DHFR/Dihydrofolate reductase; Influenza A virus
Dihydrofolate reductase (DHFR) inhibitor.

By preventing dihydrofolate (DHFR) from converting dihydrofolate to tetrahydrofolate (THF), trimethoprim prevents the body from getting folate support [1]. In E. coli, trimethoprim (3 μg/mL; 1 hour) causes significant heat shock proteins (Hsps) and protein aggregation. coli cells, indicating that protein misfolding is brought on by trimethoprim sulfate [1]. Induces the DnaK, DnaJ, GroEL, ClpB, and IbpA/B Hsps of E. coli (1.5–3 μg/mL; 1 h). coli, and the stimulated cells are subjected to heat and folic acid [1].

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Trimethoprim alone had no effect on influenza A virus infection in chick embryos; all embryos died when treated only with TMP. [4]
- In hemagglutination inhibition (HAI) assays, TMP alone showed no HAI effect (scored ++++), similar to virus-only control. [4]

Trimethoprim (10 mg/kg; intravenously; every 12 hours; 3 days) demonstrated antimicrobial activity against H. Protective effect against meningococcal infections, Streptococcus pneumoniae, influenza bacteria, and the like [2]. It can bind to thiomaltose (TM-TMP) and show stability, with an approximate half-life of one hour in complete serum and an approximate MIC value of one micromicrogram against E. coli [2].

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In chick embryo model, trimethoprim alone did not enable embryo survival after influenza virus infection at both 10 EID50 and 100 EID50 viral loads. [4]
- When combined with zinc oxide at an optimal Zn/TMP ratio of 0.18, the combination (Tri-Z) enabled >80% embryo survival even at high viral load (100 EID50). [4]

Influenza virus was isolated from patients and propagated in eggs. We determined viral load that infects 50% of eggs (50% egg lethal dose, ELD50). We introduced 10 ELD50 into embryonated eggs and repeated the experiments using 100 ELD50. A mixture of zinc oxide (Zn) and trimethoprim (TMP) (weight/weight ratios ranged from 0.01 to 0.3, Zn/TMP with increment of 0.1) was tested for embryo survival of the infection (n = 12 per ratio, in triplicates). Embryo survival was determined by candling eggs daily for 7 days. Controls of Zn, TMP, saline or convalescent serum were conducted in parallel. The effect of Tri-Z on virus binding to its cell surface receptor was evaluated in a hemagglutination inhibition (HAI) assay using chicken red cells. Tri-Z was prepared to concentration of 10 mg TMP and 1.8 mg Zn per ml, then serial dilutions were made. HAI effect was expressed as scores where ++++ = no effect; 0 = complete HAI effect.
Results: TMP, Zn or saline separately had no effect on embryo survival, none of the embryos survived influenza virus infection. All embryos treated with convalescent serum survived. Tri-Z, at ratio range of 0.15-0.2 (optimal ratio of 0.18) Zn/TMP, enabled embryos to survive influenza virus despite increasing viral load (> 80% survival at optimal ratio). At concentration of 15 µg/ml of optimal ratio, Tri-Z had total HAI effect (scored 0). However, at clinical concentration of 5 µg/ml, Tri-Z had partial HAI effect (+ +).
Conclusion: Acting on host cells, Tri-Z at optimal ratio can reduce the lethal effect of influenza A virus in chick embryo. Tri-Z has HAI effect. These findings suggest that combination of trimethoprim and zinc at optimal ratio can be provided as treatment for influenza and possibly other respiratory RNA viruses infection in man.[1]

Trimethoprim (TMP), an inhibitor of dihydrofolate reductase, decreases the level of tetrahydrofolate supplying one-carbon units for biosynthesis of nucleotides, proteins, and panthotenate. We have demonstrated for the first time that one of the effects of the TMP action in E. coli cells is protein aggregation and induction of heat shock proteins (Hsps). TMP caused induction of DnaK, DnaJ, GroEL, ClpB, and IbpA/B Hsps. Among these Hsps, IbpA/B were most efficiently induced by TMP and coaggregated with the insoluble proteins. Upon folate stress, deletion of the delta ibpA/B operon resulted in increased protein aggregation but did not influence cell viability.[1]

