Topoisomerase II

With IC50 values of 209.4 nM and 2.0 μM, respectively, amsacrine (mAMSA) supports HERG currents in HEK 293 cells and Xenopus oocytes in a concentrated form. The voltage connection between activation and inactivation is shifted negatively (-7.6 mV) by amsacrine hydrochloride (mAMSA). Amsacrine hydrochloride (mAMSA)-induced HERG current bursts and associated frequencies [1]. Ground-current studies in healthy humans using varying concentrations of mAMSA revealed elevated SCEs at the lowest concentration examined (0.005 μg; 1.5 times the normal value/mL) to 12 times the normal value (0.25 μg/mL) as well as increased levels of chromosomal aberrations, ranging from 8% to 100% [3]. The inactivation of U937 cells induced by amsacridine hydrochloride (mAMSA) is typified by the activation of caspase-9 and caspase-3, elevated intracellular Ca2+ concentration, mitochondrial demajority, and MCL1. Amsacridine lowers the stability of MCL1 to induce it. Moreover, U937 cells treated with amsacridine hydrochloride demonstrated Ca2+-mediated ERK deactivation and AKT degradation [4].

At 9 and 12 mg/kg treatment doses, the frequency of micronucleated polychromatic erythrocytes rose significantly in animals treated with varying doses of amsacrine hydrochloride (0.5–12 mg/kg). Furthermore, this work shows for the first time that nocodazole has a low clastogenicity rate and a high clastogenicity rate during internal mitosis, whereas mAMSA) has both low and high clastogenicity rates during this process. Chromosome breakage frequency [2].

1 The topoisomerase II inhibitor amsacrine is used in the treatment of acute myelogenous leukemia. Although most anticancer drugs are believed not to cause acquired long QT syndrome (LQTS), concerns have been raised by reports of QT interval prolongation, ventricular fibrillation and death associated with amsacrine treatment. Since blockade of cardiac human ether-a-go-go-related gene (HERG) potassium currents is an important cause of acquired LQTS, we investigated the acute effects of amsacrine on cloned HERG channels to determine the electrophysiological basis for its proarrhythmic potential. 2 HERG channels were heterologously expressed in human HEK 293 cells and Xenopus laevis oocytes, and the respective potassium currents were recorded using patch-clamp and two-microelectrode voltage-clamp electrophysiology. 3 Amsacrine blocked HERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner, with IC50 values of 209.4 nm and 2.0 microm, respectively. 4 HERG channels were primarily blocked in the open and inactivated states, and no additional voltage dependence was observed. Amsacrine caused a negative shift in the voltage dependence of both activation (-7.6 mV) and inactivation (-7.6 mV). HERG current block by amsacrine was not frequency dependent. 5 The S6 domain mutations Y652A and F656A attenuated (Y652A) or abolished (F656A, Y652A/F656A) HERG current blockade, indicating that amsacrine binding requires a common drug receptor within the pore-S6 region. 6 In conclusion, these data demonstrate that the anticancer drug amsacrine is an antagonist of cloned HERG potassium channels, providing a molecular mechanism for the previously reported QTc interval prolongation during clinical administration of amsacrine.[1]

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Previous studies have attributed the anticancer activity of amsacrine to its inhibitory effect on topoisomerase II. However, 9-aminoacridine derivatives, which have the same structural scaffold as amsacrine, induce cancer cell apoptosis by altering the expression of BCL2 family proteins. Therefore, in the present study, we assessed whether BCL2 family proteins mediated the cytotoxic effects of amsacrine on human leukemia U937 cells. Amsacrine-induced apoptosis of U937 cells was characterized by caspase-9 and caspase-3 activation, increased intracellular Ca2+ concentration, mitochondrial depolarization, and MCL1 down-regulation. Amsacrine induced MCL1 down-regulation by decreasing its stability. Further, amsacrine-treated U937 cells showed AKT degradation and Ca2+-mediated ERK inactivation. Blockade of ERK-mediated phosphorylation of MCL1 inhibited the effect of Pin1 on the stabilization of MCL1, and AKT degradation promoted GSK3β-mediated degradation of MCL1. Restoration of ERK phosphorylation and AKT expression abrogated amsacrine-induced MCL1 down-regulation. Moreover, MCL1 over-expression inhibited amsacrine-induced depolarization of mitochondria membrane and increased the viability of amsacrine-treated cells. Taken together, our data indicate that amsacrine abolishes ERK- and Pin1-mediated stabilization of MCL1 and promotes GSK3β-mediated degradation of MCL1, leading to activate mitochondria-mediated apoptosis pathway in U937 cells[4].

