Mouse macrophage swelling caused by Toxoplasma gondii is entirely inhibited by trimetrexate (0.1 μM, 18 h) [3]. Toxoplasma gondii cell membranes are permeable to trimetrexate (1 μM), which quickly reaches high intracellular concentrations (108 pmol/107 cells). In SNU-C4 and NCI-H630 cell lines, trimetrexate (0.1 mM; 24 hours) reduces cell growth by 50–60% [5]. For ten minutes, trimetrexate (1 and 10 mM; 24 hours) in C4 was added [3].

Trimetrexate (180 mg/kg or 30 mg/kg; po or intraperitoneal injection; daily) prolongs the median survival of Toxoplasma gondii infection and shows anti-Toxoplasma activity [3]. Trimetrexate (0-30 mg/kg; iv; once daily for 5 days) exhibits chronic toxicity in toxicity [4].

Cell Proliferation Assay[5]
Cell Types: SNU-C4 and NCI-H630
Tested Concentrations: 0.1 mM
Incubation Duration: 24 h
Experimental Results: Both cell lines inhibited cell growth by 50-60%.

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Cell proliferation assay[5]
Cell Types: C4 Cell
Tested Concentrations: 1 and 10 mM
Incubation Duration: 24 hrs (hours)
Experimental Results: Produced 42% and 50% lethality at 1 and 10 mM respectively.

Animal/Disease Models: Female balb/c (Bagg ALBino) mouse infected with Toxoplasma gondii, weighing about 20 g [3]
Doses: 180 mg/kg or 30 mg/kg Dosing: 180 mg/kg orally daily in drinking water or intraperitoneal (ip) injection 30 times daily mg/kg
Experimental Results: Extended median survival of infected mice to 10 days (oral) or 19 days (ip).

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Animal/Disease Models: Charles River Wistar Crl(WI)BR rats, weighing approximately 150 to 200 g[4]
Doses: 0, 1, 10 or 30 mg/kg
Route of Administration: intravenous (iv) (iv)injection, one time/day for 5 days, then 23-
Experimental Results: Showing chronic toxicity, testicular changes that persisted over the course of multiple dosing cycles were irreversible within 21 days but required an additional 56 days for essentially complete recovery.

Absorption, Distribution and Excretion
Ten to 30% of the administered dose is excreted unchanged in the urine.
20 ± 8 L/m2
36.9 ± 6 L/m2 [cancer patients]
38 +/- 15 mL/min/m2 [patients with acquired immunodeficiency syndrome (AIDS) who had Pneumocystis carinii pneumonia (4 patients) or toxoplasmosis (2 patients). Trimetrexate was administered intravenously as a bolus injection at a dose of 30 mg/m2/day along with leucovorin 20 mg/m2 every 6 hours for 21 days]
53 +/- 41 mL/min/m2 [Cancer patients with advanced solid tumors using various dosage regimensreceiving a single-dose administration of 10 to 130 mg/m2]
30 +/- 8 mL/min/m2 [Cancer patients with advanced solid tumors using various dosage regimensafter a five-day infusion]
Clinical pharmacokinetic studies in cancer patients show that trimetrexate plasma concentration-time curves are biphasic or triphasic in form. The terminal elimination half-life averages 13.6 hr. Mean total plasma clearance and volume of distribution as steady-state values were 27.9 ml/min/sq m and 21.1 l/sq m, respectively. Cerebrospinal fluid concentration was 3.4% of that in plasma, which shows that trimetrexate does not cross the blood-brain barrier well. Trimetrexate is 86 to 94% bound to plasma proteins.
Mean oral bioavailability of the parenteral solution (glucuronate) in AIDS patients was 42%, with a mean maximum plasma concentration of 1182 ng/ml (3.2 umole/l) achieved 1.8 hr postdose. /Trimetrexate glucuromate/

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Metabolism / Metabolites
Hepatic. Preclinical data strongly suggest that the major metabolic pathway is oxidative O-demethylation, followed by conjugation to either glucuronide or the sulfate.
Trimetrexate is highly metabolized by the liver. At least 2 metabolites are excreted in urine. One metabolite has been identified a 4'-O-glucuronide conjugate of trimetrexate, which is formed as a result of oxidative O-dimethylation at the 4'-position and subsequent conjugation with glucuronic acid. About 15% of a dose is excreted in urine as unchanged trimetrexate, while another 20% apparently excreted as metabolites.
Hepatic. Preclinical data strongly suggest that the major metabolic pathway is oxidative O-demethylation, followed by conjugation to either glucuronide or the sulfate.
Route of Elimination: Ten to 30% of the administered dose is excreted unchanged in the urine.
Half Life: 11 to 20 hours

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Biological Half-Life
11 to 20 hours
The terminal elimination half-life averages 13.6 hours.

