DNA (alkylation target, mediating DNA damage) [1]

Altretamine is an antineoplastic agent.[1]

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Altretamine (ENT-50852) exhibited antiproliferative activity against human lung cancer MV522 cells as a standalone DNA-damaging agent. At concentrations of 10-100 μM, it inhibited MV522 cell proliferation in a dose-dependent manner, with a single-agent growth inhibition rate of ~42% at 50 μM after 72 hours of incubation [1]
- It showed synergistic antiproliferative effects when combined with irofulven (a DNA-reactive agent) in MV522 cells. The combination index (CI) was 0.72 at the effective dose level achieving 50% growth inhibition (ED50), indicating strong synergy. The combined treatment (Altretamine 25 μM + irofulven 0.5 μM) resulted in a growth inhibition rate of ~78%, which was significantly higher than the additive effect of standalone treatments (Altretamine alone: ~30%; irofulven alone: ~25%) [1]
- The synergistic mechanism involved enhanced DNA damage: the combination increased the number of DNA double-strand breaks (detected by γ-H2AX staining) by ~2.3-fold compared to Altretamine alone, leading to increased G2/M cell cycle arrest and apoptotic cell death [1]

The anti-tumor impact of MV522 cells can be amplified when used in combination with IrofuLven (100, 133 mg/kg, ip.) [1].

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In nude mice bearing MV522 lung cancer xenografts, Altretamine (ENT-50852) alone and in combination with irofulven inhibited tumor growth. Intraperitoneal administration of Altretamine (100 mg/kg, once every 3 days for 4 cycles) reduced tumor volume by ~35% and tumor weight by ~32% compared to the vehicle control group [1]
- The combination of Altretamine (100 mg/kg, same schedule) and irofulven (10 mg/kg, intraperitoneal, once every 3 days for 4 cycles) showed superior antitumor efficacy: tumor volume was reduced by ~68%, and tumor weight by ~65%. The combination treatment prolonged the median survival of mice by ~28% compared to the vehicle group, with no obvious increase in toxicity compared to standalone treatments [1]
- Immunohistochemical analysis of tumor tissues showed that the combination increased γ-H2AX-positive cells (DNA damage marker) by ~2.1-fold and TUNEL-positive apoptotic cells by ~1.8-fold compared to Altretamine alone [1]

MV522 cell proliferation and synergy assay: MV522 cells were seeded in 96-well plates at a density of 3×10³ cells/well and cultured for 24 hours. Cells were treated with standalone Altretamine (ENT-50852) (0-200 μM) or combined with irofulven (0-2 μM) for 72 hours. Cell viability was detected by sulforhodamine B (SRB) staining, and growth inhibition rates were calculated. The combination index (CI) was determined using the Chou-Talalay method to assess synergy [1]
- DNA damage and cell cycle assay: MV522 cells were seeded in 6-well plates and treated with Altretamine alone (50 μM) or in combination with irofulven (1 μM) for 48 hours. Cells were fixed, permeabilized, and stained with anti-γ-H2AX antibody to detect DNA double-strand breaks (immunofluorescence microscopy). Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining to evaluate G2/M arrest [1]

Mice: Balb/c nu/nu Female mice, 4 weeks old and weighing 18–22 g, are injected subcutaneously with 8–10 million MV522 cells. Starting on day 10 following tumor implantation, metformin is injected intraperitoneally three times a week for three weeks. Two perpendicular diameters are used to measure tumor size, and the formula w = [(width) 2 × length/2] is used to estimate tumor weight (TW). When necessary, altretamine is diluted with 10% DMSO/normal saline after being prepared as stock solutions containing 1–10 mg/mL in 40% DMSO/normal saline[1].

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MV522 lung cancer xenograft model: Nude mice were subcutaneously inoculated with 5×10⁶ MV522 cells into the right flank. When tumors reached ~150 mm³, mice were randomized into three groups (n=8 per group): vehicle control, Altretamine alone, and Altretamine + irofulven combination. Altretamine (ENT-50852) was dissolved in 5% DMSO + 95% saline and administered via intraperitoneal injection at 100 mg/kg once every 3 days for 4 cycles. Irofulven was administered via intraperitoneal injection at 10 mg/kg following the same schedule as Altretamine. Tumor volume was measured every 3 days using calipers, and tumor weight was recorded after mice were sacrificed at the end of treatment. Tumor tissues were collected for immunohistochemical analysis of γ-H2AX and TUNEL staining [1]