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A bacterial uptake assay was performed using a fluorescent thiomaltose-perylene conjugate (TM-P) to demonstrate targeting specificity to bacteria over mammalian cells. Uptake in E. coli was 98-fold higher than in macrophages. A lamB mutant E. coli strain (lacking the outer membrane maltodextrin transporter) showed a 2.5-fold decrease in TM-P uptake compared to wild-type, indicating the role of maltodextrin transporters. This assay validates the targeting moiety used in the prodrug but is not a direct assay of trimethoprim. [3]

Animal/Disease Models: Female C3H/HeOuJ mouse (transurethral infection containing 1-2 × 107 in 50 μL suspension of 3% E. coli CFU in isoflurane) [2]
Doses: 10 mg/kg
Route of Administration: intravenous (iv) (iv)injection; once every 12 hrs (hrs (hours)); 3 d
Experimental Results: It has antibacterial activity against Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli and Neisseria meningitidis. The CD50 of infected patients is 150 mg respectively. /kg, 335 mg/kg, 27.5 mg/kg and 8.4 mg/kg mice.

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Animal/Disease Models: Fertilized eggs (H3N2 virus is injected into the amniotic membrane and allantoic cavity on day 8) [4]
Doses: 10 mg/mL; 0.5 mL
Route of Administration: Trimethoprim-zinc composite suspension is injected into the air sac; single dose
Experimental Results: The virus titer was diminished and the survival rate of chicken embryos was improved. Survival rates peaked at a ratio of approximately 0.18 (Zn/trimethoprim).

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Chick embryos (6-day-old, Hamburger-Hamilton stage 29) were used. Trimethoprim was suspended in sterile water at 10 mg/ml. For control groups, TMP alone (5 mg in 0.5 ml saline) was injected into the air sac. One hour later, influenza virus (10 or 100 EID50) was injected into the allantoic space. Embryo survival was monitored daily for 7 days by candling. [4]

Absorption, Distribution and Excretion
Steady-state plasma concentrations are reached approximately 3 days after repeated administration. Following a single 100 mg dose, the mean peak serum concentration is approximately 1 µg/mL (Cmax), with a time to peak concentration (Tmax) ranging from 1 to 4 hours. The pharmacokinetics of trimethoprim follow first-order kinetics; after a single 200 mg dose, the serum concentration is approximately twice that of the 100 mg dose. The steady-state AUC of orally administered trimethoprim is approximately 30 mg/L·h. Approximately 10-20% of ingested trimethoprim is metabolized, primarily in the liver, with the remainder mostly excreted unchanged in the urine. Following oral administration, 50% to 60% of trimethoprim is excreted in the urine within 24 hours, of which approximately 80% is the unchanged drug. After oral administration, trimethoprim is widely distributed in various tissues. It is well distributed in sputum, middle ear effusion, and bronchial secretions. Trimethoprim is effectively distributed in vaginal secretions at concentrations approximately 1.6 times higher than its serum concentration. It may cross the placental barrier and enter breast milk. Trimethoprim is also readily excreted in feces, significantly reducing and/or eliminating fecal flora sensitive to it. The reported renal clearance of trimethoprim after oral administration varies, ranging from 51.7 to 91.3 mL/min. Trimethoprim is widely distributed in tissues and fluids throughout the body, including aqueous humor, middle ear fluid, saliva, lung tissue, sputum, semen, prostate tissue and fluid, vaginal secretions, bile, bones, and cerebrospinal fluid. The apparent volume of distribution of trimethoprim in adults with normal renal function is 100–120 liters. …The binding rate of trimethoprim to plasma proteins is 42–46%. Trimethoprim readily crosses the placenta; it has been reported that the concentration of trimethoprim in amniotic fluid can reach 80% of the maternal serum concentration. Only a small amount of trimethoprim is excreted in feces via bile. Hemodialysis can partially remove trimethoprim. Trimethoprim is readily absorbed from the gastrointestinal tract and is almost completely absorbed. Following a single oral dose of 100, 160, and 200 mg of trimethoprim, peak serum concentrations within 1–4 hours are approximately 1, 1.6, and 2 μg/mL, respectively. After multiple oral doses, steady-state peak serum concentrations of trimethoprim are typically 50% higher than after a single dose. In adults with normal renal function, steady-state serum concentrations after an oral dose of 160 mg of trimethoprim every 12 hours range from 1.2 to 3.2 μg/mL. Trimethoprim is rapidly and extensively distributed in various tissues and fluids, including the kidneys, liver, spleen, bronchial secretions, saliva, and semen. Trimethoprim has also been detected in bile and aqueous humor; bone marrow and cancellous bone, but not compact bone. For more complete data on the absorption, distribution, and excretion of trimethoprim (12 types), please visit the HSDB records page.

