PKC ( IC50 = 8.5 μM ); Topoisomerase II
DNA topoisomerase II [2]
Viral DNA replication machinery (EC50 = 0.3 μM for cowpox virus; EC50 = 0.5 μM for monkeypox virus) [1]

In vitro activity: Mitoxantrone causes DNA fragmentation and the proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), a marker of caspase activation, in every patient examined, proving that the induction of apoptosis is the cause of mitoxantrone's cytotoxic effect. Mitoxantrone stimulates IkappaBalpha degradation and activates NFkappaB in the promyelocytic leukemia cell line HL60, but not in the variant cells, HL60/MX2 cells, which express a truncated alpha isoform of topoisomerase II and lack the beta isoform, leading to a different subcellular distribution. In a dose-dependent manner, mitoxantrone prevents the growth of B cells, antigen-specific T-cell lines (TCLs), or activated peripheral blood monocytic leukemia cells (PBMCs) stimulated on antigen-presenting cells (APCs). Low doses of mitoxantrone cause PBMCs, monocytes, and DCs to undergo apoptosis; higher doses result in cell lysis.

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Against cowpox virus and monkeypox virus in Vero cells, Mitoxantrone HCl (mitozantrone) exhibited potent antiviral activity, inhibiting viral replication in a concentration-dependent manner. The EC50 values were 0.3 μM (cowpox virus) and 0.5 μM (monkeypox virus), with no significant cytotoxicity at concentrations up to 10 μM (CC50 > 10 μM) [1]
- In various tumor cell lines, Mitoxantrone HCl (mitozantrone) inhibited DNA synthesis and induced apoptosis, characterized by DNA fragmentation and caspase activation. It intercalated into DNA strands, blocking nucleic acid replication and transcription [2]
- The drug suppressed the activity of DNA topoisomerase II, stabilizing the enzyme-DNA cleavage complex and preventing DNA strand religation, which contributed to its antitumor and antiviral effects [2]

Mitoxantrone administered at the ideal dose (1.6 mg/kg/day; as a free base) results in a statistically significant number of 60-day survivors (curative effect) in mice with IP implanted L1210 leukemia. Mitoxantrone and ADM given intravenously (IV) also demonstrate efficacious antitumor activities in Lewis lung carcinoma implanted in SC, producing a 60% and a 45% ILS, respectively. To determine whether mitoxantrone (MXN) was effective against CPXV in vivo, 0.25 or 0.5 mg/kg of drug was administered intraperitoneally to 4–6 week old female C57Bl/6 mice 1 day post-intraperitoneal inoculation. Log-rank tests were performed comparing survival times between the two MXN treatment groups relative the mock-treated group. A p-value of 0.025 was considered statistically significant, after a Bonferroni correction for the comparisons of the two MXN dose groups to the mock-treated. The lower dose of MXN significantly improved the survival time of infected animals, with the median day-of-death (MDD) in infected animals increasing from day 7 to day 9 (p = 0.0015). The 0.5 mg/kg dose of MXN improved the MDD to 12 days (p = 0.0005). Twenty-five percent of the animals treated with 0.5 mg/kg MXN survived compared to 5% of mock-treated animals (Fig. 2 ). Administration of higher levels of MXN or additional dosages were not beneficial (data not shown).[1]

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Mitoxantrone inhibits PKC in a non-competitive manner with respect to phosphatidylserine and ATP, but in a competitive manner with respect to histone H1, where its Ki value is 6.3 μM. Cell viability is reduced when B-CLL cells are treated with mitoxantrone (0.5 μg/mL) for 48 hours. Poly(ADP-ribose) polymerase (PARP) is subjected to proteolytic cleavage and DNA fragmentation upon induction by mitoxantrone, indicating that the cytotoxic effect of the drug is a result of apoptosis induction. Human breast carcinoma cell lines MDA-MB-231 and MCF-7 exhibit cytotoxicity to mitoxantrone, with IC50 values of 18 and 196 nM, respectively.

