PKC ( IC50 = 8.5 μM ); Topoisomerase II

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].

[1]. Curr Opin Rheumatol . 1999 May;11(3):226-32.

[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.

The folate antagonist methotrexate (MTX) is extensively used in graft-versus-host disease, rheumatoid arthritis, and other chronic inflammatory disorders. In addition to its antiinflammatory activity associated with increased release of adenosine, MTX exerts antiproliferative properties by inhibition of dihydrofolate reductase and other folate-dependent enzymes. However, the mechanisms of immunosuppressive properties associated with low-dose MTX treatments are still elusive. We report here that MTX (0.1-10 microM) induces apoptosis of in vitro activated T cells from human peripheral blood. PBL exposed to MTX for 8 h, then activated in drug-free medium, underwent apoptosis, which was completely abrogated by addition of folinic acid or thymidine. Apoptosis of activated T cells did not require interaction between CD95 (Fas, APO-1) and its ligand, and adenosine release accounted for only a small part of this MTX activity. Apoptosis required progression of activated T cells to the S phase of the cell cycle, as it was prevented by drugs or antibodies that interfere with IL-2 synthesis or signaling pathways. MTX achieved clonal deletion of activated T cells in mixed lymphocyte reactions. Finally, in vitro activation of PBL taken from rheumatoid arthritis patients after MTX injection resulted in apoptosis. Altogether, the data demonstrate that MTX can selectively delete activated peripheral blood T cells by a CD95-independent pathway. This property could be used as a new pharmacological end point to optimize dosage and timing of MTX administration. It may account for the immunosuppressive effects of low-dose MTX treatments.[5]

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

163.18

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

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; 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.)