The mechanism of genotoxic potential of the cancer chemotherapeutic drugs amsacrine and nocodazole in mouse bone marrow was investigated using a micronucleus test complemented by fluorescence in situ hybridization assay with mouse centromeric and telomeric DNA probes. In animals treated with different doses of amsacrine (0.5-12 mg kg(-1) ), the frequencies of micronucleated polychromatic erythrocytes increased significantly after treatment with 9 and 12 mg kg(-1) . A statistically significant increase in micronuclei frequency was also detected for 75 mg kg(-1) nocodazole (two exposures, spaced 24 h apart). Both compounds caused significant suppressions of erythroblast proliferation at higher doses. Furthermore, the present study demonstrated for the first time that amsacrine has high incidences of clastogenicity and low incidences of aneugenicity whereas nocodazole has high incidences of aneugenicity and low incidences of clastogenicity during mitotic phases in vivo. The assay also showed that chromosomes can be enclosed in the micronuclei before and after centromere separation. Therefore, the clinical use of these genotoxic drugs must be weighed against the risks of the development of chromosomal aberrations in cancer patients and medical personnel exposed to drug regimens that include these chemicals.[2]

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Amsacrine (m-AMSA) is presently being utilized in phase I-II studies at the Medicine Branch, National Cancer Institute, National Institutes of Health (Bethesda, MD), and is being administered as a continuous infusion to patients with progressive malignancy after conventional therapy. In the present study, we examined the effects of this drug, in vivo and in vitro, on chromosomal morphology and the frequency of sister chromatid exchange (SCE) induction in human peripheral blood lymphocytes. In the in vivo studies, eight patients receiving 30 mg/m2/day of m-AMSA by continuous infusion showed increased levels of chromosomal aberrations, up to a maximum of 14% (median; range, 10%-24%) at 96 hours compared to 1% (median; range, 0%-4%) in the control group; no increase was noted in SCE frequencies.[3]

Absorption, Distribution and Excretion
Poorly absorbed
Volume of distribution (VolD) -- 1.67 L/kg. Amsacrine does not significantly penetrate into the CNS
Elimination: Renal: 35% of the dose is excreted by the kidneys within 72 hours after administration (20% as intact drug). Biliary: Amsacrine is also eliminated by biliary excretion.
In cancer patients, amsacrine undergoes biphasic elimination, with a distribution half-life of 0.25-1.6 hours and an elimination half-time of 4.7-9 hours. The total plasma clearance rate is 200-300 ml/min per sq m, and the apparent distribution volume is 70-110 l/sq m, suggesting concentration in tissues. During a 1 hour injusion of amasacrine at 90-200 mg/sq m, the peak plasma concentration was 10-15 umol/l.
Although not fully reported, early trials in which amsacrine was given orally failed to reach the maximum tolerated dose, as shown by lack of toxicity even at doses as high as 500 mg/sq m per day, suggesting incomplete or erratic absorption. In subsequent studies, the intravenous route was used, with which the maximum tolerated dose in patients with solid tumors is 100-150 mg/sq m when administered over 1-3 hours.
After intravenous administration of (14)C amsacrine to mice and rats, > 50% of the radiolabel was excreted in bile within the first 2 hours, and the bile:plasma ratio was > 400:1; 74% of an intravenous dose was excreted in the feces of mice with 72 hours. These studies demonstrate the importance of the liver in clearance of amsacrine.