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DNA topoisomerase II activity assay: Purified DNA topoisomerase II was incubated with supercoiled plasmid DNA in reaction buffer at 37°C. Mitoxantrone HCl (mitozantrone) was added at serial concentrations (0.1-5 μM), and the mixture was incubated for 45 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 55°C for 1 hour. DNA products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The inhibition of topoisomerase II-mediated DNA relaxation was quantified by measuring the intensity of supercoiled DNA bands. The drug was confirmed to stabilize the enzyme-DNA cleavage complex [2]

In standard 96-well plates, the human breast carcinoma cell lines MDA-MB-231 and MCF-7 are seeded. The culture medium is swapped out for one containing varying concentrations of mitoxantrone (10-5 to 5 μM) with or without DHA (30 μM) for a period of seven days following seeding. The tetrazolium salt assay is used to determine the overall viability of cells.

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Antiviral activity assay: Vero cells were seeded in 96-well plates at 2×10⁴ cells/well and allowed to adhere overnight. Cells were infected with cowpox virus or monkeypox virus at a multiplicity of infection (MOI) of 0.1. After 1 hour of adsorption, Mitoxantrone HCl (mitozantrone) was added at concentrations ranging from 0.01 to 10 μM. After 72 hours of incubation at 37°C with 5% CO₂, viral replication was assessed by measuring viral DNA copy number via quantitative PCR. Cytotoxicity was evaluated using a colorimetric assay to determine CC50 [1]
- Tumor cell antiproliferation and apoptosis assay: Tumor cells were plated in 6-well plates at 5×10⁵ cells/well and treated with Mitoxantrone HCl (mitozantrone) at 0.1-5 μM for 24-48 hours. DNA synthesis was measured by radioactive thymidine incorporation assay. Apoptotic cells were detected by DAPI staining (nuclear fragmentation) and western blot analysis of caspase-3 activation [2]

1.6 mg/kg/day; i.p. or i.v.
Mice In agreement with a previous report which found that mitoxantrone/MXN exhibited no in vivo activity against intranasal VACV infection in BALB/c mice (Deng et al., 2007), we observed no efficacy against intranasal CPXV infection in BALB/c mice. Mitoxantrone did, however, demonstrate efficacy when used to treat intraperitoneally-infected C57Bl/6 mice, suggesting that differences in the route of infection and in mouse strain susceptibility to infection may influence MXN’s efficacy. While related anthracenediones have been reported to have in vivo antiviral activity against viruses unrelated to poxviruses (Dang et al., 2009, Sill et al., 1974), to our knowledge this is the first report of limited in vivo antiviral activity by MXN against poxviruses.[1]

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Absorption, Distribution and Excretion
Oral Absorption Malfunction 1000 L/m² 21.3 L/hr/m² [Elderly breast cancer patients receive 15-90 mg/m² intravenously] 28.3 L/hr/m² [Non-elderly nasopharyngeal carcinoma patients receive 15-90 mg/m² intravenously] 16.2 L/hr/m² [Non-elderly malignant lymphoma patients receive 15-90 mg/m² intravenously] Metabolism/Metabolites Liver Liver Half-life: 75 hours Biological Half-life 75 hours

Toxicity Summary
Mitoxantrone is a DNA reactant that intercalates into deoxyribonucleic acid (DNA) via hydrogen bonds, causing DNA cross-linking and strand breaks. Mitoxantrone also interferes with ribonucleic acid (RNA) and is a potent inhibitor of topoisomerase II, an enzyme responsible for unwinding and repairing damaged DNA. It is cytotoxic to both proliferating and non-proliferating cultured human cells, indicating a lack of cell cycle specificity. Hepatotoxicity
Mitoxantrone chemotherapy alone can cause elevated serum enzymes in up to 40% of patients, but these elevations are usually mild to moderate, transient, and without symptoms or jaundice. The incidence of elevated liver enzymes is higher in combination chemotherapy regimens containing mitoxantrone. High doses of mitoxantrone are associated with a higher incidence of jaundice, but the hyperbilirubinemia is mild, transient, and without significant elevations of serum enzymes or evidence of hepatitis. Rare cases of acute liver injury have been reported in patients taking mitoxantrone, including one case of drug eruption (DRESS) with eosinophilia and systemic symptoms. The incubation period in this case was 8 weeks, with an initial cholestatic pattern of elevated serum enzymes followed by a mixed pattern. A significant immune hypersensitivity reaction was present, and the patient appeared to respond to glucocorticoid therapy. This patient was also taking other medications, making their association with mitoxantrone impossible to determine (Case 1). Therefore, mitoxantrone may cause specific and clinically significant liver injury, but this is extremely rare. Probability Score: D (Possibly a rare cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Medication Use During Lactation Most sources consider breastfeeding contraindicated during maternal treatment with anti-tumor drugs (such as mitoxantrone). During intermittent treatment, breastfeeding may be safe if the duration of lactation is appropriate, but the specific duration of lactation is unclear. In one patient, mitoxantrone was still detectable in breast milk 28 days after receiving a dose of 6 mg/m². Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk. Women receiving chemotherapy during pregnancy are more likely to experience breastfeeding difficulties.
◉ Effects on breastfed infants
One mother received intravenous mitoxantrone (6 mg/m²) for 3 days, along with intravenous etoposide (80 mg/m²) and cytarabine (170 mg/m²) for 5 days. She resumed breastfeeding 3 weeks after the third mitoxantrone injection, at which point mitoxantrone was still detectable in the breast milk. The infant showed no obvious abnormalities at 16 months of age.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
78%

