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
10% 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) and Pneumocystis pneumonia (4 cases) or toxoplasmosis (2 cases)]. Trimethoprim was administered intravenously as a bolus injection of 30 mg/m²/day, simultaneously with 20 mg/m² of leucovorin intravenously every 6 hours for 21 days.
53 ± 41 mL/min/m² [Advanced solid tumor cancer patients receiving a single dose of 10 to 130 mg/m², using different dosing regimens]
30 ± 8 mL/min/m² [Advanced solid tumor cancer patients receiving 5 days of infusion, using different dosing regimens]
Clinical pharmacokinetic studies in cancer patients showed that trimesartan's plasma concentration-time curves were biphasic or triphasic. The mean terminal elimination half-life was 13.6 hours. The mean total plasma clearance and steady-state volume of distribution were 27.9 ml/min/m² and 21.1 l/m², respectively. The cerebrospinal fluid concentration was 3.4% of the plasma concentration, indicating that trimesartan does not readily cross the blood-brain barrier. Trimetasartan's binding to plasma proteins ranged from 86% to 94%.
In HIV patients, the mean oral bioavailability of the parenteral solution (glucuronic acid) was 42%, and the mean maximum plasma concentration of 1182 ng/ml (3.2 μmol/L) was reached 1.8 hours after administration. /Trimethasone Glucuronic Acid/

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Metabolic/Metabolic Substances
Hepatic metabolism. Preclinical data strongly suggest that its primary metabolic pathway is oxidative O-demethylation, followed by conjugation with glucuronic acid or sulfate.
Trimethasone is primarily metabolized in the liver. At least two metabolites are excreted in the urine. One metabolite has been identified as the 4'-O-glucuronide conjugate of trimesone, which is formed by the conjugation of trimesone with glucuronic acid following oxidative O-dimethylation at the 4' position. Approximately 15% of the dose is excreted unchanged in the urine, and another 20% is excreted as metabolites.
Hepatic metabolism. Preclinical data strongly suggest that its primary metabolic pathway is oxidative O-demethylation, followed by conjugation with glucuronide or sulfate.
Elimination pathway: 10% to 30% of the administered dose is excreted unchanged in the urine.
Half-life: 11 to 20 hours
Biological half-life
11 to 20 hours
Terminal elimination half-life averages 13.6 hours.

Toxicity Summary
In vitro studies have shown that trimesartan is a competitive inhibitor of dihydrofolate reductase (DHFR) from bacteria, protozoa, and mammals. DHFR catalyzes the reduction of intracellular dihydrofolate to the active coenzyme tetrahydrofolate. Inhibition of DHFR leads to the depletion of this coenzyme, directly interfering with thymidine biosynthesis and inhibiting folate-dependent formylate transferase, indirectly inhibiting prn biosynthesis. Ultimately, this results in disordered DNA, RNA, and protein synthesis, leading to cell death. Hepatotoxicity
When not protected with folinic acid calcium, trimesartan treatment is associated with moderate elevations in serum enzymes, with up to 20% of patients experiencing serum ALT or AST levels exceeding five times the upper limit of normal. However, when used in combination with folinic acid calcium, trimesartan has fewer side effects, but elevations in serum enzymes may still occur. In clinical trials of HIV-infected patients with Pneumocystis carinii pneumonia, 1% to 8% of patients experienced ALT elevations exceeding five times the upper limit of normal, but this was generally not more common than with standard therapy using sulfamethoxazole-trimethoprim. These elevations were usually transient, asymptomatic, or without jaundice, and would subside or improve with continued treatment. No clinically significant acute liver injury caused by trimex has been reported in the literature. Furthermore, trimex has not been confirmed to be associated with hepatic sinusoidal obstruction syndrome or hepatitis B virus reactivation. Nevertheless, trimex may be hepatotoxic, but due to its limited use, short treatment duration, and need for combination therapy with leucovorin, there is no conclusive evidence that it is associated with clinically significant liver injury with jaundice. Probability Score: E (Unproven but suspected cause of liver injury). Protein binding rate: 95% (concentration range: 18.75 to 1000 ng/mL) Toxicity data: LD50: 62 mg/kg (intravenous injection, mice) (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.

