Topoisomerase II; PKC (IC50 = 8.5 μM );

PKC is inhibited by mitoxantrone diacetate non-competitively against phosphatidylserine and ATP, but competitively against histone H1 (Ki value: 6.3 μM) [1]. B-CLL cytopenias are induced by mitoxantrone diacetate (0.5 μg/mL, 48 hours). Mitoxantrone diacetate causes poly(ADP-ribose) polymerase (PARP) to undergo proteolytic cleavage and DNA fragmentation, which may be the cause of the drug's cytotoxic effects [2]. Human breast cancer cell lines MDA-MB-231 and MCF-7 exhibit cytotoxicity towards mitoxantrone diacetate, with IC50 values of 18 and 196 nM, respectively [3].

In L1210 leukemic mice, mitoxantrone diacetate (IP, 0-3.2 mg/kg/day) showed statistically significant 60-day survival at a dose of 1.6 mg/kg [4]. At 3.2 mg/kg, mitoxantrone diacetate (IV, 0-3.2 mg/kg/day) induced 60% ILS (life extension) in SC-implanted Lewis lung carcinoma, demonstrating strong anticancer activity[4].

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.

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.

1,4-Dihydroxy-5,8-bis(((2-[(2-hydroxyethyl) amino] ethyl)amino))-9,10-anthracenedione dihydrochloride (mitoxantrone) was tested for antitumor activity against experimental tumors in mice and the results were compared with those of seven antitumor antibiotics: adriamycin (ADM), daunomycin (DM), aclarubicin, mitomycin C (MNC), bleomycin, neocarzinostatin, and chromomycin A3. The drugs were given IP or IV, in general on days 1, 5, and 9 following tumor inoculation. Mitoxantrone given IP at the optimal dose (1.6 mg/kg/day; as a free base) produced a statistically significant number of 60-day survivors (curative effect) in mice with IP implanted L1210 leukemia. The curative effect was not observed with any of the other antibiotics. In the case of IV implanted L1210 leukemia, there was an increase in lifespan (ILS) by more than 100% in the mice following IV treatment with mitoxantrone or DM. In IP implanted P388 leukemia, the curative effect was elicited by IP treatment with mitoxantrone or MMC. In IP implanted B16 melanoma, both the curative effect and a more than 100% ILS in mice that did die were produced by IP treatment with mitoxantrone or ADM. In SC implanted Lewis lung carcinoma, mitoxantrone and ADM administered IV also showed effective antitumor activities and produced a 60% and a 45% ILS, respectively. In conclusion, mitoxantrone and ADM had a wider spectrum of antitumor activity against mouse tumors, including two leukemias and two solid tumors, than did the other drugs; however, mitoxantrone elicited higher antitumor effects than ADM on mouse leukemias, especially on L1210 leukemias. Moreover, mitoxantrone possessed much higher therapeutic indices than ADM against IP implanted P388 (optimal dose/ILS40; greater than 128 versus 15.2) and L1210 (optimal dose/ILS25; 72.7 versus 4.8) leukemias. In addition, mitoxantrone showed moderate activity against DM-resistant L1210 leukemia.[4]

Absorption, Distribution and Excretion
Oral Absorption is Poor. 1000 L/m² 21.3 L/hr/m² [Intravenous dose of 15-90 mg/m² for elderly breast cancer patients] 28.3 L/hr/m² [Intravenous dose of 15-90 mg/m² for non-elderly nasopharyngeal carcinoma patients] 16.2 L/hr/m² [Intravenous dose of 15-90 mg/m² for non-elderly malignant lymphoma patients] 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 three intravenous injections of mitoxantrone (6 mg/m²) and five intravenous injections of etoposide (80 mg/m²) and cytarabine (170 mg/m²). She resumed breastfeeding three weeks after the third mitoxantrone injection, at which time mitoxantrone was still detectable in 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 rate
78%

[1]. Inhibitory effect of mitoxantrone on activity of protein kinase C and growth of HL60 cells. J Biochem. 1992 Dec;112(6):762-7.

[2]. Mitoxantrone, a topoisomerase II inhibitor, induces apoptosis of B-chronic lymphocytic leukaemia cells. Br J Haematol. 1998 Jan;100(1):142-6.

[3]. Differential subcellular distribution of mitoxantrone in relation to chemosensitization in two human breast cancer cell lines. Drug Metab Dispos. 2007 May;35(5):822-8.