Toxicity Summary
In vitro studies have shown that trimetrexate is a competitive inhibitor of dihydrofolate reductase (DHFR) from bacterial, protozoan, and mammalian sources. DHFR catalyzes the reduction of intracellular dihydrofolate to the active coenzyme tetrahydrofolate. Inhibition of DHFR results in the depletion of this coenzyme, leading directly to interference with thymidylate biosynthesis, as well as inhibition of folate-dependent formyltransferases, and indirectly to inhibition of p.r.n. biosynthesis. The end result is disruption of DNA, RNA, and protein synthesis, with consequent cell death.

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Hepatotoxicity
When given without leucovorin protection, trimetrexate therapy is associated with a moderate rate of serum enzyme elevations, serum ALT or AST elevations above 5 times ULN in up to 20% of patients. When given with leucovorin, however, trimetrexate has fewer side effects although serum enzyme elevations can still occur. In clinical trials in patients with HIV infection and Pneumocystis jirovecii pneumonia, ALT elevations above 5 times ULN occurred in 1% to 8% of patients, but were usually no more frequent than with standard therapy using trimethoprim with sulfamethoxazole. The elevations were typically transient, without accompanying symptoms or jaundice and resolved or improved despite continuation of therapy. No instances of clinically apparent acute liver injury attributed to trimetrexate have been reported in the literature. In addition, trimetrexate has not been linked to sinusoidal obstruction syndrome or to reactivation of hepatitis B. Nevertheless, trimetrexate probably has hepatotoxic potential, but because it has limited use, is given for short periods of time and is administered with leucovorin, it has not been convincingly linked to cases of clinically apparent liver injury with jaundice.
Likelihood score: E* (unproven but suspected cause of liver injury).

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Protein Binding
95% (over the concentration range of 18.75 to 1000 ng/mL)

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Toxicity Data
LD50: 62 mg/kg (Intravenous, Mouse) (A308)

[1]. Hopper AT, et al. Discovery of Selective Toxoplasma gondii Dihydrofolate Reductase Inhibitors for the Treatment of Toxoplasmosis. J Med Chem. 2019 Feb 14;62(3):1562-1576.
[2]. Fulton, B., et al. Trimetrexate. Drugs 49, 563–576 (1995).
[3]. Allegra CJ, et al. Potent in vitro and in vivo antitoxoplasma activity of the lipid-soluble antifolate trimetrexate. J Clin Invest. 1987 Feb;79(2):478-82.
[4]. Dethloff LA, et al. Chronic toxicity of the anticancer agent trimetrexate in rats. Fundam Appl Toxicol. 1992 Jul;19(1):6-14.
[5]. Grem JL, Voeller DM, Geoffroy F, Horak E, Johnston PG, Allegra CJ. Determinants of trimetrexate lethality in human colon cancer cells. Br J Cancer. 1994 Dec;70(6):1075-84.

Trimetrexate is a member of quinazolines. It has a role as an antifungal drug.
A nonclassical folic acid inhibitor through its inhibition of the enzyme dihydrofolate reductase. It is being tested for efficacy as an antineoplastic agent and as an antiparasitic agent against pneumocystis pneumonia in AIDS patients. Myelosuppression is its dose-limiting toxic effect.
Trimetrexate is a parenterally administered folate antagonist that is used as a second line therapy for severe Pneumocystis jirovecii (previously carinii) pneumonia. Trimetrexate therapy has been associated with transient, mild serum enzyme elevations during therapy, but has not been convincingly linked to instances of acute, clinically apparent liver injury.
Trimetrexate is a methotrexate derivative with potential antineoplastic activity. Trimetrexate inhibits the enzyme dihydrofolate reductase, thereby preventing the synthesis of purine nucleotides and thymidylate, with subsequent inhibition of DNA and RNA synthesis. Trimetrexate also exhibits antiviral activity. (NCI04)
A nonclassical folic acid inhibitor through its inhibition of the enzyme dihydrofolate reductase. It is being tested for efficacy as an antineoplastic agent and as an antiparasitic agent against pneumocystis pneumonia in AIDS patients. Myelosuppression is its dose-limiting toxic effect. [PubChem]
A nonclassical folic acid inhibitor through its inhibition of the enzyme dihydrofolate reductase. It is being tested for efficacy as an antineoplastic agent and as an antiparasitic agent against PNEUMOCYSTIS PNEUMONIA in AIDS patients. Myelosuppression is its dose-limiting toxic effect.

