Aminopterin is a potential antagonist to folic acid. [4]

After 72 hours of exposure, aminopterin (4-aminofolate) had an IC50 value of 4.4 nM against CCRF-CEM cells [2]. At low quantities, aminopterin (4-aminofolate) can severely impair human leukemia leukocytes' ability to undergo mitosis [4].

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Aminopterin inhibited the growth of Streptococcus faecalis R in low concentrations, and this inhibition was reversible by folic acid. [4]
When added to human leukemic leukocytes in vitro, Aminopterin produced a marked inhibition of mitosis at low concentrations. Initial experiments with a concentration of 55 µg/ml (about 1.2 × 10⁻⁴ M) showed no significant difference in total and differential counts compared to controls and no evidence of direct toxic action on mature or immature cells, but there was a complete absence of mitotic figures at all culture stages. [4]
In 24-hour cultures, mitotic inhibition was practically complete at Aminopterin concentrations down to and including 5 × 10⁻⁷ M. At 5 × 10⁻⁴ M, a 60-70% inhibition was regularly obtained. Lower concentrations showed lesser degrees of inhibition. [4]
Aminopterin proved equally effective whether added immediately or up to eight hours after culture setup, indicating it could inhibit mitosis both before its inception and after it was fully underway. [4]
Attempts to counteract the effects of small quantities of Aminopterin (5 × 10⁻⁸ M) with folic acid yielded varying results. In four experiments, folic acid at concentrations 1 to 10 times that of Aminopterin were apparently inert, but concentrations 100-1000 times stronger produced a partial restoration of mitotic activity. In two other experiments (using cells from patients in the terminal phase of leukemia), even high concentrations of folic acid were ineffective in countering the inhibitory action of Aminopterin. [4]

Administration of Aminopterin to chicks, weanling rats, and mice produced slowing of growth and depression of red and white blood cell counts. These effects were produced by doses little short of toxic and could be counteracted only partially by folic acid. [4]
In mice, a diet containing 0.3 parts per million of Aminopterin produced partially reversible effects, whereas 1 part per million invariably caused death within a few days, regardless of the quantity of folic acid given. [4]
Aminopterin was found to be active against Rous sarcoma in chicks, but was so toxic that only adult birds could tolerate it. Toxicity and tumor inhibition could be neutralized in baby chicks by simultaneous injection of pteroylglutamic acid. Tumors in successfully treated birds showed cytological degeneration and possible disruption of mitotic figures. [4]
In a group of 16 children with acute and subacute leukemia treated with Aminopterin, clinical and hematologic remissions were observed in 10, a rate regarded as greatly exceeding spontaneous remission rates. Results in adults were less predictable, and toxic effects such as hemorrhage or aplastic anemia were common. [4]

Leukemic leukocytes were obtained from the peripheral blood of patients with chronic myeloid leukemia. Cells were cultured using a modified method based on Osgood and Brownlee's technique. In brief, leukocytes isolated from peripheral blood were suspended in 50% fresh homologous human serum or plasma and incubated. Total, differential, and mitotic cell counts were performed at suitable intervals. For experiments with Aminopterin, the drug was added to cultures at the time of setup unless otherwise stated. Cultures were typically carried on for 24 or 48 hours. The effects of Aminopterin were assessed by comparing total, differential, and particularly mitotic counts with those of untreated control cultures. To test reversibility, cultures were pre-incubated for one hour in the presence of folic acid before adding Aminopterin, and mitotic activity was subsequently assessed. [4]

The information is presented as observed results from prior studies. For example, in mice, Aminopterin was incorporated into the diet at concentrations of 0.3 or 1 part per million. In studies with Rous sarcoma in chicks, the drug was administered, and toxicity/tumor inhibition was assessed, with pteroylglutamic acid used as a potential neutralizing agent. [4]

Aminopterin is extremely toxic in animal models. In mice, dietary concentrations of 1 part per million almost always lead to death within days. In chicks, its toxicity is so high that only adult chicks can tolerate it. [4]
When used clinically in humans to treat leukemia, toxic reactions such as bleeding or aplastic anemia are common, especially in adult patients. [4]

[1]. Aminopterin, a folic acid antagonist, in the treatment of leukemia. Am J Clin Pathol. 1949 Feb;19(2):119-26.

[2]. Synthesis and in vitro antifolate activity of rotationally restricted aminopterin and methotrexate analogues. J Med Chem. 2004 Dec 30;47(27):6958-63.

[3]. The influence of aminopterin on limb regeneration in Ambystoma mexicanum. J Embryol Exp Morphol. 1966 Aug;16(1):143-58.

[4]. THE EFFECT OF 4-AMINO-PTEROYLGLUTAMIC ACID (AMINOPTERIN) ON HUMAN LEUKEMIC LEUKOCYTES IN VITRO. Blood (1950) 5 (2): 161-166.