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Metabolism / Metabolites
Extensive, primarily hepatic, converted to glutathione conjugate.
Oxidative metabolism of the anti-cancer drug amsacrine 4'-(9-acridinylamino) methane-sulphan-m-anisidide has been suggested to account for its cytotoxicity. However, enzymes capable of oxidizing it in non-hepatic tissue have yet to be identified. A potential candidate, that may be relevant to the metabolism of amsacrine in blood and its action in myeloid leukaemias and myelosuppression, is the haem enzyme myeloperoxidase. We have found that the purified human enzyme oxidizes amsacrine to its quinone diimine, either directly or through the production of hypochlorous acid. In comparison, the 4-methyl-5-methylcarboxamide derivative of amsacrine, CI-921 9-[[2-methoxy-4[(methylsulphonyl)-amino]phenyl]amino)-N, 5-dimethyl-4-acridine carboxamide, reacted poorly with myeloperoxidase, although it was oxidized by hypochlorous acid. Detailed studies of the mechanism by which myeloperoxidase oxidizes amsacrine revealed that the semiquinone imine free radical is a likely intermediate in this reaction. Oxidation of amsacrine analogues indicated that factors other than their reduction potential determine how readily they are metabolized by myeloperoxidase. Both amsacrine and CI-921 inhibited production of hypochlorous acid by myeloperoxidase. CI-921 acted by trapping the enzyme as the inactive redox intermediate compound II. Amsacrine inhibited by a different mechanism that may involve conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl-. The susceptibility of amsacrine to oxidation by myeloperoxidase indicates that this reaction may contribute to the cytotoxicity of amsacrine toward neutrophils, monocytes and their precursors.
In mouse bile, 5'- and 6'-glutathione conjugates were present in roughly equal amounts and accounted for 70% of the excreted biliary radiolabel after administration of radiolabelled amsacrine. In rats, the principal biliary metabolite was the 5'-gutathione conjugate, which accounted for 80% of the excreted radiolabel within the first 90 minutes and > 50% of the administered dose over 3 hours. The 6'-conjugate was also subsequently identified in rat bile. In rat liver microsomes and human neutrophils, intermediate oxidation products have been identified as N1'-methanesulfonyl-N4'-(9-acridinyl)-3'-methoxy-2',5'-cyclohexadience-1',4'-dii mine and 3'-methoxy-4'-(9-acridinylamino-2'5'-cyclohexadien-1'-one.
Extensive, primarily hepatic, converted to glutathione conjugate.
Half Life: 8-9 hours

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Biological Half-Life
8-9 hours

Toxicity Summary
Amsacrine binds to DNA through intercalation and external binding. It has a base specificity for A-T pairs. Rapidly dividing cells are two to four times more sensitive to amsacrine than are resting cells. Amsacrine appears to cleave DNA by inducing double stranded breaks. Amsacrine also targets and inhibits topoisomerase II. Cytotoxicity is greatest during the S phase of the cell cycle when topoisomerase levels are at a maximum.

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Evidence for Carcinogenicity
Evaluation: There is inadequate evidence in humans for the carcinogenicity of amsacrine. There is sufficient evidence in experimental animals for the carcinogenicity of amsacrine. Overall evaluation: Amsacrine is possibly carcinogenic to humans (Group 2B).

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mouse LD50 oral 181 mg/kg National Cancer Institute Screening Program Data Summary, Developmental Therapeutics Program., JAN1986
mouse LD50 intraperitoneal 20560 ug/kg National Cancer Institute Screening Program Data Summary, Developmental Therapeutics Program., JAN1986
mouse LD50 subcutaneous 110 mg/kg National Cancer Institute Screening Program Data Summary, Developmental Therapeutics Program., JAN1986

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Protein Binding
96-98%

[1]. Inhibition of cardiac HERG currents by the DNA topoisomerase II inhibitor amsacrine: mode of action. Br J Pharmacol. 2004 Jun;142(3):485-94.

[2]. Attia SM. Molecular cytogenetic evaluation of the mechanism of genotoxic potential of amsacrine and nocodazole in mouse bone marrow cells. J Appl Toxicol. 2013 Jun;33(6):426-33.

[3]. Cytogenetic effects of amsacrine on human lymphocytes in vivo and in vitro. Cancer Treat Rep. 1984 Jul-Aug;68(7-8):989-97.

[4]. Amsacrine-induced apoptosis of human leukemia U937 cells is mediated by the inhibition of AKT- and ERK-induced stabilization of MCL1. Apoptosis. 2017 Mar;22(3):406-420.