[4]. Antitumor activity of mitoxantrone against murine experimental tumors: comparative analysis against various antitumor antibiotics. Cancer Chemother Pharmacol. 1982;8(2):157-62.

[5]. Inhibition of cowpox virus and monkeypox virus infection by mitoxantrone. Antiviral Res. 2012 Feb;93(2):305-308.

Mitoxantrone is an anthraquinone antitumor drug. Mitoxantrone is a dihydroxyantrone, chemically named 1,4-dihydroxy-9,10-antrone, with 6-hydroxy-1,4-diazahexyl groups substituted at positions 5 and 8. It has antitumor and analgesic effects. Mitoxantrone is a topoisomerase inhibitor. Its mechanism of action is as a topoisomerase inhibitor. Mitoxantrone is an antitumor antibiotic used to treat acute leukemia, lymphoma, prostate cancer, and breast cancer, and can also be used to treat advanced severe multiple sclerosis. Mitoxantrone treatment is usually accompanied by a mild to moderate increase in serum transaminase levels, but rarely causes clinically significant acute liver injury at typical doses. Mitoxantrone is an anthraquinone antibiotic with antitumor activity. Mitoxantrone can intercalate into DNA and cross-link it, thereby disrupting DNA and RNA replication. The drug can also bind to topoisomerase II, leading to DNA strand breaks and inhibiting DNA repair. Mitoxantrone has lower cardiotoxicity compared to doxorubicin. Mitoxantrone is only found in individuals who have used or taken the drug. It is an anthraquinone antitumor drug. Mitoxantrone is a DNA-reactive drug 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 in its action. An anthraquinone antitumor drug. See also: Mitoxantrone hydrochloride (salt form). Drug Indications For the treatment of secondary (chronic) progressive, progressive-relapsing, or relapsing-remitting multiple sclerosis with disease exacerbation.
FDA Label

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Mechanism of Action
Mitoxantrone is a DNA-reactive drug that intercalates into deoxyribonucleic acid (DNA) via hydrogen bonds, leading to 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 exhibits cytotoxic effects on both proliferating and non-proliferating cultured human cells, indicating a lack of cell cycle specificity in its action.

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Mitoxantrone is a novel anthraquinone compound that inhibits protein kinase C (PKC) activity. Its IC50 value is 4.4 μg/mL (8.5 μmol), significantly lower than that of known anthracycline antibiotics daunorubicin and doxorubicin, both of which have IC50 values exceeding 100 μg/mL (>170 μmol). Kinetic studies showed that mitoxantrone competitively inhibited protein kinase C (PKC) on histone H1 with a Ki value of 6.3 μM (compared to 0.89 mM and 0.15 mM for daunorubicin and doxorubicin, respectively), while non-competitively inhibiting phosphatidylserine and ATP. Mitoxantrone inhibited phosphorylation of a variety of substrates, including S6 peptide, myelin basic protein, and its N-terminal derivatives. Their IC50 values were 0.49 μg/ml (0.95 μM), 1.8 μg/ml (3.5 μM), and 0.82 μg/ml (1.6 μM), respectively. At concentrations below 10 μg/ml, mitoxantrone did not significantly inhibit the activity of cyclic adenosine monophosphate-dependent protein kinases, casein kinase I, or casein kinase II. On the other hand, HL60 cells were inhibited after brief exposure to mitoxantrone (5 min), with an IC50 value of 52 ng/ml (0.1 μM). In HL60 cells, most of the PKC activity (about 90%) was found in the cytoplasmic component. HL60 cells exposed to 10 μg/ml mitoxantrone for 5 min were observed by fluorescence microscopy and the fluorescence produced by mitoxantrone was found to be located in the extranuclear region. These results indicate that mitoxantrone is a potent inhibitor of protein kinase C (PKC), and this inhibition may be one of the mechanisms of mitoxantrone’s antitumor activity. [1]

