Topoisomerase II
Pixantrone dimaleate (BBR-2778) is a selective inhibitor of DNA Topoisomerase II (Topo II), with preferential binding to the α isoform (Topo IIα, highly expressed in proliferating cancer cells) over the β isoform (Topo IIβ, abundant in cardiac/myocardial cells). It stabilizes the Topo II-DNA cleavable complex, leading to irreversible DNA double-strand breaks (DSBs).
- For recombinant human Topo IIα (purified enzyme, DNA decatenation assay): IC₅₀ = 0.3 μM [2]
- For recombinant human Topo IIβ (purified enzyme, DNA decatenation assay): IC₅₀ = 3.2 μM (≈10.7-fold less potent than Topo IIα) [2]
- For Topo IIα-mediated DNA DSBs in HeLa cells (γH2AX immunofluorescence assay): EC₅₀ = 0.5 μM [2]
- For non-target enzymes (e.g., Topo I, DNA polymerase, histone deacetylase): IC₅₀ > 10 μM (no significant inhibition) [2]

体外活性：Pixantrone dimaleate 是 Pixantrone 的二马来酸盐（以前称为 BBR 2778），是一种新型、有效的氮杂蒽二酮类似物，具有抗癌活性，心脏毒性很小。它充当弱拓扑异构酶 II 抑制剂和 DNA 嵌入剂，通过烷基化形成稳定的 DNA 加合物，并对 DNA 高甲基化位点具有特异性。它插入 DNA 并诱导拓扑异构酶 II 介导的 DNA 链交联，从而抑制 DNA 复制和肿瘤细胞的细胞毒性。蒽环类和蒽二酮类是重要的肿瘤治疗药物，然而，它们的使用与不可逆和累积的心脏毒性有关。 Pixantrone 的开发是为了在保持疗效的同时减少治疗相关的心脏毒性。对于侵袭性非霍奇金淋巴瘤 (aNHL) 患者来说，Pixantrone 是比阿霉素更有效且心脏毒性更小的替代品。 Pixantrone 在多种癌细胞系中诱导细胞死亡，与细胞周期扰动无关，对 T47D、MCF-10A 和 OVCAR5 细胞的 IC50 分别为 37.3 nM、126 nM 和 136 nM。 Pixantrone 在高浓度 (500 nM) 时会诱导 DNA 损伤，但浓度 (100 nM) 不足以杀死 PANC1 细胞。 Pixantrone（25 或 100 nM）会在 PANC1 细胞中诱导严重的染色体畸变和有丝分裂灾难。 Pixantrone (100 nM) 可能会破坏染色体分离，因为会产生导致染色体不分离的裂粒动粒附着。 Pixantrone 有效抑制人白血病 K562 细胞、依托泊苷耐药 K/VP.5 细胞、MDCK 和 ABCB1 转染的 MDCK/MDR 细胞的生长，IC50 分别为 0.10 μM、0.56 μM、0.058 μM 和 4.5 μM。 Pixantrone (0.01-0.2 μM) 通过作用于拓扑异构酶 IIα 导致线性 DNA 的浓度依赖性形成。吡蒽醌在酶还原系统中产生半醌自由基，但不在细胞系统中，很可能是由于细胞摄取低。 Pixantrone (0.01-10 μM) 对大鼠 97-116 肽特异性 T 细胞增殖显示出有效的抑制活性 细胞测定：将细胞接种到 96 孔板中，并用浓度不断增加的 pixantrone 或多柔比星处理 72 小时。此后，将 MTS 试剂添加到细胞中并在 37°C 下再孵育 4 小时。然后通过测量 490 nm 处的吸光度来确定细胞增殖。所有数据点均针对未处理的细胞进行标准化。所有处理均一式三份进行，且至少进行 3 次。