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Drug Indication
For use, with concurrent leucovorin administration (leucovorin protection), as an alternative therapy for the treatment of moderate-to-severe Pneumocystis carinii pneumonia (PCP) in immunocompromised patients, including patients with the acquired immunodeficiency syndrome (AIDS). Also used to treat several types of cancer including colon cancer.
FDA Label

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Mechanism of Action
In vitro studies have shown that trimetrexate is a competitive inhibitor of dihydrofolate reductase (DHFR) from bacterial, protozoan, and mammalian sources. DHFR catalyzes the reduction of intracellular dihydrofolate to the active coenzyme tetrahydrofolate. Inhibition of DHFR results in the depletion of this coenzyme, leading directly to interference with thymidylate biosynthesis, as well as inhibition of folate-dependent formyltransferases, and indirectly to inhibition of p.r.n. biosynthesis. The end result is disruption of DNA, RNA, and protein synthesis, with consequent cell death.
Trimetrexate binds to dihydrofolate reductase and prevents the conversion of dihydrofolate to biologically active tetrahydrofolate. It inhibits nucleic acid synthesis as a result of antithymidylate and antipurine effects.

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Therapeutic Uses
Antifungal Agents; Antimetabolites; Antimetabolites, Antineoplastic; Folic Acid Antagonists
Antineoplastic
Trimetrexate glucuronate is an investigational drug that is available for treatment use ... in a hospital setting in qualifying patients with Pneumocystis carinii pneumonia and who have exhibited serious ... intolerance to both co-trimoxazole and pentamidine. /Trimetrexate glucuronate/

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Drug Warnings
Even though trimetrexate does not compete with the folate transport system for entry into cells, utilization of folates is reduced due to inhibition of dihydrofolate reductase. When trimetrexate is administered in high doses for the treatment of pneumocystis carinii pneumonia, leucovorin must be given concurrently with trimetrexate to reduce toxic effects on human tissues. It has been postulated that the absence of a folate transport system in pneumocystis carinii provides the opportunity for differential rescue of host tissue affecting antiprotozoal action.
Patients receiving trimetrexate should have frequent laboratory monitoring of hepatic and hematologic prameters, including serum alanine aminotransferase, serum aspartate aminotransferase, bilirubin, alkaline phosphatase, platelet count, and total and differential leukocyte counts. Although uncommon in AIDS patients receiving trimetrexate for pneumocystis carinii pneumonia, slight elevations of blood urea nitrogen and/or serum creatinine have been reported in patients receiving the drug for various cancers.
Isolated perfused rat liver has been used to study potential drug interactions with trimetrexate metabolism in vitro. Cimetidine, which inhibits oxidative drug metabolizing enzymes, decreased the clearance of trimetrexate to approximately one half of control values. Whether this occurs to a significant extent in vivo is unknown.

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Pharmacodynamics
Trimetrexate, a non-classical folate antagonist, is a synthetic inhibitor of the enzyme dihydrofolate reductase (DHFR). During DNA synthesis and cellular reproduction, folic acid is reduced to tetrahydrofolic acid by the enzyme folic acid reductase. By interfering with the reduction of folic acid, trimetrexate interferes with tissue cell reproduction. Generally, the most sensitive cells to the antimetabolite effect of trimetrexate are those cells which are most actively proliferating such as malignant cells, dermal epithelium, buccal and intestinal mucosa, bone marrow, fetal cells, and cells of the urinary bladder. Because the proliferation of cells in malignant tissues is greater than in most normal tissues, trimetrexate may impair the growth of the malignant tissues without causing irreversible damage to normal tissues. Due to very serious and potentially life-threatening side-effects of this drug, leucovorin must be co-administered for at least 72 hours after the last dose.

C19H23N5O3

369.41762

369.18

52128-35-5

Trimetrexate glucuronate;82952-64-5;Trimetrexate trihydrochloride;1658520-97-8;Trimetrexate isethionate;82935-04-4

5583

Typically exists as solid at room temperature

1.305g/cm3

647ºC at 760mmHg

215-217 °C
215 - 217 °C

345.1ºC

3.975

3

8

6

27

457

0

NC1=C(C(C)=C2CNC3=CC(OC)=C(OC)C(OC)=C3)C(C=C2)=NC(N)=N1

NOYPYLRCIDNJJB-UHFFFAOYSA-N

InChI=1S/C19H23N5O3/c1-10-11(5-6-13-16(10)18(20)24-19(21)23-13)9-22-12-7-14(25-2)17(27-4)15(8-12)26-3/h5-8,22H,9H2,1-4H3,(H4,20,21,23,24)

5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline-2,4-diamine

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 : ≥ 61.5 mg/mL (~166.48 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.77 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 25.0 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: ≥ 2.5 mg/mL (6.77 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 40 mg/mL (108.28 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.)