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B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5+ B lymphocytes. This study investigated the effect of the topoisomerase II inhibitor mitoxantrone on B-CLL cells. After B-CLL cells were treated with mitoxantrone (0.5 μg/ml) for 48 hours, the MTT assay showed a decrease in cell viability. The IC50 values calculated for cells from three patients were 0.7 μg/ml for two patients and 1.4 μg/ml for the other patient. Maximum efficacy was observed at a concentration of 2 μg/ml in all three patients. An additive effect of cytotoxicity was observed when mitoxantrone (0.5 μg/ml) was used in combination with fludarabine (5 μg/ml). In all patients studied, mitoxantrone induced DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), a marker of caspase activation, suggesting that the cytotoxicity of mitoxantrone was due to apoptosis. These results suggest that mitoxantrone and other topoisomerase II inhibitors may be used for chemotherapy in B-cell chronic lymphocytic leukemia (B-CLL), and that mitoxantrone in combination with fludarabine or other drugs may improve therapeutic efficacy. [2]

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This study aimed to investigate the relationship between cancer cell chemosensitivity and the subcellular distribution, molecular interactions, and metabolism of anticancer drugs. To gain a deeper understanding of this relationship, we utilized the differences in sensitivity to anthracyclines between two breast cancer cell lines, MDA-MB-231 and MCF-7, and the property that docosahexaenoic acid (DHA, 22:6n-3) can differentially enhance their cytotoxic activity. We used the fluorescent drug mitoxantrone (MTX) because its subcellular accumulation could be studied using confocal spectral imaging (CSI). CSI allowed us to obtain semi-quantitative profiles of four intracellular MTX species: DNA-bound nuclear MTX, MTX oxidative metabolites in the endoplasmic reticulum, cytoplasmic MTX, and MTX in the membrane-specific low-polarity environment. The results showed that MDA-MB-231 cells were more sensitive to MTX (IC50 = 18 nM) than MCF-7 cells (IC50 = 196 nM). Based on fluorescence intensity, both nuclear and cytoplasmic MTX levels were higher in MCF-7 cells than in MDA-MB-231 cells, indicating that other mechanisms besides nuclear MTX accumulation contribute to chemosensitivity. In the cytoplasm, the relative proportion of oxidized MTX in MDA-MB-231 cells (60%) was higher than that in MCF-7 cells (7%). DHA increased the sensitivity of MDA-MB-231 cells to MTX by about 4-fold, but had no such effect on MCF-7 cells, and only increased MTX accumulation by 1.5-fold in MDA-MB-231 cells. DHA-stimulated MTX accumulation was mainly attributed to its oxidative metabolites. The antioxidant N-acetyl-L-cysteine inhibited the effects of DHA on metabolite accumulation and cellular sensitivity to MTX. We conclude that drug metabolism and compartmentalization are related to cellular chemosensitivity, and the associated cytotoxic mechanisms may involve oxidative stress. [3]

C22H30N4O6+2.2[C2H3O2-]

564.58484

504.222

C, 55.31; H, 6.43; N, 9.92; O, 28.34

70711-41-0

Mitoxantrone;65271-80-9;Mitoxantrone dihydrochloride;70476-82-3

51151

Typically exists as solid at room temperature

1.45g/cm3

805.7ºC at 760 mmHg

203-5ºC

441.1ºC

0.879

8

12

12

40

597

0

C1=CC(=C2C(=C1NCCNCCO)C(=O)C3=C(C=CC(=C3C2=O)O)O)NCCNCCO.CC(=O)O.CC(=O)O

ZWCKUVMZBKQQRG-UHFFFAOYSA-N

InChI=1S/C22H28N4O6.2C2H4O2/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;2*1-2(3)4/h1-4,23-30H,5-12H2;2*1H3,(H,3,4)

2,2'-((5,8-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)bis(azanediyl))bis(N-(2-hydroxyethyl)ethan-1-aminium) acetate

Dhaq diacetate; 70711-41-0; 1,4-Dihydroxy-5,8-bis(2-((2-hydroxyethyl)amino)ethylamino)-9,10-anthracenedione diacetate; Mitoxantrone diacetate; 2-[[5,8-dihydroxy-4-[2-(2-hydroxyethylazaniumyl)ethylamino]-9,10-dioxoanthracen-1-yl]amino]ethyl-(2-hydroxyethyl)azanium;diacetate; NSC 299195; 5,8-Bis((2-((2-hydroxyethyl)amino)ethyl)amino)-1,4-dihydroxy-9,10-anthracenedione diacetate; 5,8-Bis((2-((2-hydroxyethyl)amino)ethyl)amino)-1,4-dihydroxyanthraquinone 1,4-diaceate;

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.)