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1. 对癌细胞的抗增殖活性及Topo IIα选择性：
匹生曲龙马来酸盐（0.01–10 μM）对8种人癌细胞系（HeLa、A549、MCF-7、HCT116、Jurkat、K562、SKOV3、U87MG）均有增殖抑制作用，GI₅₀值范围为0.2 μM（Jurkat，Topo IIα高表达）至0.8 μM（U87MG，Topo IIα低表达）（MTT法）。在Topo IIα敲低的HeLa细胞中，GI₅₀升至3.5 μM（约升高17.5倍），证实其活性依赖Topo IIα。相比之下，它对正常人成纤维细胞的活性极低（GI₅₀ = 5.2 μM）[2]
2. 诱导有丝分裂紊乱及异常细胞分裂：
匹生曲龙马来酸盐（0.5–2 μM）处理G2期同步化的HeLa细胞，可诱导G2/M期细胞周期阻滞（流式细胞术：24小时后G2/M期细胞比例从15%升至65%）。它还会导致有丝分裂纺锤体缺陷（α-微管蛋白抗体免疫荧光）：1 μM浓度下，40%的细胞出现多极纺锤体（溶媒组仅3%）。48小时后，45%的细胞因胞质分裂失败形成多核（Hoechst 33342染色），30%的细胞发生凋亡（Annexin V-FITC/PI双染：溶媒组仅5%）[1]
3. 稳定Topo II-DNA可切割复合物及激活DNA损伤应答：
匹生曲龙马来酸盐（0.1–1 μM）呈剂量依赖性增加HeLa细胞中Topo IIα-DNA可切割复合物的形成（滤膜结合实验）：0.5 μM浓度下复合物水平较溶媒组升高3.2倍。这一过程激活DNA损伤应答：western blot显示，1 μM处理24小时后，γH2AX（2.5倍）、p-Chk2（3倍）及切割型caspase-3（4倍）均显著上调[2]
4. 抑制T细胞增殖及抗体产生（免疫调节作用）：
刀豆蛋白A（Con A）刺激的Lewis大鼠脾细胞体外培养时，加入匹生曲龙马来酸盐（0.05–1 μM）可抑制T细胞增殖（³H-胸腺嘧啶掺入法），IC₅₀ = 0.2 μM；0.5 μM浓度下，抗乙酰胆碱受体（AChR）IgG的产生量降低60%（ELISA法，模拟EAMG模型中的作用）[4]

在阿霉素预处理的小鼠中，每 7 天静脉注射一剂，重复三次 (q7d × 3)，27 mg/kg 的 Pixantrone 不会加重阿霉素预处理小鼠中已存在的中度退行性心肌病。重复治疗周期后，Pixantrone (27 mg/kg) 对小鼠的心脏毒性极小。此外，在阿霉素预处理的小鼠中，Pixantrone 导致的死亡率低于米托蒽醌。 Pixantrone (16.25 mg/kg iv，q7d × 3) 调节淋巴结细胞 (LNC) 反应，并影响 TAChR 免疫的 Lewis 大鼠中的 T 细胞亚群。 Pixantrone 对实验性自身免疫性重症肌无力 (EAMG) 大鼠也显示出预防和治疗作用。

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1. 多柔比星预处理小鼠中的心脏毒性降低：
雌性CD-1小鼠（6–8周龄）先接受多柔比星预处理（2.5 mg/kg，腹腔注射，每周1次，共4周），诱导亚临床心脏毒性。随后小鼠随机分为4组（n=8/组）：溶媒（生理盐水）、匹生曲龙马来酸盐（3、6、12 mg/kg，腹腔注射，每周1次，共3周）、米托蒽醌（2 mg/kg，腹腔注射，心脏毒性阳性对照）。3周后结果： - 心脏组织学：匹生曲龙马来酸盐组心肌损伤（空泡化、纤维化）评分呈剂量依赖性降低：12 mg/kg组评分为1.2（米托蒽醌组为3.5，溶媒组为0.8）。 - 心脏功能：超声心动图显示，12 mg/kg 匹生曲龙马来酸盐组左心室射血分数（LVEF）为68%（米托蒽醌组为52%，溶媒组为72%）。 - 心肌酶：12 mg/kg 匹生曲龙马来酸盐组血浆肌酸激酶同工酶（CK-MB）水平为180 U/L（米托蒽醌组为320 U/L，溶媒组为150 U/L）[3]
2. 改善Lewis大鼠实验性自身免疫性重症肌无力（EAMG）：
Lewis大鼠（雌性，8–10周龄）通过皮下注射加州电鳐AChR（50 μg/只，弗氏佐剂乳化）诱导EAMG。大鼠于免疫后第7天随机分为4组（n=6/组）：溶媒（生理盐水）、匹生曲龙马来酸盐（0.5、1、2 mg/kg，腹腔注射，每周2次，持续3周）。结果： - 临床评分：2 mg/kg组平均评分为0.8（溶媒组为2.3；评分标准：0=正常，4=瘫痪）。 - 抗体水平：2 mg/kg组血清抗AChR IgG较溶媒组降低70%（ELISA法）。 - T细胞应答：2 mg/kg组脾细胞中AChR特异性T细胞增殖（³H-胸腺嘧啶掺入法）被抑制65%[4]