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Metabolic/Metabolic Substances
Trimethoprim is oxidatively metabolized to produce a variety of metabolites, the most abundant of which are demethylated 3'- and 4'-metabolites, accounting for approximately 65% and 25% of total metabolite production, respectively. Minor metabolites include N-oxide metabolites (<5%) and less abundant benzyl metabolites. The parent drug is considered to be the therapeutically active form. Biotransformation of trimethoprim mainly involves CYP2C9 and CYP3A4 enzymes, with a smaller contribution from CYP1A2. Trimethoprim is metabolized in the liver to oxidized and hydroxylated metabolites… This study investigated the pharmacokinetics of two groups of six healthy pigs after a single oral administration of either sulfadiazine (SDM) or sulfamethoxazole (SMX) in combination with trimethoprim (TMP) (25 mg + 5 mg/kg body weight). The elimination half-lives of SMX and TMP were very similar (2–3 hours); the half-life of SDM was relatively longer, at 13 hours. Both sulfonamides (S) are primarily metabolized to N4-acetyl derivatives, but to different degrees. The main metabolic pathway for TMP is O-demethylation and subsequent conjugation. Furthermore, plasma concentrations of these drugs and their main metabolites were determined at different feed addition concentrations. The feed drug (S:TMP) concentrations were 250:50, 500:100, and 1000:200 mg/kg. Steady-state concentrations were reached within 48 hours of feed addition, and were achieved with twice-daily (SDM+TMP) or three-daily (SMX+TMP) administration. SDM and its metabolites showed high protein binding (>93%), while SMX, TMP, and their metabolites showed moderate protein binding (48-75%). Adding 500 ppm of sulfonamides and 100 ppm of trimethoprim (TMP) to the feed resulted in a minimum steady-state plasma concentration (C(ss,min)) higher than the concentration required to inhibit the growth of 90% of Actinobacillus pleuropneumoniae strains (n=20).

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Biological half-life
The half-life of trimethoprim is 8-10 hours, but may be prolonged in patients with renal insufficiency.
In adults with normal renal function, the serum half-life of trimethoprim is approximately 8-11 hours. In adults with creatinine clearance of 10-30 or 0-10 ml/min, the serum half-life of the drug may be prolonged to 15 hours or >26 hours, respectively.
The serum half-life of trimethoprim in children under 1 year of age and children aged 1 to 10 years has been reported to be approximately 7.7 hours and 5.5 hours, respectively.

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In humans, the effect of trimethoprim on folic acid metabolism has been described as transient and mostly subclinical. [4]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Due to the very low levels of trimethoprim in breast milk, the amount ingested by infants is minimal, and no adverse effects are expected on breastfed infants.
◉ Effects on Breastfed Infants
In one study, no adverse reactions were observed in infants 4 days after mothers took trimethoprim-sulfamethoxazole.
In a telephone follow-up study, 12 breastfeeding mothers reported taking trimethoprim-sulfamethoxazole (dosage not specified). Two of these mothers reported feeding difficulties in their infants. No cases of diarrhea were reported among the exposed infants.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.
Protein Binding
Trimethoprim binds to 44% of plasma proteins, but the specific proteins it binds to are not yet identified.
In humans, the effects of trimethoprim on DNA synthesis are transient and subclinical. [4]
The efficacy of trimethoprim is limited by its dose-limiting toxicities (not specified). [3]
-In humans, the effects of trimethoprim on folic acid metabolism have been described as transient and mostly subclinical. [3]

[1]. Trimethoprim induces heat shock proteins and protein aggregation in E. coli cells. Curr Microbiol, 2003. 47(4): p. 286-9.