Aminopterin to an independent committee of scientific and health experts, aminopterin may cause developmental toxicity and female reproductive toxicity. It is a cluster of yellow, needle-like plants. It has been used as a rodenticide, medicine, and rodenticide. It is not registered as a rodenticide in the United States (EPA, 1998). 4-Aminofolate is a dicarboxylic acid. It has mutagenic and EC 1.5.1.3 (dihydrofolate reductase) inhibitory effects. It is functionally related to folic acid. Aminopterin is an amino derivative of folic acid and was once used as an anti-tumor drug to treat childhood leukemia. Production of aminopterin was discontinued in the 1950s and replaced by methotrexate, which is less potent but also less toxic. Aminopterin was also used to treat psoriasis, but this was off-label use. Clinicians need to be aware of the characteristic teratogenic effects of aminopterin and methotrexate. Aminopterin is a synthetic pterin derivative with antitumor and immunosuppressive properties. As a folic acid analog, aminopterin competes with dihydrofolate reductase for the folic acid binding site, thereby blocking the synthesis of tetrahydrofolate, leading to the depletion of nucleotide precursors and inhibiting the synthesis of DNA, RNA, and proteins. (NCI04)
A folic acid derivative used as a rodenticide, it has been shown to be teratogenic.

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Drug Indications
Before its withdrawal from the market, aminopterin was initially used to treat childhood leukemia, particularly for inducing remission. Later, aminopterin was used off-label in the United States for the treatment of psoriasis, with significant lesion clearance. Due to its better therapeutic index, methotrexate was later replaced by it for the treatment of cancer. Aminopterin (and methotrexate) was also explored as an abortifacient. However, they were associated with severe congenital malformations and teratogenic effects, known as fetal aminopterin syndrome.

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Mechanism of Action
Aminopterin is an amino derivative of folic acid that competitively binds to dihydrofolate reductase, thereby blocking the synthesis of tetrahydrofolate. Tetrahydrofolate is essential for the synthesis of purines and pyrimidines, and its deficiency leads to reduced DNA, RNA and protein synthesis. It is an antimetabolite that antagonizes the body's utilization of folic acid. Aminopterin was once a commonly used drug that inhibits the digestive and absorptive mechanisms by inhibiting folic acid transport and cell proliferation. /Excerpt from Table/

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Therapeutic Uses It was used to treat childhood leukemia, sometimes in sodium form. /Aminopterin Sodium/

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Drug Warnings The known effects of aminopterin on humans (early embryonic development) are well documented and well-documented: multiple malformations, craniofacial malformations, miscarriage, intrauterine growth retardation. /Excerpt from Table/

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Aminopterin was initially synthesized as a potential folic acid antagonist. [4]
The observation that folic acid intake sometimes accelerates the progression of human leukemia led to the idea that folic acid antagonists might slow its progression, prompting the investigation of aminopterin. [4]
This paper discusses the mechanism by which aminopterin inhibits mitosis in vitro. Although aminopterin is a folic acid antagonist, attempts to reverse its effects with folic acid have not always been successful, raising questions about whether its mechanism of action might differ from simple folic acid antagonism. [4]
Another study reported that normal human myeloid cells underwent erythrocyte phagocytosis after the addition of aminopterin in vitro, but this phenomenon was not observed under the experimental conditions of this study. [4]

C19H20N8O5

440.4127

440.155

54-62-6

58602-66-7 (sodium);54-62-6 (free acid);

169371

Light yellow to yellow solid powder

1.6±0.1 g/cm3

228-235 °C (dec.)

1.762

-1.21

6

12

9

32

674

1

C1=CC(=CC=C1C(=O)N[C@@H](CCC(=O)O)C(=O)O)NCC2=CN=C3C(=N2)C(=NC(=N3)N)N

TVZGACDUOSZQKY-LBPRGKRZSA-N

InChI=1S/C19H20N8O5/c20-15-14-16(27-19(21)26-15)23-8-11(24-14)7-22-10-3-1-9(2-4-10)17(30)25-12(18(31)32)5-6-13(28)29/h1-4,8,12,22H,5-7H2,(H,25,30)(H,28,29)(H,31,32)(H4,20,21,23,26,27)/t12-/m0/s1

(4-(((2,4-diaminopteridin-6-yl)methyl)amino)benzoyl)-L-glutamic acid

4-amino Folic Acid; 4-amino PGA; NSC-739; NSC 739; NSC739; APGA; BRN 0069045; CCRIS 5856; ENT 26079; BRN0069045; CCRIS5856; ENT26079; BRN-0069045; CCRIS-5856; ENT-26079

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)

DMSO : ~14.71 mg/mL (~33.40 mM)
H2O : ~5 mg/mL (~11.35 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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