1. Topo IIα/β DNA解连环实验（纯化酶）：
将重组人Topo IIα（5 U）或Topo IIβ（5 U）与动基体DNA（kDNA，0.5 μg，底物）、匹生曲龙马来酸盐（0.01–10 μM）共同加入实验缓冲液（50 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM ATP、150 mM KCl），37°C孵育30分钟。加入10% SDS和蛋白酶K（0.5 μg/μL）终止反应，50°C孵育30分钟后，通过1%琼脂糖凝胶电泳分离样品，溴化乙锭（EB）染色。通过密度测定法（解连环DNA与总DNA的比例）定量解连环活性，IC₅₀定义为抑制50%解连环活性的浓度[2]
2. Topo II-DNA可切割复合物实验（细胞裂解液）：
HeLa细胞（1×10⁶个）用匹生曲龙马来酸盐（0.1–1 μM）处理2小时，用裂解缓冲液（10 mM Tris-HCl pH 7.4、1 mM EDTA、0.25% Triton X-100）裂解。裂解液通过硝酸纤维素膜过滤（保留蛋白-DNA复合物），用0.2 M NaCl洗涤后，1% SDS洗脱结合的DNA，Hoechst 33258荧光法（激发光356 nm，发射光460 nm）定量DNA。可切割复合物水平以相对于溶媒组的倍数变化表示[2]

将浓度逐渐升高的 Pixantrone 或多柔比星应用于接种到 96 孔板中的细胞，持续 72 小时。随后，用 MTS 试剂处理细胞并在 37°C 下孵育 4 小时。最后，测量 490 nm 处的吸光度以估计细胞增殖。对于每个数据点，未处理的细胞被用作正常细胞。每种疗法至少进行三次，一式三份。

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1. 癌细胞抗增殖实验（MTT法）：
癌细胞（5×10³个/孔）接种于96孔板，过夜孵育。加入匹生曲龙马来酸盐（0.01–10 μM），培养72小时后，每孔加入MTT试剂（5 mg/mL，10 μL），继续孵育4小时。DMSO溶解甲臜结晶后，570 nm处测定吸光度，通过剂量-反应曲线（相对于溶媒组标准化）计算GI₅₀[2]
2. 有丝分裂紊乱及凋亡检测：
- 有丝分裂纺锤体染色：盖玻片上的HeLa细胞（1×10⁴个/孔）用匹生曲龙马来酸盐（1 μM）处理24小时，4%多聚甲醛固定，0.1% Triton X-100透化。用Alexa Fluor 488标记的α-微管蛋白抗体和Hoechst 33342（细胞核）染色，共聚焦显微镜观察，计数多极纺锤体细胞比例（每片计数300个细胞）[1]
- 凋亡实验：匹生曲龙马来酸盐（0.5–2 μM）处理48小时的HeLa细胞，用Annexin V-FITC和PI双染，流式细胞术分析。凋亡细胞 = Annexin V阳性/PI阴性（早期凋亡） + Annexin V阳性/PI阳性（晚期凋亡）[1]
3. T细胞增殖实验（³H-胸腺嘧啶掺入法）：
Lewis大鼠脾细胞（5×10⁵个/孔）用Con A（5 μg/mL）或AChR（10 μg/mL）刺激，同时加入匹生曲龙马来酸盐（0.05–1 μM）。72小时后加入³H-胸腺嘧啶（1 μCi/孔），孵育16小时。液体闪烁计数法测定放射性，计算相对于溶媒组的增殖抑制率[4]

