DHFR/Dihydrofolate reductase; Influenza A virus

By preventing dihydrofolate reductase (DHFR) from functioning, trimethoprim disrupts the metabolism of folate and converts dihydrofolate to tetrahydrofolate (THF) [1]. In E. coli, trimethoprim (3 μg/mL; 1 h) causes significant heat shock proteins (Hsps) and protein aggregation. coli cells, indicating that protein misfolding is brought on by trimethoprim sulfate [1]. E. coli produces DnaK, DnaJ, GroEL, ClpB, and IbpA/B Hsps when treated with trimethoprim (1.5–3 μg/mL; 1 hour). Coli cells subjected to heat stress and folate [1].

Trimethoprim (10 mg/kg; IV; every 12 hours; 3 days) demonstrated antibacterial activity against Neisseria meningitidis, Escherichia coli, Haemophilus influenzae, and Streptococcus pneumoniae in infected mice[2]. Trimethoprim has a half-life of about an hour in full serum and a MIC value of about 1 μM against E. coli. It can be associated with thiomaltose (TM-TMP) and shows stability. Coli [2]. The injection of trimethoprim-zinc mixed suspension (10 mg/mL; 0.5 mL) lowers virus titers and increases the survival rate of chicken embryos [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]

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

---

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
Steady-state plasma concentrations are reached approximately 3 days after repeated administration. Following a single 100 mg dose, the mean peak serum concentration (Cmax) is reached within 1 to 4 hours (Tmax), approximately 1 µg/mL. The pharmacokinetics of trimethoprim appear to follow first-order kinetics; after a single 200 mg dose, serum concentrations are approximately twice that of the 100 mg dose. The steady-state AUC of oral trimethoprim is approximately 30 mg/L·h.
Elimination Route
Approximately 10-20% of the ingested dose of trimethoprim is metabolized, primarily in the liver, with the remainder largely 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.
Volume of Distribution
After oral administration, trimethoprim is widely distributed in various tissues. It is well distributed in sputum, middle ear effusion, and bronchial secretions. Trimethoprim is efficiently distributed in vaginal secretions at concentrations approximately 1.6 times higher than its serum concentration. It can 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.
Clearance
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; its concentration in amniotic fluid has been reported to be approximately 80% of the maternal serum concentration.
Only a small amount of trimethoprim is excreted in the bile and feces. Hemodialysis can partially remove trimethoprim.
Trimethoprim is readily absorbed from the gastrointestinal tract and is almost completely absorbed. After a single oral dose of 100 mg, 160 mg, and 200 mg of trimethoprim, the peak serum concentrations within 1–4 hours are approximately 1 μg/mL, 1.6 μg/mL, and 2 μg/mL, respectively. After multiple oral doses, the steady-state peak serum concentration of trimethoprim is typically 50% higher than that after a single dose. In adults with normal renal function, the steady-state serum concentration range after an oral dose of 160 mg of trimethoprim every 12 hours is 1.2–3.2 μg/mL.
Trimethoprim is rapidly and widely distributed in various tissues and fluids, including the kidneys, liver, spleen, bronchial secretions, saliva, and semen. Trimethoprim is also present in bile and aqueous humor; bone marrow and cancellous bone, but not compact bone.

---

View More

Metabolism/Metabolites
Trimethoprim is oxidatively metabolized to produce various metabolites, the most abundant of which are demethylated 3'- and 4'-metabolites, accounting for approximately 65% and 25% of total metabolite production, respectively. Minor products include N-oxide metabolites (<5%) and less abundant benzyl metabolites. The parent drug is considered to be the therapeutically active form. The 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 sulfadimidine (SDM) or sulfamethoxazole (SMX) combined with trimethoprim (TMP) (25 mg + 5 mg/kg body weight) after a single oral administration to six healthy pigs in two groups. 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, the 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, respectively. Steady-state concentrations were reached within 48 hours after feed addition, and steady-state concentrations were also achieved with twice-daily (SDM+TMP) or three-daily (SMX+TMP) administration. SDM and its metabolites have high protein binding rates (>93%), while SMX, TMP, and their metabolites have moderate protein binding rates (48-75%). Adding 500 ppm sulfonamides and 100 ppm trimethoprim (TMP) to the feed resulted in minimum steady-state plasma concentrations (C(ss,min)) higher than those required to inhibit the growth of 90% of Actinobacillus pleuropneumoniae strains (n=20). Mengelers MF et al.; Vet Res Commun 25 (6): 461-481. 2001.

---

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 ml/min or 0–10 ml/min, the serum half-life of this drug may be prolonged to 15 hours or >26 hours, respectively. It has been reported that the serum half-life of trimethoprim is approximately 7.7 hours in children under 1 year of age and approximately 5.5 hours in children aged 1 to 10 years.

Effects during pregnancy and lactation
◉ Overview of medication use during lactation
Due to the low levels of trimethoprim in breast milk, the amount ingested by infants is very small, 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 lactating mothers reported taking trimethoprim-sulfamethoxazole (dosage not specified). Two of these mothers reported feeding difficulties in their infants. No diarrhea was reported in exposed infants.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

---

Drug interactions
Concomitant use of trimethoprim or trimethoprim/sulfamethoxazole with methotrexate may increase myelosuppression, possibly due to the additive antifolate effects.
Concomitant use of trimethoprim or between courses of treatment with other folic acid antagonists (such as methotrexate or pyrimethamine) is not recommended, as it may increase the incidence of megaloblastic anemia.
Trimethoprim may inhibit the metabolism of phenytoin, prolonging its half-life by up to 50% and reducing its clearance by 30%.

---

Non-human toxicity values
Oral LD50 in mice: 7000 mg/kg
Oral LD50 in rats: 200 mg/kg
Oral LD50 in mice: 3960 mg/kg

---

Protein binding
Trimethoprim binds to 44% of plasma proteins, but the specific proteins it binds to have not been identified.

[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.

See also: Trimethoprim (with active ingredient).

C14H18N4O3.HCL

326.77866

326.115

60834-30-2

Trimethoprim;738-70-5;Trimethoprim lactate;23256-42-0;Trimethoprim-d3;1189923-38-3;Trimethoprim sulfate;56585-33-2;Trimethoprim-13C3;1189970-95-3

173769

White to off-white solid

526ºC at 760 mmHg

271.9ºC

1.919

3

7

5

22

307

0

Cl.COC1=CC(CC2=CN=C(N)N=C2N)=CC(OC)=C1OC

YLCCEQZHUHUYPA-UHFFFAOYSA-N

InChI=1S/C14H18N4O3.ClH/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);1H

5-[(3,4,5-trimethoxyphenyl)methyl]pyrimidine-2,4-diamine;hydrochloride

TRIMETHOPRIM HYDROCHLORIDE; Trimethoprim HCl; Trimplex; Primsol; 60834-30-2; Trimplex 200; UNII-9XE000OU9B; 9XE000OU9B;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (~153.0 mM; with ultrasonication and warming by heating to 60°C)

Solubility in Formulation 1: ≥ 5 mg/mL (15.30 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 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 DMSO stock solution (50.0 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and 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.

---

Solubility in Formulation 2: ≥ 5 mg/mL (15.30 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 DMSO stock solution (50.0 mg/mL) to 900 μL of 20% SBE-β-CD saline and mix well.
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.

---

Solubility in Formulation 3: ≥ 5 mg/mL (15.30 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 DMSO stock solution (50.0 mg/mL) to 900 μL of corn oil and mix well.

---

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