An aminoacridine derivative that intercalates into DNA and is used as an antineoplastic agent.
Amsacrine can cause cancer according to California Labor Code.
Amsacrine is a sulfonamide that is N-phenylmethanesulfonamide substituted by a methoxy group at position 3 and an acridin-9-ylamino group at position 4. It exhibits antineoplastic activity. It has a role as an antineoplastic agent and an EC 5.99.1.3 [DNA topoisomerase (ATP-hydrolysing)] inhibitor. It is a sulfonamide, a member of acridines and an aromatic ether.
Aminoacridine derivative that is a potent intercalating antineoplastic agent. It is effective in the treatment of acute leukemias and malignant lymphomas, but has poor activity in the treatment of solid tumors. It is frequently used in combination with other antineoplastic agents in chemotherapy protocols. It produces consistent but acceptable myelosuppression and cardiotoxic effects.
Amsacrine has been reported in Cystodytes with data available.
Amsacrine is an aminoacridine derivative with potential antineoplastic activity. Although its mechanism of action is incompletely defined, amsacrine may intercalate into DNA and inhibit topoisomerase II, resulting in DNA double-strand breaks, arrest of the S/G2 phase of the cell cycle, and cell death. This agent's cytotoxicity is maximal during the S phase of the cell cycle when topoisomerase levels are greatest. In addition, amsacrine may induce transcription of tumor promoter p53 protein and block p53 ubiquitination and proteasomal degradation, resulting in p53-dependent tumor cell apoptosis.
Aminoacridine derivative that is a potent intercalating antineoplastic agent. It is effective in the treatment of acute leukemias and malignant lymphomas, but has poor activity in the treatment of solid tumors. It is frequently used in combination with other antineoplastic agents in chemotherapy protocols. It produces consistent but acceptable myelosuppression and cardiotoxic effects.
An aminoacridine derivative that intercalates into DNA and is used as an antineoplastic agent.

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Drug Indication
For treatment of acute myeloid leukaemia.

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Mechanism of Action
Amsacrine binds to DNA through intercalation and external binding. It has a base specificity for A-T pairs. Rapidly dividing cells are two to four times more sensitive to amsacrine than are resting cells. Amsacrine appears to cleave DNA by inducing double stranded breaks. Amsacrine also targets and inhibits topoisomerase II. Cytotoxicity is greatest during the S phase of the cell cycle when topoisomerase levels are at a maximum.
Amsacrine binds to DNA through intercalation and external binding and has base specificity for A-T pairs. Cycling cells are two to four times more sensitive to amsacrine than are resting cells. Cells initially in S and G2 phases are grossly delayed in their capacity for normal progression, leading to an accumulation of cells in the S phase, followed at later times by arrest in the G2 phase.
Cytotoxicity of several classes of antitumor DNA intercalators is thought to result from disturbance of DNA metabolism following trapping of the nuclear enzyme DNA topoisomerase II as a covalent complex on DNA. Here, molecular interactions of the potent antitumor drug amsacrine (m-AMSA), an inhibitor of topoisomerase II, within living K562 cancer cells have been studied using surface-enhanced Raman (SER) spectroscopy. The work is based on data of the previously performed model SER experiments dealing with amsacrine/DNA, drug/topoisomerase II and drug/DNA/topoisomerase II complexes in aqueous buffer solutions. The SER data indicated two kinds of amsacrine interactions in the model complexes with topoisomerase II alone or within ternary complex: non-specific (via the acridine moiety) and specific to the enzyme conformation (via the side chain of the drug). These two types of interactions have been both revealed by the micro-SER spectra of amsacrine within living K562 cancer cells. Our data suppose the specific interactions of amsacrine with topoisomerase II via the side chain of the drug (particular feature of the drug/topoisomerase II and ternary complexes) to be crucial for its inhibitory activity.

C21H20CLN3O3S

429.9198

429.091

C, 58.67; H, 4.69; Cl, 8.25; N, 9.77; O, 11.16; S, 7.46

54301-15-4

Amsacrine;51264-14-3

148673

Orange to red solid powder

563ºC at 760 mmHg

197-199ºC

294.3ºC

6.54

3

6

5

29

601

0

Cl[H].S(C([H])([H])[H])(N([H])C1C([H])=C([H])C(=C(C=1[H])OC([H])([H])[H])N([H])C1C2=C([H])C([H])=C([H])C([H])=C2N=C2C([H])=C([H])C([H])=C([H])C=12)(=O)=O

WDISRLXRMMTXEV-UHFFFAOYSA-N

InChI=1S/C21H19N3O3S.ClH/c1-27-20-13-14(24-28(2,25)26)11-12-19(20)23-21-15-7-3-5-9-17(15)22-18-10-6-4-8-16(18)21;/h3-13,24H,1-2H3,(H,22,23);1H

N-[4-(acridin-9-ylamino)-3-methoxyphenyl]methanesulfonamide;hydrochloride

Amsacrine hydrochloride; 54301-15-4; m-Amsa hydrochloride; M-Amsacrine; Amsacrine HCl; Amsacrine (hydrochloride); NCI-C03190; UNII-U66HX4K4CO;

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)

DMSO : ~62.5 mg/mL (~145.38 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)