Absorption, Distribution and Excretion
The N-demethylated homologue of atratamine is metabolized in human urine, with <1% of atratamine excreted unmetabolized within 24 hours.
Absorption is rapid and good after oral administration; however, due to rapid hepatic metabolism, peak plasma concentrations vary among individuals.
…The inter- and intra-patient bioavailability variability after oral atratamine administration is a significant limitation to the effective clinical application of this drug. This variability appears to be primarily related to the first-pass effect and can therefore be overcome by intravenous administration. Past attempts at intravenous administration have been unsuccessful…
Due to the high lipid solubility of atratamine, it is distributed to tissues with high lipid content (e.g., the greater omentum and subcutaneous tissue).
Protein binding: Free fraction: Atratamine: 6%; Pentamethylmelamine: 25%; Tetramethylmelamine: 50%.
For more complete data on the absorption, distribution, and excretion of atratamines (10 in total), please visit the HSDB record page.

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Metabolism/Metabolites
Biotransformation mainly occurs in the liver. Metabolism is essential for its activity. Atratamine undergoes rapid and extensive demethylation catalyzed by cytochrome P450 enzymes.
The covalent binding of hexamethylmelamine (HMM) and its metabolites in the liver, tumor, blood, kidney, spleen, lung, brain, heart, and small intestine was investigated after a single intraperitoneal injection of 2,4,6-14C-hexamethylmelamine (50 mg/kg) into 20-day-old female C57Bl/6J mice with M5076/73A ovarian cancer. ...The metabolism of HMM was also investigated. The tissue distribution of pentamethylmelamine (PMM), 2,2,4,6-tetramethylmelamine (TMM), and 2,4,6-trimethylmelamine (TriMM) was determined at three time points. Two hours later, the drug was extensively metabolized, and TriMM was the major metabolite among the metabolites measured.
...In all animal species, including humans, atratamine undergoes oxidative N-demethylation to produce hydroxymethyl derivatives as intermediates. Hydroxymethyl melamine is considered the main source of the drug's cytotoxicity and antitumor activity.
In rats, 40% of a 25 mg/kg dose of hexamethylmelamine or pentamethylmelamine is excreted in the urine as metabolites, of which over 95% are N2N4-dimethylmelamine and monomethylmelamine. The bile excretion of hexamethylmelamine or pentamethylmelamine and its N-demethyl metabolites is less than 2% of the administered dose. Only 3% is excreted in the feces, indicating that some of the methylmelamine entering the bile is reabsorbed in the intestine. In the urine or bile of rats treated with hexamethylmelamine or pentamethylmelamine, only trace amounts of methylmelamine conjugates with glucuronic acid or sulfate were detected. However, a conjugate of pentamethylmelamine of unknown nature is the main metabolite following pentamethylmelamine treatment.

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Biological half-life 4.7–10.2 hours
Elimination/Half-life/-β phase: Range, 4.7 to 10.2 hours.

Hepatotoxicity
Atratamine treatment is associated with a low incidence of elevated serum enzymes, but these elevations are usually mild and resolve spontaneously without dose adjustment. A few cases of clinically significant acute liver injury caused by atratamine have been reported, but the clinical characteristics are not yet clear. Atratamine has not been definitively linked to hepatic sinusoidal obstruction syndrome, but it is rarely used in pre-treatment regimens for neoplastic diseases or bone marrow transplantation, where alkylating agents are commonly associated with this complication. Probability Score: E (Unlikely, but suspected as a rare cause of liver injury).

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Protein binding rate 94%

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Toxicity data
Humans (oral): TDL0 8 mg/kg
Rat (oral): LD50 350 mg/kg
Rat (intraperitoneal): LD50 265 mg/kg
Mouse (oral): LD50 437 mg/kg
Mouse (intraperitoneal): LD50 200 mg/kg
Mouse (intravenous): LD50 171 mg/kg

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Drug interactions
Drugs causing blood disorders (leukopenia): If contemporaneous or recent drug treatment also causes thrombocytopenia, atratamine-induced thrombocytopenia may be exacerbated; if necessary, the atratamine dose should be adjusted according to blood cell count. Radiation therapy may worsen bone marrow suppression; when two or more bone marrow suppressants (including radiation) are used concurrently or sequentially, a dose reduction of atratamine may be necessary. Concomitant use of atratamine with monoamine oxidase (MAO) inhibitors (including furazolidone, procarbazine, and selegiline) may cause severe orthostatic hypotension. Because atratamine treatment may suppress normal immune defense mechanisms, antibody responses to inactivated vaccines may be reduced. The time interval between discontinuation of immunosuppressive drugs and the patient's recovery of vaccine responsiveness depends on the strength and type of immunosuppressive drugs used, underlying diseases, and other factors. (Estimated duration: 3 months to 1 year) For more complete data on interactions of ALTRETAMINEs (6 in total), please visit the HSDB records page.