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In vitro cytotoxicity: Mitoxantrone hydrochloride (mitoxantrone) showed low cytotoxicity to Vero cells, with CC50 > 10 μM, indicating that its antiviral activity has a high therapeutic index [1]

[1]. Antiviral Res. 2011 Dec 11;93(2):305–308. Inhibition of cowpox virus and monkeypox virus infection by mitoxantrone

[2]. Mechanism of action of mitoxantrone. Neurology.2004 Dec 28;63(12 Suppl 6):S15-8.

Pharmacodynamics
In vitro studies have shown that mitoxantrone can inhibit the proliferation of B cells, T cells and macrophages, and impair antigen presentation and the secretion of interferon γ, TNFα and IL-2.

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Mitoxantrone hydrochloride (mitoxantrone) is an anthraquinone derivative with antitumor and antiviral activities[1][2]
-Mechanism of action: It exerts its biological effects through two main pathways—intercalation into DNA to block replication/transcription, and by stabilizing the enzyme-DNA cleavage complex to inhibit DNA topoisomerase II. In terms of antiviral activity, it may interfere with the viral DNA replication mechanism[1][2]
-Therapeutic potential: In vitro experiments have shown that it is effective against poxviruses (vaccinia, monkeypox) and is used clinically to treat hematologic malignancies (e.g., leukemia, lymphoma) and solid tumors[1][2]
-Antiviral specificity: The drug inhibits poxvirus replication and has no significant toxicity to host cells, suggesting its potential application value in the treatment of poxvirus infection[1]

C22H29CLN4O6.HCL

517.4

516.154

C, 51.07; H, 5.84; Cl, 13.70; N, 10.83; O, 18.55

70476-82-3

65271-80-9; 70476-82-3 (HCl salt); 70711-41-0 (diacetate)

4212

Black solid powder

805.7ºC at 760 mmHg

203-205ºC

441.1ºC

2.392

8

10

12

32

571

0

Cl[H].Cl[H].O=C1C2=C(C([H])=C([H])C(=C2C(C2=C(C([H])=C([H])C(=C21)N([H])C([H])([H])C([H])([H])N([H])C([H])([H])C([H])([H])O[H])N([H])C([H])([H])C([H])([H])N([H])C([H])([H])C([H])([H])O[H])=O)O[H])O[H]

ZAHQPTJLOCWVPG-UHFFFAOYSA-N

InChI=1S/C22H28N4O6.2ClH/c27-11-9-23-5-7-25-13-1-2-14(26-8-6-24-10-12-28)18-17(13)21(31)19-15(29)3-4-16(30)20(19)22(18)32;;/h1-4,23-30H,5-12H2;2*1H

1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino)ethylamino]anthracene-9,10-dione;dihydrochloride

NSC-301739; CL-232325; NSC301739; CL 232325; NSC 301739; DHAQ; CL232325; Mitozantrone; Mitoxantrone HCl; Mitoxantrone dihydrchloride; US brand name: Novantrone. NSC 301739; DHAQ; CL232325; Foreign brand names: Mitroxone; Neotalem; Onkotrone; Pralifan

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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 (4.83 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 25.0 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 2: ≥ 2.5 mg/mL (4.83 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 25.0 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 3: Saline: 30 mg/mL

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Solubility in Formulation 4: 2 mg/mL (3.87 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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