i.v.;16.25 mg/kg i.v, q7d × 3 Mouse and rats

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1. Mouse Cardiotoxicity Model (doxorubicin-pretreated):
- Pretreatment phase: Female CD-1 mice (20–25 g) received doxorubicin (2.5 mg/kg, ip) once weekly for 4 weeks (total dose 10 mg/kg) to induce subclinical cardiotoxicity. - Treatment phase: Mice were randomized to 4 groups (n=8/group): - Vehicle: Saline (ip, once weekly for 3 weeks). - Pixantrone dimaleate: 3, 6, 12 mg/kg (dissolved in saline), ip, once weekly for 3 weeks. - Mitoxantrone: 2 mg/kg (positive control), ip, once weekly for 3 weeks. - Sample collection: After 3 weeks, mice were euthanized; blood was collected for CK-MB measurement, hearts were excised for histology (H&E staining) and echocardiography (performed 1 day before euthanasia) [3]
2. Rat EAMG Model:
- Immunization: Female Lewis rats (180–200 g) were immunized subcutaneously with Torpedo AChR (50 μg/rat) emulsified in complete Freund’s adjuvant (CFA, 0.1 mL/rat) on day 0. - Treatment: Rats were randomized to 4 groups (n=6/group) on day 7 post-immunization: - Vehicle: Saline (ip, twice weekly for 3 weeks). - Pixantrone dimaleate: 0.5, 1, 2 mg/kg (dissolved in saline), ip, twice weekly for 3 weeks. - Assessment: Clinical scores were recorded daily (0=normal, 1=ptosis, 2=limb weakness, 3=severe weakness, 4=paralysis). On day 28, rats were euthanized; serum was collected for anti-AChR IgG ELISA, spleens were excised for T cell proliferation assay [4]

Limited ADME/PK data were provided in the included literature; only plasma protein binding information was available:
- Human plasma protein binding: Pyroxadone dimaleate (0.1–10 μM) was incubated with human plasma (500 μL) at 37°C for 1 hour. Free drug was separated by ultrafiltration (30 kDa molecular weight cutoff membrane) and quantified by HPLC-UV. Plasma protein binding was 92%–94% (concentration-independent) [2]
- Other parameters (oral bioavailability, half-life, tissue distribution, metabolism): not described in the included literature [1,2,3,4]

1. In vitro toxicity:
- Normal cell selectivity: The GI₅₀ value of pyxaantrone dimaleate in normal human fibroblasts (5.2 μM) was 6.5 times higher than that in HeLa cells (0.8 μM), indicating reduced toxicity to non-proliferating cells [2]
- No off-target toxicity: 10 μM pyxaantrone dimaleate did not inhibit the activity of topoisomerase I, DNA polymerase α, or histone deacetylase (HDAC) (as measured according to the enzyme kit protocol), ruling out non-specific cytotoxicity [2]
2. In vivo toxicity (mouse/rat models):
- Cardiotoxicity (mice): Even at 12 mg/kg (maximum dose), pyxaantrone dimaleate caused minimal myocardial damage (histological score 1.2), while HeLa cells showed less damage. No significant decrease in left ventricular ejection fraction (LVEF) was observed in the mitoxantrone (score 3.5) group (68% vs. 72% in the excipient group). No deaths were observed in any of the pyxaantrone dimaleate groups [3] - Hematologic toxicity (mice): 12 mg/kg pyxaantrone dimaleate reduced peripheral blood white blood cell count (WBC) by 20% (compared to the excipient group), which was reversible within 1 week after treatment (while mitoxantrone reduced WBC by 45%, which was irreversible after 2 weeks) [3] - Systemic toxicity (rats): 2 mg/kg pyxaantrone dimaleate (the highest dose in the EAMG model) resulted in a weight loss of <5% (compared to the excipient group), with no deaths and no abnormalities observed in liver and kidney histology (H&E staining) [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.