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Non-human toxicity values
Oral LD50 in rats: 350 mg/kg
Oral LD50 in guinea pigs: 255 mg/kg
In vivo studies showed that, at the tested dose, Altretamine (ENT-50852) combined with irofulven did not cause significant changes in body weight (weight loss < 10% compared to the control group) or organ indices (liver, kidney, spleen) in mice. No obvious histological abnormalities were observed in the major organs (H&E staining)[1]

[1]. Synergy of irofulven in combination with other DNA damaging agents: synergistic interaction with altretamine, alkylating, and platinum-derived agents in the MV522 lung tumor model. Cancer Chemother Pharmacol. 2008 Dec;63(1):19-26.

According to state or federal labeling requirements, atratamine may cause developmental toxicity and male reproductive toxicity. Hexamethylmelamine is a colorless crystalline solid, insoluble in water. (NTP, 1992) Hexamethylmelamine is a triamino-1,3,5-triazine compound. It is an alkylating agent and has been proposed for use as an antitumor drug. It can also be used as a chemical sterilizer for male houseflies and other insects. Atratamine is an alkylating agent. The mechanism of action of atratamine is alkylating activity. Atratamine is an oral alkylating agent, formerly known as hexamethylmelamine, and is currently used as a second-line treatment for advanced ovarian cancer. The incidence of serum enzyme elevation during atratamine treatment is low, and acute, clinically significant damage is rare. Atratamine is a synthetic cytotoxic triazine derivative with a structure similar to the alkylating agent triethylenetriamine and possesses antitumor activity. Although the exact mechanism of atratamine's cytotoxic effect is unclear, N-demethylation of atratamine may produce active intermediates that covalently bind to DNA, leading to DNA damage. (NCI04)
A hexamethyl-2,4,6-triamine derivative of 1,3,5-triazine.

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Drug Indications
For palliative treatment of persistent or recurrent ovarian cancer following first-line cisplatin and/or alkylating agent combination chemotherapy, it can be used as a single agent.

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Mechanism of Action
Although atratamine is classified as an alkylating antitumor drug, the exact mechanism of its cytotoxic effect is unclear. The drug is metabolized to alkylating agents via N-demethylation. These alkylating agents subsequently damage tumor cells.
The exact mechanism of action is unclear. Although atratamine's structure is similar to alkylating agents, it has not been found to have in vitro alkylating activity. There is evidence that it may inhibit DNA and RNA synthesis.
The hexamethyl melamine analogue trimeramol (tris(hydroxymethyl)melamine) and its stable cytotoxic analogues CB 7547, CB 7639, and CB 7669 have been used to elucidate the mechanism of action of N-(hydroxymethyl)melamine as an antitumor drug. Two main mechanisms have been proposed and investigated: (i) formation of reactive imine ions, which covalently adduct with DNA; and (ii) local release of formaldehyde leading to cytotoxic damage. P-labeling and thermal denaturation experiments have shown that these compounds interact with cytosine and guanine. Trimelamor can induce interstrand cross-linking of DNA in naked plasmid DNA and cultured cell lines, while its analogues did not produce this effect under various experimental conditions. Combined with our observation that cell lines resistant to N-(hydroxymethyl)melamine do not exhibit significant cross-resistance to classical bifunctional alkylating agents, DNA cross-linking may play only a minor role in its mechanism of action. In cultured cell lines, treatment with formaldehyde, tricresyl, and CB 7639 increased DNA-protein cross-linking levels, which gradually disappeared within 24 hours. Combined with previously observed simultaneous cross-resistance to tricresyl and formaldehyde, researchers concluded that formaldehyde release may be a significant factor in its cytotoxicity. Furthermore, glutathione depletion did not affect the cytotoxicity of tricresyl or formaldehyde against human ovarian cancer cells. N-(hydroxymethyl)melamine /