[2]. Trimethoprim: a review of its antibacterial activity, pharmacokinetics and therapeutic use in urinary tract infections. Drugs, 1982. 23(6): p. 405-30.

[3]. A Trimethoprim Conjugate of Thiomaltose Has Enhanced Antibacterial Efficacy In Vivo. Bioconjug Chem. 2018 May 16;29(5):1729-1735.

[4]. El Habbal MH. Combination therapy of zinc and trimethoprim inhibits infection of influenza A virus in chick embryo. Virol J. 2021 Jun 3;18(1):113.

Trimethoprim is an odorless white powder with a bitter taste. (NTP, 1992)
Trimethoprim is an aminopyrimidine antibiotic whose structure consists of a pyrimidine-2,4-diamine and a 1,2,3-trimethoxyphenyl moiety linked by a methylene bridge. It has multiple functions, including as an EC 1.5.1.3 (dihydrofolate reductase) inhibitor, a xenobiotic, an environmental pollutant, a drug allergen, an antibacterial agent, and a diuretic. It belongs to the methoxybenzene and aminopyrimidine classes of compounds.
Trimethoprim is an antifolate antibacterial agent that works by inhibiting bacterial dihydrofolate reductase (DHFR). DHFR is a key enzyme that catalyzes the production of tetrahydrofolate (THF). Trimethoprim inhibits DHFR, thereby preventing bacterial DNA synthesis and ultimately leading to bacterial death. Due to the complementary and synergistic effects of trimethoprim and sulfamethoxazole, it is often used in combination with sulfamethoxazole, but it can also be used alone to treat and/or prevent urinary tract infections. Its structure and chemical properties are related to pyrimethamine (another antifolate-containing antibacterial drug used to treat Plasmodium infection). Trimethoprim is a dihydrofolate reductase inhibitor. Its mechanism of action is as a dihydrofolate reductase inhibitor, a cytochrome P450 2C8 inhibitor, and an organic cation transporter 2 inhibitor. Trimethoprim is a synthetic derivative of trimethoxybenzylpyrimidine and possesses antibacterial and antiprotozoal properties. As a pyrimidine inhibitor of bacterial dihydrofolate reductase, trimethoprim binds tightly to the bacterial enzyme, blocking the conversion of dihydrofolate to tetrahydrofolate. Sulfonamides can enhance its antibacterial activity. (NCI04)
It is a pyrimidine dihydrofolate reductase inhibitor, belonging to the pyrimethamine class of antibacterial drugs. Sulfonamides can enhance its antibacterial activity; trimethoprim/sulfamethoxazole combination preparations are the most commonly used dosage form. It can also sometimes be used alone as an antimalarial drug. There have been reports of trimethoprim resistance. See also: sulfamethoxazole; trimethoprim (ingredient); trimethoprim hydrochloride (salt form); trimethoprim lactate (active ingredient)... See more...

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Drug Indications
Trimethoprim monotherapy is used to treat acute uncomplicated urinary tract infections caused by susceptible bacteria (including Escherichia coli, Klebsiella pneumoniae, etc.). Streptococcus pneumoniae, Enterobacter spp., Proteus mirabilis, and coagulase-negative Staphylococcus spp. are also indicated. Trimethoprim, in combination with sulfamethoxazole, is indicated for the treatment of infections caused by the following bacteria that have been shown to be susceptible to trimethoprim: urinary tract infections, acute otitis media in children (when clinically necessary), acute exacerbations of chronic bronchitis in adults, enteritis caused by susceptible Shigella, prevention and treatment of Pneumocystis pneumonia, and traveler's diarrhea caused by enterotoxigenic Escherichia coli. Trimethoprim is an ophthalmic solution used in combination with polymyxin B to treat acute bacterial conjunctivitis, blepharitis, and palpebral conjunctivitis caused by susceptible bacteria.
FDA Label

