DHFR/Dihydrofolate reductase; Influenza A virus

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].

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].

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]

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).

Absorption, Distribution and Excretion
Steady-state concentrations are achieved after approximately 3 days of repeat administration. Average peak serum concentrations of approximately 1 µg/mL (Cmax) are achieved within 1 to 4 hours (Tmax) following the administration of a single 100mg dose. Trimethoprim appears to follow first-order pharmacokinetics, as a single 200mg dose results in serum concentrations approximately double that of a 100mg dose. The steady-state AUC of orally administered trimethoprim is approximately 30 mg/L·h.
Approximately 10-20% of an ingested trimethoprim dose is metabolized, primarily in the liver, while a large portion of the remainder is excreted unchanged in the urine. Following oral administration, 50% to 60% of trimethoprim is excreted in the urine within 24 hours, approximately 80% of which is unchanged parent drug.
Trimethoprim is extensively distributed into various tissues following oral administration. It distributes well into sputum, middle ear fluid, and bronchial secretions. Trimethoprim distributes efficiently into vaginal fluids, with observed concentrations approximately 1.6-fold higher than those seen in the serum. It may pass the placental barrier and into breast milk. Trimethoprim is also sufficiently excreted in the feces to markedly reduce and/or eliminate trimethoprim-susceptible fecal flora.
Following oral administration, the renal clearance of trimethoprim has been variably reported between 51.7 - 91.3 mL/min.
Trimethoprim is widely distributed into body tissues & fluids including the aqueous humor, middle ear fluid, saliva, lung tissue, sputum, seminal fluid, prostatic tissue & fluid, vaginal secretions, bile, bone, & /cerebrospinal fluid/. The apparent volume of distribution of trimethoprim in adults with normal renal function ranges from 100-120 l. ... Trimethoprim is 42-46% bound to plasma proteins. Trimethoprim readily crosses the placenta, & amniotic fluid concns are reported to be 80% of concurrent maternal serum concns.
Only small amounts of trimethoprim are excreted in feces via biliary elimination. Trimethoprim may be moderately removed by hemodialysis.
Trimethoprim is readily & almost completely absorbed from the GI tract. Peak serum concns of approx 1, 1.6, & 2 ug/ml are reached in 1-4 hr after single 100-, 160-, & 200 mg oral doses of trimethoprim. Following multiple-dose oral admin, steady-state peak serum concns of trimethoprim usually are 50% greater than those obtained after single-dose admin of the drug. Steady-state serum concns range from 1.2-3.2 ug/ml following oral admin of 160 mg of trimethoprim every 12 hr in adults with renal function.
Rapidly and widely distributed to various tissues and fluids, including kidneys, liver, spleen, bronchial secretions, saliva, and seminal fluid. Trimethoprim has also been demonstrated in bile; aqueous humor; bone marrow and spongy, but not compact, bone.
For more Absorption, Distribution and Excretion (Complete) data for TRIMETHOPRIM (12 total), please visit the HSDB record page.

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Metabolism / Metabolites
Trimethoprim undergoes oxidative metabolism to a number of metabolites, the most abundant of which are the demethylated 3'- and 4'- metabolites, accounting for approximately 65% and 25% of the total metabolite formation, respectively. Minor products include N-oxide metabolites (<5%) and benzylic metabolites in even smaller quantities. The parent drug is considered to be the therapeutically active form. The majority of trimethoprim biotransformation appears to involve CYP2C9 and CYP3A4 enzymes, with CYP1A2 contributing to a lesser extent.
Trimethoprim is metabolized in the liver to oxide and hydroxylated metabolites ... .
The pharmacokinetics were studied of sulfadimethoxine (SDM) or sulfamethoxazole (SMX) in combination with trimethoprim (TMP) administered as a single oral dose (25 mg + 5 mg/kg bw) to 2 groups of 6 healthy pigs. The elimination half-lives of SMX & TMP were quite similar (2-3 hr); SDM had a relatively long half-life of 13 hr. Both sulfonamides (S) were exclusively metabolized to N4-acetyl derivatives but to different extents. The main metabolic pathway for TMP was O-demethylation & subsequent conjugation. In addition, the plasma concns of these drugs & their main metabolites after medication with different in-feed concns were determined. The drug (S:TMP) concns in the feed were 250:50, 500:100, & 1000:200 mg/kg. Steady-state concns were achieved within 48 hr of feed medication, twice daily (SDM+TMP) or 3 times/day (SMX+TMP). Protein binding of SDM & its metabolite was high (>93%), whereas SMX, TMP & their metabolites showed moderate binding (48-75%). Feed medication with 500 ppm sulfonamide combined with 100 ppm TMP provided minimum steady-state plasma concns (C(ss,min)) higher than the concn required for inhibition of the growth of 90% of Actinobacillus pleuropneumoniae strains (n=20).

