN-UHFFFAOYSA-N

InChI=1S/C17H19N5O2.ClH/c18-4-7-21-12-1-2-13(22-8-5-19)15-14(12)16(23)10-3-6-20-9-11(10)17(15)24;/h1-3,6,9,21-22H,4-5,7-8,18-19H2;1H

6,9-bis(2-aminoethylamino)benzo[g]isoquinoline-5,10-dione;hydrochloride

Pixantrone monohydrochloride; BBR 2778 HCl; BBR-2778; BBR-2778 HCl; Pixantrone hydrochloride [MI]; UNII-FC6HVZ9K78; FC6HVZ9K78;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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