PKC ( IC50 = 8.5 μM ); Topoisomerase II

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.[8]

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Mitoxantrone temporarily reduces the growth rate of HID xenografts in mice, but PAC120 xenografts are unaffected.[4] In rats that develop spontaneous hypertension, mitoxantrone increases the severity of cardiac lesions, nephropathy, and intestinal toxicity. Mitoxantrone and iron(III) combine to form a potent 2:1 complex, wherein mitoxantrone might be functioning as a tridentate ligand.[5]

Mitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50 value was 4.4 micrograms/ml (8.5 microM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100 micrograms/ml (> 170 microM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its Ki value was 6.3 microM (Ki values of daunorubicin and doxorubicin were 0.89 and 0.15 mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50 values were 0.49 microgram/ml (0.95 microM), 1.8 micrograms/ml (3.5 microM), and 0.82 microgram/ml (1.6 microM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10 micrograms/ml. On the other hand, brief exposure (5 min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50 value of 52 ng/ml (0.1 microM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10 micrograms/ml mitoxantrone for 5 min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.[7]

Cell preparation and culture.[5]
PBL were collected from healthy donors in the presence of sodium citrate. Blood was defibrinated, and then mononuclear cells were isolated by centrifugation on a layer of Histopaque®. Those cell suspensions, referred to as PBL, contained 1.860.4% monocytes, as defined by CD14 expression. PBL were resuspended in Rosewell Park Memorial Institute culture medium, supplemented with 10% FCS or TCH medium, 2 mM L-glutamine, and antibiotics (penicillin 100 U/ml, streptomycin 100 mg/ml). Cultures were maintained at 378C in a humid atmosphere containing 5% CO2. During the last 8 h of incubation they were pulsed with (methyl-3 H)thymidine at 0.5 mCi/well. 3 H-TdR uptake was measured using a Packard direct beta counte after harvesting. For mixed lymphocyte reactions (MLR), the human B lymphoma cell lines RAJI and DAKIKI were used as stimulators. Stimulator cells were treated for 1 h at 378C with 25 mg/ml of mitomycin C, extensively washed, and then mixed with PBL at a ratio of 1 B cell for 10 PBL.

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Measurement of nuclear apoptosis.[5]
After 3 d of culture, PHAactivated PBL were harvested. Dead cells were removed by centrifugation on a layer of Histopaque®. Viable cells (106 /ml) were washed in HBSS, and then incubated in 96-well microplates with MTX. In other experiments, PBL were either incubated for 1–24 h in the presence of MTX, and then activated with PHA for 24 to 72 h, or MTX and PHA were added together at the onset of the culture. Cell death was evaluated by fluorescence microscopy after staining with Hoechst 33342 at 10 mg/ml after previously described methods. Apoptosis was also measured by flow cytometry after addition of biotinylated annexin V and by TdT-mediated dUTP–FITC nick end labeling (TUNEL), as previously described, using reagents from Boehringer Mannheim. Samples were analyzed by flow cytrometry on a FACScan®. Nuclear fragmentation and/or marked condensation of the chromatin with reduction of nuclear size were considered as typical features of apoptotic cells. Based on these measurements, results were expressed as percentage of apoptotic cells or percentage of specific apoptosis according to the following formula: specific apoptosis 5 (T 2 C)/(100 2 C), where T stands for % of apoptotic-treated cells and C for % of apoptotic control cells. The morphological features of the cells after MTX treatment were also observed by transmission electronic microscopy, as previously described. For DNA fragmentation assay, cells were incubated in RPMI medium for 12 h with MTX, and DNA preparations were obtained and processed for electrophoresis in 2% agarose gel after previously described methods.

Mice: Mitoxantrone is tested for antitumor activity against experimental tumors in mice and the results are compared with those of seven antitumor antibiotics. The drugs are given IP or IV, in general on days 1, 5, and 9 following tumor inoculation. Mitoxantrone is given IP at the optimal dose (1.6 mg/kg/day; as a free base)[8].

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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.[1]

Absorption, Distribution and Excretion
Poorly absorbed following oral administration
1000 L/m2
21.3 L/hr/m2 [Elderly patients with breast cancer receiving IV administration of 15-90 mg/m2]
28.3 L/hr/m2 [Non-elderly patients with nasopharyngeal carcinoma receiving IV administration of 15-90 mg/m2]
16.2 L/hr/m2 [Non-elderly patients with malignant lymphoma receiving IV administration of 15-90 mg/m2]

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Metabolism / Metabolites
Hepatic
Hepatic
Half Life: 75 hours

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Biological Half-Life
75 hours

Toxicity Summary
Mitoxantrone, a DNA-reactive agent that intercalates into deoxyribonucleic acid (DNA) through hydrogen bonding, causes crosslinks and strand breaks. Mitoxantrone also interferes with ribonucleic acid (RNA) and is a potent inhibitor of topoisomerase II, an enzyme responsible for uncoiling and repairing damaged DNA. It has a cytocidal effect on both proliferating and nonproliferating cultured human cells, suggesting lack of cell cycle phase specificity.