Trimetaxel belongs to the quinazoline class of drugs and is an antifungal agent. It acts as a non-classical folate inhibitor by inhibiting dihydrofolate reductase. Its efficacy as an antitumor and antiparasitic drug for treating Pneumocystis carinii pneumonia in HIV patients is currently being tested. Bone marrow suppression is its dose-limiting toxicity. Trimetaxel is a parenteral folate antagonist used as a second-line treatment for severe Pneumocystis carinii pneumonia (formerly known as Pneumocystis jirovecii pneumonia). Transient, mild elevations in serum enzymes have occurred during Trimetaxel treatment, but there is no conclusive evidence linking it to acute, clinically significant liver injury. Trimetaxel is a derivative of methotrexate and has potential antitumor activity. Trimetaxel works by inhibiting dihydrofolate reductase, thereby preventing the synthesis of purine nucleotides and thymidine phosphates, and consequently inhibiting DNA and RNA synthesis. Trimetaxel also has antiviral activity. (NCI04)
Trimetaxel is a non-classical folate inhibitor whose mechanism of action is through the inhibition of dihydrofolate reductase. Currently, its efficacy as an antitumor and antiparasitic drug for treating Pneumocystis carinii pneumonia in AIDS patients is being tested. Myelosuppression is its dose-limiting toxicity. [PubChem]
Trimetaxel is a non-classical folate inhibitor whose mechanism of action is through inhibition of dihydrofolate reductase. Currently, its efficacy as an antitumor and antiparasitic drug for treating Pneumocystis carinii pneumonia in AIDS patients is being tested. Myelosuppression is its dose-limiting toxicity.

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Drug Indications
Used in combination with folinic acid (folinic acid protection), as an alternative therapy for moderate to severe Pneumocystis carinii pneumonia (PCP) in immunocompromised patients, including those with acquired immunodeficiency syndrome (AIDS). It can also be used to treat a variety of cancers, including colon cancer.
FDA Label

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Mechanism of Action
In vitro studies have shown that trimetaxel 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 dihydrofolate reductase (DHFR) leads to the depletion of this coenzyme, directly interfering with the biosynthesis of thymidine and inhibiting folate-dependent formylate transferase, indirectly inhibiting the biosynthesis of purine nucleotides (prn). Ultimately, this results in disordered DNA, RNA, and protein synthesis, leading to cell death. Trimetaxel binds to dihydrofolate reductase, preventing the conversion of dihydrofolate to biologically active tetrahydrofolate. It inhibits nucleic acid synthesis through its anti-thymidine and anti-purine effects.
Therapeutic Uses
Antifungal; antimetabolite; antitumor antimetabolite; folate antagonist
Antitumor drug
Trimetaxel gluconate is an investigational drug for the treatment of eligible patients with Pneumocystis carinii pneumonia who have shown severe intolerance to trimethoprim-sulfamethoxazole and pentamidine in a hospital setting. /Trimetaxel Glucuronate/

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Drug Warning
Although trimetaxel does not compete with the folic acid transport system for cell entry, folic acid utilization is reduced due to inhibition of dihydrofolate reductase. When using high doses of trimetaxel to treat Pneumocystis carinii pneumonia, folinic acid calcium must be administered concurrently to reduce toxicity to human tissues. Some studies speculate that Pneumocystis carinii lacks a folic acid transport system, enabling it to differentially repair host tissues, thus affecting antiprotozoal activity. Patients receiving trimetaxel should undergo frequent laboratory monitoring of liver function and hematological parameters, including serum alanine aminotransferase, serum aspartate aminotransferase, bilirubin, alkaline phosphatase, platelet count, and total and differential white blood cell counts. Although uncommon in HIV patients receiving trimetaxel for Pneumocystis carinii pneumonia, mild elevations in blood urea nitrogen and/or serum creatinine have been reported in patients receiving the drug for various cancers. Ex vivo perfusion of rat liver has been used to study potential interactions between the drug and trimetaxel metabolism in vitro. Cimetidine inhibits oxidative drug-metabolizing enzymes, reducing trimesat's clearance to about half the control value. Whether this phenomenon is significant in vivo is unclear.

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Pharmacodynamics
Trimesat is a non-classical folic acid antagonist, an inhibitor of dihydrofolate reductase (DHFR) synthesis. During DNA synthesis and cell proliferation, folic acid is reduced to tetrahydrofolate by folate reductase. Trimeesat interferes with the reduction of folic acid, thereby interfering with the proliferation of tissue cells. Typically, the cells most sensitive to trimesat's antimetabolite effects are those with the most active proliferation, such as malignant cells, dermal epithelial cells, oral and intestinal mucosal cells, bone marrow cells, fetal cells, and bladder cells. Because cells in malignant tissues proliferate at a higher rate than in most normal tissues, trimesat may inhibit the growth of malignant tissues without harming normal tissues. Due to the very serious and potentially life-threatening side effects of this drug, leucovorin calcium must be taken concurrently 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

Light yellow to green yellow solid powder

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.)