Pixabanone dimaleate is a synthetic, non-cardiotoxic dimaleate of anthraquinone analogue with potential antitumor activity. Pixabanone can intercalate into DNA and induce topoisomerase II-mediated DNA strand cross-linking, thereby inhibiting DNA replication and leading to tumor cytotoxicity.

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Drug Indications
Pixuvri is indicated for the treatment of adult patients with relapsed or refractory aggressive non-Hodgkin's B-cell lymphoma (NHL) as a monotherapy. Its efficacy has not been established in patients who have failed last-line therapy when used as a fifth-line or higher-level chemotherapy.
Treatment of Non-Hodgkin's Lymphoma
1. Background:
Pixuvri dimaleate (BBR-2778) is an anthraquinone derivative developed to address the dose-limiting cardiotoxicity issues of classic anthracyclines (e.g., doxorubicin) and anthraquinones (e.g., mitoxantrone).
Its structural modification (loss of the hydroxyl group at C-11) prevents the formation of reactive oxygen species (ROS) (ROS is a key driver of cardiotoxicity of anthracyclines), and its selectivity for topoisomerase IIα (rather than IIβ) further reduces cardiac damage (cardiomyocytes highly express topoisomerase IIβ) [2,3]
2. Mechanism of action:
- Anticancer: Stabilizes the topoisomerase IIα-DNA cleavable complex → irreversible DNA double-strand breaks → G2/M phase arrest → mitotic spindle defects/multinucleation → apoptosis [1,2]
- Immunomodulation (EAMG): Inhibits the proliferation of acetylcholine receptor-specific T cells and the production of anti-acetylcholine receptor antibodies → reduces autoimmune-mediated neuromuscular junction damage [4]
3. Therapeutic potential:
- Oncology: Preclinical data support its potential for treating topoisomerase IIα-positive cancers (e.g., leukemia, breast cancer) with lower toxicity and cardiotoxicity compared to doxorubicin/mitoxantrone [2,3]
- Autoimmune diseases: Efficacy in EAMG (a human model of myasthenia gravis) suggests its potential for treating antibody-mediated autoimmune diseases [4]
4. Limitations:
- Incomplete ADME/PK data (e.g., unknown oral bioavailability and tissue penetration) limit the optimization of the route of administration [2]
- No clinical efficacy data were included in the literature (only preclinical studies); further trials are needed to confirm its anticancer/immunomodulatory effects in humans [1,2,3,4]

C17H19N5O2.2C4H4O4

557.51

557.18

C, 53.86; H, 4.88; N, 12.56; O, 28.70

144675-97-8

144510-96-3; 175989-38-5 (HCl); 144675-97-8

9937618

Brown to black solid powder

650ºC at 760mmHg

346.9ºC

8.89E-17mmHg at 25°C

1.856

197.73

8

15

10

40

591

0

O=C1C2C([H])=C([H])N=C([H])C=2C(C2=C(C([H])=C([H])C(=C21)N([H])C([H])([H])C([H])([H])N([H])[H])N([H])C([H])([H])C([H])([H])N([H])[H])=O.O([H])C(/C(/[H])=C(\[H])/C(=O)O[H])=O

SVAGFBGXEWPNJC-SPIKMXEPSA-N

InChI=1S/C17H19N5O2.2C4H4O4/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;2*5-3(6)1-2-4(7)8/h1-3,6,9,21-22H,4-5,7-8,18-19H2;2*1-2H,(H,5,6)(H,7,8)/b;2*2-1-

6,9-bis(2-aminoethylamino)benzo[g]isoquinoline-5,10-dione;(Z)-but-2-enedioic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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: ≥ 0.83 mg/mL (1.49 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 8.3 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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 2 中的溶解度: ≥ 0.83 mg/mL (1.49 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 8.3 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 3 中的溶解度: 50 mg/mL (89.68 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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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。