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Biological Half-Life
Trimethoprim half-life ranges from 8-10 hours, but may be prolonged in patients with renal dysfunction.
Trimethoprim has a serum half-life of approx 8-11 hr in adults with normal renal function. In adults with creatinine clearances of 10-30 or 0-10 ml/min, serum half-life of the drug may incr to 15 hr or >26 hr, respectively. Trimethoprim serum half-lives of about 7.7 & 5.5 hr have been reported in children <1 yr of age & between 1 & 10 yr of age, respectively.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Because of the low levels of trimethoprim in breastmilk, amounts ingested by the infant are small and would not be expected to cause any adverse effects in breastfed infants.
◉ Effects in Breastfed Infants
In one study, no adverse effects were noted in infants during 4 days of maternal therapy with co-trimoxazole.
In a telephone follow-up study, 12 nursing mothers reported taking co-trimoxazole (dosage unspecified). Two mothers reported poor feeding in their infants. Diarrhea was not reported among the exposed infants.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Trimethoprim is 44% bound to plasma proteins, though the specific proteins to which it binds have not been elucidated.

[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. Bitter taste. (NTP, 1992)
Trimethoprim is an aminopyrimidine antibiotic whose structure consists of pyrimidine 2,4-diamine and 1,2,3-trimethoxybenzene moieties linked by a methylene bridge. It has a role as an EC 1.5.1.3 (dihydrofolate reductase) inhibitor, a xenobiotic, an environmental contaminant, a drug allergen, an antibacterial drug and a diuretic. It is a member of methoxybenzenes and an aminopyrimidine.
Trimethoprim is an antifolate antibacterial agent that inhibits bacterial dihydrofolate reductase (DHFR), a critical enzyme that catalyzes the formation of tetrahydrofolic acid (THF) - in doing so, it prevents the synthesis of bacterial DNA and ultimately continued bacterial survival. Trimethoprim is often used in combination with [sulfamethoxazole] due to their complementary and synergistic mechanisms but may be used as a monotherapy in the treatment and/or prophylaxis of urinary tract infections. It is structurally and chemically related to [pyrimethamine], another antifolate antimicrobial used in the treatment of plasmodial infections.
Trimethoprim is a Dihydrofolate Reductase Inhibitor Antibacterial. The mechanism of action of trimethoprim is as a Dihydrofolate Reductase Inhibitor, and Cytochrome P450 2C8 Inhibitor, and Organic Cation Transporter 2 Inhibitor.
Trimethoprim is a synthetic derivative of trimethoxybenzyl-pyrimidine with antibacterial and antiprotozoal properties. As a pyrimidine inhibitor of bacterial dihydrofolate reductase, trimethoprim binds tightly to the bacterial enzyme, blocking the production of tetrahydrofolic acid from dihydrofolic acid. The antibacterial activity of this agent is potentiated by sulfonamides. (NCI04)
A pyrimidine inhibitor of dihydrofolate reductase, it is an antibacterial related to PYRIMETHAMINE. It is potentiated by SULFONAMIDES and the TRIMETHOPRIM, SULFAMETHOXAZOLE DRUG COMBINATION is the form most often used. It is sometimes used alone as an antimalarial. TRIMETHOPRIM RESISTANCE has been reported.
See also: Sulfamethoxazole; Trimethoprim (component of); Trimethoprim Hydrochloride (has salt form); Trimethoprim lactate (is active moiety of) ... View More ...

