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
Malabsorption
Volume of Distribution (VolD) -- 1.67 L/kg. Amaracidine does not readily cross the blood-brain barrier to enter the central nervous system.
Elimination: Renal: Within 72 hours after administration, 35% of the dose is excreted via the kidneys (20% of which is intact). Bile: Amaracidine is also excreted via bile.
In cancer patients, the elimination of amaracidine is biphasic, with a distribution half-life of 0.25–1.6 hours and an elimination half-life of 4.7–9 hours. Total plasma clearance is 200–300 ml/min/m², and the apparent volume of distribution is 70–110 L/m², indicating high tissue concentrations. Peak plasma concentrations are 10–15 μmol/L following a 1-hour injection of amaracidine at a dose of 90–200 mg/m².
Although not fully reported, early oral trials of amaracidine failed to reach the maximum tolerated dose, with no toxicity observed even at doses as high as 500 mg/m²/day, suggesting incomplete or unstable absorption. Subsequent studies used intravenous administration, with the maximum tolerated dose for patients with solid tumors being 100-150 mg/m² 1-3 hours after intravenous administration.
In mice and rats, over 50% of the radiolabeled amaracidine was excreted in bile within the first 2 hours, with a bile-to-plasma ratio > 400:1; 74% of the intravenously administered dose was excreted in mouse feces within 72 hours. These studies demonstrate the importance of the liver in amaracidine clearance.
Metabolism/Metabolites

Extensively metabolized, primarily in the liver, it is converted to glutathione conjugates. The oxidative metabolism of the anticancer drug acridine (4'-(9-acridylamino)methane-thio-m-anisidine) is considered to be the cause of its cytotoxicity. However, no enzyme capable of oxidizing acridine in non-hepatic tissues has been identified. Heme enzyme myeloperoxidase, potentially related to the metabolism of acridine in the blood and its role in myeloid leukemia and bone marrow suppression, is a potential candidate enzyme. We found that purified human myeloperoxidase can oxidize acridine to its quinone diimine directly or by producing hypochlorous acid. In contrast, the 4-methyl-5-methylformamide derivative of acridine, CI-921 (9-[[2-methoxy-4-[(methanesulfonyl)-amino]phenyl]amino)-N,5-dimethyl-4-acridylformamide, reacts weakly with myeloperoxidase, although it can be oxidized by hypochlorous acid. Detailed studies on the mechanism of myeloperoxidase oxidation of acridine suggest that semiquinone imine radicals may be an intermediate in this reaction. The oxidation of acridine analogs indicates that factors other than reduction potential determine the ease with which they are metabolized by myeloperoxidase. Both acridine and CI-921 inhibit myeloperoxidase production of hypochlorous acid. The mechanism of action of CI-921 involves capturing the enzyme as an inactive redox intermediate, compound II. The inhibitory mechanism of acridine differs from that of myeloperoxidase and may involve the conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl⁻. The susceptibility of acridine to myeloperoxidase oxidation suggests that this reaction may contribute to the cytotoxicity of acridine to neutrophils, monocytes, and their precursor cells. In mouse bile, the concentrations of 5'- and 6'-glutathione conjugates are approximately equal, accounting for 70% of the radiolabeled acridine excreted in bile after administration. In rats, the main bile metabolite is 5'-glutathione conjugate, accounting for 80% of the excreted radiolabeled substance within 90 minutes of administration and more than 50% of the administered dose within 3 hours. Subsequently, 6'-conjugate was also detected in rat bile. Intermediate oxidation products, N1'-methanesulfonyl-N4'-(9-acridinyl)-3'-methoxy-2',5'-cyclohexadiene-1',4'-diimide and 3'-methoxy-4'-(9-acridinyl)amino-2',5'-cyclohexadiene-1'-one, have been identified in rat liver microsomes and human neutrophils. These are primarily converted to glutathione conjugate in the liver. Half-life: 8-9 hours.

Toxicity Overview
Acridine binds to DNA through intercalation and extrinsic binding. It is specific for AT base pairs. Rapidly dividing cells are 2 to 4 times more sensitive to acridine than quiescent cells. Acridine appears to cleave DNA by inducing double-strand breaks. Acridine also targets and inhibits topoisomerase II. Cytotoxicity is maximal during the S phase of the cell cycle, when topoisomerase levels peak.