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Toxicity Summary
Mitoxantrone, a DNA-reactive agent that intercalates into deoxyribonucleic acid (DNA) through hydrogen bonding, causes crosslinks and strand breaks. Mitoxantrone also interferes with ribonucleic acid (RNA) and is a potent inhibitor of topoisomerase II, an enzyme responsible for uncoiling and repairing damaged DNA. It has a cytocidal effect on both proliferating and nonproliferating cultured human cells, suggesting lack of cell cycle phase specificity.

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Hepatotoxicity
Chemotherapy with mitoxantrone alone is associated with serum enzyme elevations in up to 40% of patients, but these elevations are generally mild-to-moderate in severity, transient and not accompanied by symptoms or jaundice. Higher rates of liver enzyme elevations have been reported with combination chemotherapeutic regimens that include mitoxantrone. In high doses, mitoxantrone has been associated with a high rate of jaundice, but the degree of hyperbilirubinemia has been mild, transient and not associated with significant serum enzyme elevations or evidence of hepatitis. Rare instances of acute liver injury have been reported in patients taking mitoxantrone, including a single case of drug-rash with eosinophilia and systemic symptoms (DRESS). The latency to onset was 8 weeks and the pattern of serum enzyme elevations was cholestatic and later mixed. Immunoallergic features were prominent and appeared to respond to corticosteroid therapy. Other drugs were being taken and the association with mitoxantrone was not definite (Case 1). Thus, idiosyncratic and clinically apparent liver injury from mitoxantrone may occur but is quite rare.
Likelihood score: D (possible rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Most sources consider breastfeeding to be contraindicated during maternal antineoplastic drug therapy, such as mitoxantrone. It might be possible to breastfeed safely during intermittent therapy with an appropriate period of breastfeeding abstinence, but the duration of abstinence is not clear. In one patient, mitoxantrone was still detectable in milk 28 days after a dose of 6 mg per square meter. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.
◉ Effects in Breastfed Infants
One mother received 3 daily doses of 6 mg/sq. m. of mitoxantrone intravenously along with 5 daily doses of etoposide 80 mg/sq. m. and cytarabine 170 mg/sq. m. intravenously. She resumed breastfeeding her infant 3 weeks after the third dose of mitoxantrone at a time when mitoxantrone was still detectable in milk. The infant had no apparent abnormalities at 16 months of age.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
78%

[1]. Cancer Chemother Pharmacol. 1982;8(2):157-62.

[2]. Arthritis Rheum. 1989 Sep;32(9):1065-73.

[3]. Semin Arthritis Rheum. 1990 Dec;20(3):190-200.

[4]. Arthritis Rheum. 1989 Sep;32(9):1065-73.

[5]. J Clin Invest. 1998 Jul 15;102(2):322-8.

[6]. J Clin Invest. 1993 Dec;92(6):2675-82.

[7]. J Biochem. 1992 Dec;112(6):762-7.

[8]. Cancer Chemother Pharmacol. 1982;8(2):157-62.

Pharmacodynamics
Mitoxantrone has been shown in vitro to inhibit B cell, T cell, and macrophage proliferation and impair antigen presentation, as well as the secretion of interferon gamma, TNFa, and IL-2.

C22H28N4O6

444.48

444.2

C, 59.45; H, 6.35; N, 12.61; O, 21.60

65271-80-9

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

4212

Brown to black solid powder

1.5±0.1 g/cm3

805.7±65.0 °C at 760 mmHg

170-174ºC

441.1±34.3 °C

0.0±3.0 mmHg at 25°C

1.709

0.45

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]

KKZJGLLVHKMTCM-UHFFFAOYSA-N

InChI=1S/C22H28N4O6/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

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

NSC-301739; DHAQ; CL-232325; NSC301739; 65271-80-9; Mitoxanthrone; Mitoxantron; DHAQ; Mitoxantrona; Mitoxantronum; CL 232325; NSC 301739; CL232325; Mitozantrone; Mitoxantrone HCl; Mitoxantrone dihydrchloride; US brand name: Novantrone. 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: This product requires protection from light (avoid light exposure) during transportation and storage.

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: ≥ 2.08 mg/mL (4.68 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 20.8 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: ≥ 2.08 mg/mL (4.68 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)