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Drug Indication
As a monotherapy, trimethoprim is indicated for the treatment of acute episodes of uncomplicated urinary tract infections caused by susceptible bacteria, including _E. coli._, _K. pneumoniae_, _Enterobacter spp._, _P. mirabilis_, and coagulase-negative _Staphylococcus_ species. In various formulations in combination with [sulfamethoxazole], trimethoprim is indicated for the following infections caused by bacteria with documented susceptibility: urinary tract infections, acute otitis media in pediatric patients (when clinically indicated), acute exacerbations of chronic bronchitis in adults, enteritis caused by susceptible _Shigella_, prophylaxis and treatment of _Pneumocystis jiroveci_ pneumonia, and travelers' diarrhea caused by enterotoxigenic _E. coli_. Trimethoprim is available as an ophthalmic solution in combination with [polymyxin B] for the treatment of acute bacterial conjunctivitis, blepharitis, and blepharoconjunctivitis caused by susceptible bacteria.
FDA Label

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Mechanism of Action
Trimethoprim is a reversible inhibitor of dihydrofolate reductase, one of the principal enzymes catalyzing the formation of tetrahydrofolic acid (THF) from dihydrofolic acid (DHF). Tetrahydrofolic acid is necessary for the biosynthesis of bacterial nucleic acids and proteins and ultimately for continued bacterial survival - inhibiting its synthesis, then, results in bactericidal activity. Trimethoprim binds with a much stronger affinity to bacterial dihydrofolate reductase as compared to its mammalian counterpart, allowing trimethoprim to selectively interfere with bacterial biosynthetic processes. Trimethoprim is often given in combination with sulfamethoxazole, which inhibits the preceding step in bacterial protein synthesis - given together, sulfamethoxazole and trimethoprim inhibit two consecutive steps in the biosynthesis of bacterial nucleic acids and proteins. As a monotherapy trimethoprim is considered bacteriostatic, but in combination with sulfamethoxazole is thought to exert bactericidal activity.
Trimethoprim is a bacteriostatic lipophilic weak base structurally related to pyrimethamine. It binds to and reversibly inhibits the bacterial enzyme dihydrofolate reductase, selectively blocking conversion of dihydrofolic acid to its functional form, tetrahydrofolic acid. This depletes folate, an essential cofactor in the biosynthesis of nucleic acids, resulting in interference with bacterial nucleic acid and protein production. Bacterial dihydrofolate reductase is approximately 50,000 to 60,000 times more tightly bound by trimethoprim than is the corresponding mammalian enzyme.
To determine the incidence & severity of hyperkalemia during trimethoprim therapy, 30 consecutive patients with acquired immunodeficiency syndrome receiving high-dose (20 mg/kg/day) trimethoprim were studied; in addition, the mechanism of trimethoprim-induced hyperkalemia was investigated in rats. Trimethoprim increased serum potassium concn by 0.6 mmol/l despite normal adrenocortical function & glomerular filtration rate. Serum potassium levels >5 mmol/l were observed during trimethoprim treatment in 15 of 30 patients. In rats, iv trimethoprim inhibited renal potassium excretion by 40% & increased sodium excretion by 46%. It was concluded that trimethoprim blocks apical membrane sodium channels in the mammalian distal nephron. As a consequence, the transepithelial voltage is reduced & potassium secretion is inhibited. Decreased renal potassium excretion secondary to these direct effects on kidney tubules leads to hyperkalemia in a substantial number of patients being treated with trimethoprim-containing drugs.

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.)