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Carcinogenicity Evidence
Assessment: There is insufficient evidence to suggest that acridine is carcinogenic to humans. In laboratory animals, there is sufficient evidence to suggest that acridine is carcinogenic. Overall Assessment: Acridine is likely carcinogenic to humans (Group 2B).

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Oral LD50 in mice: 181 mg/kg, Summary of Data from the National Cancer Institute Screening Program, Drug Development Project, January 1986
Intraperitoneal LD50 in mice: 20560 μg/kg, Summary of Data from the National Cancer Institute Screening Program, Drug Development Project, January 1986
Subcutaneous LD50 in mice: 110 mg/kg, Summary of Data from the National Cancer Institute Screening Program, Drug Development Project, January 1986
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 can intercalate into DNA is used as an antitumor drug. According to California labor law, acridine may be carcinogenic. Acridine is a sulfonamide compound with the structure N-phenylmethanesulfonamide, substituted with a methoxy group at the 3-position and an acridine-9-ylamino group at the 4-position. It possesses antitumor activity. It is both an antitumor drug and an EC 5.99.1.3 [DNA topoisomerase (ATP hydrolysis)] inhibitor. It is a sulfonamide compound, belonging to the acridine class of compounds, and is also an aromatic ether. Acridine is a potent intercalating antitumor drug. It is effective in treating acute leukemia and malignant lymphoma, but less effective in treating solid tumors. It is often used in combination with other antitumor drugs in chemotherapy regimens. It produces stable but acceptable myelosuppression and cardiotoxicity. Acridine has been reported in cysticercosis, and relevant data are available.
Acridine is an aminoacridine derivative with potential antitumor activity. Although its mechanism of action is not fully elucidated, acridine may intercalate into DNA and inhibit topoisomerase II, leading to DNA double-strand breaks, cell cycle S/G2 phase arrest, and cell death. The drug's cytotoxicity reaches its maximum during the S phase of the cell cycle, when topoisomerase levels are highest. Furthermore, acridine may induce transcription of the tumor-promoting factor p53 protein and block p53 ubiquitination and proteasome degradation, thereby leading to p53-dependent apoptosis in tumor cells.
A potent intercalational antitumor drug belonging to the aminoacridine derivative family. It is effective in treating acute leukemia and malignant lymphoma, but less effective in treating solid tumors. It is often used in combination with other antitumor drugs in chemotherapy regimens. It produces stable but acceptable myelosuppression and cardiotoxicity.
An aminoacridine derivative that can intercalate into DNA and is used as an antitumor drug.

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Indications
For the treatment of acute myeloid leukemia.

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Mechanism of Action
Acridine binds to DNA through intercalation and external binding. It is specific for AT base pairs. Rapidly dividing cells are 2 to 4 times more sensitive to acridine than quiescent cells. Acridine appears to cleave DNA by inducing double-strand breaks. Acridine also targets and inhibits topoisomerase II. Cytotoxicity is greatest during the S phase of the cell cycle, when topoisomerase levels peak.
Acridine binds to DNA through intercalation and external binding and is specific for AT base pairs. Proliferating cells are 2 to 4 times more sensitive to acridine than quiescent cells. The normal progression of cells initially in S and G2 phases is significantly delayed, leading to S-phase cell accumulation followed by arrest in G2 phase.
The cytotoxicity of several classes of antitumor DNA intercalators is thought to be due to the capture of DNA topoisomerase II in a covalent complex onto DNA, thereby disrupting DNA metabolism. This study investigated the molecular interactions of the potent antitumor drug acridine (m-AMSA, a topoisomerase II inhibitor) in in vivo K562 cancer cells using surface-enhanced Raman spectroscopy (SER). This research builds upon previous simulated SER experiments investigating acridine/DNA, drug/topoisomerase II, and drug/DNA/topoisomerase II complexes in aqueous buffer solutions. SER data revealed two types of interactions between acridine and topoisomerase II in model complexes, either alone or within ternary complexes: non-specific interactions (via the acridine moiety) and enzyme conformation-specific interactions (via the drug side chain). Both interactions were confirmed in the micro-region SER spectra of acridine in in vivo K562 cancer cells. Our data suggest that the specific interaction between acridine and topoisomerase II via the drug side chain (a characteristic feature of both drug/topoisomerase II and ternary complexes) is crucial to 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.)