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Mechanism of Action
Trimethoprim is a reversible inhibitor of dihydrofolate reductase, one of the main enzymes catalyzing the conversion of dihydrofolate (DHF) to tetrahydrofolate (THF). Tetrahydrofolate is essential for bacterial nucleic acid and protein biosynthesis and ultimately for bacterial survival—therefore, inhibiting its synthesis produces a bactericidal effect. Compared to mammalian dihydrofolate reductase, trimethoprim has a stronger affinity for bacterial dihydrofolate reductase, thus selectively interfering with bacterial biosynthesis. Trimethoprim is often used in combination with sulfamethoxazole, which inhibits a preceding step in bacterial protein synthesis—the combination of trimethoprim and sulfamethoxazole inhibits two consecutive steps in bacterial nucleic acid and protein biosynthesis. Trimethoprim monotherapy is considered a bacteriostatic agent, but its combination with sulfamethoxazole is considered to have bactericidal activity. Trimethoprim is a bacteriostatic, lipophilic, weak base with a structure similar to pyrimethamine. It binds to and reversibly inhibits the bacterial enzyme dihydrofolate reductase, selectively blocking the conversion of dihydrofolate to its functional form, tetrahydrofolate. This depletes folate (an essential cofactor in nucleic acid biosynthesis), thereby interfering with the production of bacterial nucleic acids and proteins. The binding affinity of bacterial dihydrofolate reductase to trimethoprim is approximately 50,000 to 60,000 times stronger than that of the corresponding mammalian enzyme. To determine the incidence and severity of hyperkalemia during trimethoprim treatment, we studied 30 patients with acquired immunodeficiency syndrome (AIDS) receiving high-dose (20 mg/kg/day) trimethoprim; additionally, we investigated the mechanism of trimethoprim-induced hyperkalemia in rats. Despite normal adrenal cortical function and glomerular filtration rate, trimethoprim increased serum potassium concentration by 0.6 mmol/L. In 15 of the 30 patients, serum potassium levels >5 mmol/L were observed during trimethoprim treatment. In rats, intravenous administration of trimethoprim inhibited renal potassium excretion by 40% and increased sodium excretion by 46%. It was concluded that trimethoprim blocked sodium channels on the apical membrane of the distal renal tubules in mammals. As a result, the transepithelial voltage decreased and potassium secretion was inhibited. Due to these direct effects on the renal tubules, the reduction in renal potassium excretion led to a significant proportion of patients receiving trimethoprim-containing drugs developing hyperkalemia. Trimethoprim is a dihydrofolate reductase inhibitor that affects the nuclear DNA and cell wall integrity of bacteria. In humans, its effects are mild and transient. [4] Trimethoprim has been reported to be beneficial against DNA viruses, but ineffective against RNA viruses such as influenza when used alone. [4] When used in combination with zinc at the optimal ratio (0.18 Zn/TMP), trimethoprim inhibited hemagglutination and protected chicken embryos from influenza virus infection, possibly by interfering with the binding of the virus to the host cell sialic acid receptor. [4]

C14H18N4O3

290.32

290.137

C, 57.92; H, 6.25; N, 19.30; O, 16.53

738-70-5

Trimethoprim lactate;23256-42-0;Trimethoprim-d9;1189460-62-5;Trimethoprim-d3;1189923-38-3;Trimethoprim sulfate;56585-33-2;Trimethoprim hydrochloride;60834-30-2;Trimethoprim-13C3;1189970-95-3

5578

White to off-white solid powder

1.3±0.1 g/cm3

405.2±55.0 °C at 760 mmHg

199-203 °C

198.8±31.5 °C

0.0±0.9 mmHg at 25°C

1.600

0.38

2

7

5

21

307

0

IEDVJHCEMCRBQM-UHFFFAOYSA-N

InChI=1S/C14H18N4O3/c1-19-10-5-8(6-11(20-2)12(10)21-3)4-9-7-17-14(16)18-13(9)15/h5-7H,4H2,1-3H3,(H4,15,16,17,18)

5-(3,4,5-Trimethoxybenzyl)pyrimidine-2,4-diamine

Proloprim; Monotrim; Monotrimin; Trimopan; Trimethoprim; Trimpex, Monotrim, Triprim among others

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 : ~50 mg/mL (~172.22 mM)
H2O : ~0.67 mg/mL (~2.31 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.61 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 (8.61 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.61 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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 (Please use freshly prepared in vivo formulations for optimal results.)