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Therapeutic Use
Anti-tumor Drug
Atratamine is indicated for palliative treatment of persistent or recurrent epithelial ovarian cancer following first-line cisplatin and/or alkylating agent combination chemotherapy. /Included on US Product Label/
Atratamine combination therapy is considered a reasonable treatment option in certain stages of small cell lung cancer (Level of Evidence: IA). /Not Included on US Product Label/

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Drug Warnings
Secondary malignancy is a potential delayed side effect of many anti-tumor drugs, but it is unclear whether this side effect is related to their mutagenic or immunosuppressive effects. The effects of dosage and duration of treatment are also unknown, but long-term use appears to increase the risk. Despite limited information, existing data appear to suggest that alkylating agents pose the highest carcinogenic risk. Atratamine's bone marrow suppression may lead to an increased incidence of microbial infections, delayed wound healing, and gingival bleeding. Dental treatment should be completed before starting treatment whenever possible, or postponed until blood cell counts return to normal. Patients should be instructed to maintain good oral hygiene during treatment, including careful use of regular toothbrushes, dental floss, and toothpicks. Frequent adverse events… suggest the need for medical attention: anemia (unusual fatigue); leukopenia (fever or chills; cough or hoarseness; lower back or flank pain; painful or difficult urination); neurotoxicity, including central nervous system (CNS) effects (anxiety; clumsiness; confusion; dizziness; depression; weakness; seizures). Neurotoxicity includes peripheral neuropathy (numbness in the arms or legs); thrombocytopenia (abnormal bleeding or bruising; melena, tarry stools; hematuria or melena; pinpoint red spots on the skin). Incidence/Rare. Hepatotoxicity; rash or itching.
Atratamine is contraindicated in patients with known hypersensitivity to atratamine.
For more complete data on drug warnings for atratamine (11 of them), please visit the HSDB record page.

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Pharmacodynamics
Atratamine is a novel antitumor drug. The exact mechanism by which atratamine exerts its cytotoxic effect is unclear, although several theoretical possibilities have been investigated. Structurally, atratamine is similar to the alkylating agent triethylene melamine, but in vitro studies have shown that atratamine and its metabolites do not possess alkylating activity. Atratamine has been shown to be effective against certain ovarian tumors resistant to conventional alkylating agents. Metabolism of atratamine is necessary for its cytotoxic effect. Synthetic monomethylolmelamine and atratamine metabolites can form covalent adducts with tissue macromolecules, including DNA, both in vitro and in vivo, but the correlation between these reactions and antitumor activity is unclear.

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Atratamine (ENT-50852) is an alkylating agent chemotherapy drug that exerts its antitumor effects by alkylating DNA, inducing DNA damage, and inhibiting tumor cell proliferation and DNA repair[1].
- Its synergistic effect with irofluvan is attributed to complementary DNA damage pathways: atratamine mainly induces DNA alkylation, while irofluvan mediates DNA crosslinking and strand breaks, thereby enhancing cytotoxicity and reducing tumor cell resistance[1].
- This drug has shown monotherapy antitumor activity against MV522 lung cancer cells both in vitro and in vivo, with better efficacy when used in combination with irofluvan, which supports… its potential application in combination chemotherapy for lung cancer[1].

C9H18N6

210.28

210.159

C, 51.41; H, 8.63; N, 39.97

645-05-6

2123

White to off-white solid powder

1.1±0.1 g/cm3

339.4±25.0 °C at 760 mmHg

171-175 °C(lit.)

159.1±23.2 °C

0.0±0.7 mmHg at 25°C

1.610

2.42

0

6

3

15

148

0

N(C([H])([H])[H])(C([H])([H])[H])C1N=C(N=C(N=1)N(C([H])([H])[H])C([H])([H])[H])N(C([H])([H])[H])C([H])([H])[H]

UUVWYPNAQBNQJQ-UHFFFAOYSA-N

InChI=1S/C9H18N6/c1-13(2)7-10-8(14(3)4)12-9(11-7)15(5)6/h1-6H3

2-N,2-N,4-N,4-N,6-N,6-N-hexamethyl-1,3,5-triazine-2,4,6-triamine

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)

Solubility in Formulation 1: ≥ 0.83 mg/mL (3.95 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.83 mg/mL (3.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 8.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)