Dihydrofolate reductase, Bacteria[1] Influenza A virus[4]

Dihydrofolase reductase (DHFR), which converts dihydrofolate to tetrahydrofolate (THF), is inhibited by trimethoprim, which stops the metabolism of folate[1]. Heat shock proteins (Hsps) and protein aggregation are induced in E. coli by trimethoprim (3 μg/mL; 1 h). coli cells, suggesting that protein misfolding is caused by the presence of trimethoprim sulfate[1]. DnaK, DnaJ, GroEL, ClpB, and IbpA/B Hsps are all inducible in E when treated with trimethoprim (1.5–3 μg/mL; 1 h). Coli cells subjected to heat stress and folate[1].

Trimethoprim (10 mg/kg; iv; once every 12 h; 3 d) demonstrates antibacterial action in infected mice against H. influenzae, S. pneumoniae, E. coli, and N. meningitidis[2]. Trimethoprim has a MIC value of approximately 1 μM against E. coli and can be linked to thiomaltose (TM-TMP). It also exhibits stability with a half-life of approximately 1 hour in full serum[2]. Trimethoprim (10 mg/mL; 0.5 mL; inject with Trimethoprim-Zn mixed suspension) raises the survival rate of chicken embryos while lowering the viral titer[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 (transurethrally infected with a 50 μL suspension containing 1-2×107 CFU of E. coli under 3% isoflurane)[2]
Doses: 10 mg/kg
Route of Administration: iv; once every 12 h; for 3 d
Experimental Results: demonstrated antibacterial activity against H. influenzae , S. pneumoniae, E. coli and N. meningitidis with CD50s of 150 mg/kg, 335 mg/kg, 27.5 mg/kg and 8.4 mg/kg, respectively in infected mice.

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Animal/Disease Models: Fertilized eggs (injected H3N2 virus into amniotic and allantoic space at day 8)[4]
Doses: 10 mg/mL; 0.5 mL
Route of Administration: The Trimethoprim-Zn combined suspension was injected into the air sac; single dosage
Experimental Results: diminished the virus titer and increased the survival rate of chicken embryo. The survival rate peaked at ratio about 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.

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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.

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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 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.

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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%.

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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

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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 Oct;47(4):286-9.

[2]. Trimethoprim: A Review of Its Antibacterial Activity, Pharmacokinetics and Therapeutic Use in Urinary Tract Infections. Drugs. 1982 Jun;23(6):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 a tasteless white powder with a bitter taste. National Toxicology Program (NTP), Institute of Environmental Health Sciences, National Institutes of Health, 1992. National Toxicology Program Chemical Repository Database. Research Triangle Park, North Carolina.

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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, exogenous substance, environmental pollutant, drug allergen, antibacterial agent, and diuretic. It belongs to the methoxybenzene and aminopyrimidine classes of compounds.
Trimethoprim is an antifolate antibacterial agent that inhibits bacterial dihydrofolate reductase (DHFR), a key enzyme catalyzing the production of tetrahydrofolate (THF). By inhibiting DHFR, trimethoprim can prevent bacterial DNA synthesis, ultimately inhibiting bacterial survival. Because trimethoprim and sulfamethoxazole have complementary and synergistic effects, trimethoprim is often used in combination with sulfamethoxazole, but it can also be used alone to treat and/or prevent urinary tract infections. The structure and chemical properties of trimethoprim are similar to pyrimethamine, another antifolate-resistant antibacterial agent 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. Trimethoprim is a pyrimidine inhibitor of dihydrofolate reductase and belongs to the pyrimethamine class of antibacterial drugs. Sulfonamides can enhance their antibacterial activity, and trimethoprim-sulfamethoxazole combination formulations are the most commonly used dosage forms. It can also sometimes be used alone as an antimalarial drug. Trimethoprim resistance has been reported.

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Drug Indications
Trimethoprim, as monotherapy, is indicated for the treatment of acute uncomplicated urinary tract infections caused by susceptible bacteria, including Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Proteus mirabilis, and coagulase-negative staphylococci. Trimethoprim, in combination with sulfamethoxazole in various formulations, is indicated for the treatment of infections caused by bacteria to which it has been shown to be susceptible: urinary tract infections, acute otitis media in children (when clinically indicated), acute exacerbations of chronic bronchitis in adults, enteritis caused by susceptible Shigella, prevention and treatment of Pneumocystis carinii pneumonia, and traveler's diarrhea caused by enterotoxigenic Escherichia coli. Trimethoprim is an ophthalmic solution used in combination with polymyxin B for the treatment of acute bacterial conjunctivitis, blepharitis, and palpebral conjunctivitis caused by susceptible bacteria.

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Therapeutic Uses
Anti-infective, urinary tract; antimalarial; antimetabolite; folic acid antagonist
Trimethoprim alone is also effective for urinary tract infections, but the emergence of drug-resistant bacteria limits the effectiveness of this treatment.
Trimethoprim is indicated for the treatment of primary, uncomplicated urinary tract infections caused by trimethoprim-sensitive Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Enterobacter spp., and coagulase-negative staphylococci (including saprophytic staphylococci). (This information is included on the US product label.)
Trimethoprim is used for the prevention of bacterial urinary tract infections.

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Drug Warnings
Because trimethoprim may interfere with folic acid metabolism, breastfeeding women should use this medication with caution.
Approximately 6% of patients taking trimethoprim experience gastrointestinal adverse reactions, which may include upper abdominal discomfort, nausea, vomiting, glossitis, and dysgeusia. Elevated serum transaminase and bilirubin levels have been reported in patients taking this medication, but the clinical significance of these findings is unclear. Cholestatic jaundice has been rarely reported.
The most common adverse reactions to trimethoprim are rash and pruritus. Mild to moderate rashes have been reported in 2.9% to 6.7% of patients taking 200 mg of trimethoprim daily, 7 to 14 days after starting treatment. The rash is typically a maculopapular rash, measles-like, and accompanied by pruritus. Rash has been reported in up to 24% of patients receiving 400 mg or more of trimethoprim for 14 days. Photosensitivity reactions (e.g., erythematous phototoxic rash followed by hyperpigmentation of sun-exposed areas) have also been reported. Rare cases of exfoliative dermatitis, toxic epidermal necrolysis (Lyer's syndrome), erythema multiforme, and Stevens-Johnson syndrome have been reported in patients treated with this drug. Allergic reactions are also rare. The safety and efficacy of trimethoprim in infants under 2 months of age, and its efficacy as a monotherapy in children under 12 years of age, have not been established. Trimethoprim should be used with caution in children with Fragile X syndrome (associated with intellectual disability) because folic acid deficiency may exacerbate psychomotor developmental regression associated with this condition. Drug Tolerance: Most Gram-negative and Gram-positive bacteria are sensitive to trimethoprim, but resistance may develop when used alone. Although resistance to trimethoprim-sulfamethoxazole is lower than resistance to either drug alone, bacterial resistance to combination formulations is rapidly increasing. Drug resistance is often due to the acquisition of plasmids encoding altered dihydrofolate reductase. The development of resistance is a challenge in the treatment of many different bacterial infections.

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Pharmacodynamics
Trimethoprim exerts its antibacterial effect by inhibiting a key step in bacterial nucleic acid and protein synthesis. It shows activity against a wide range of Gram-negative bacteria and coagulase-negative staphylococci. Resistance to trimethoprim can develop through various mechanisms, including alterations to the bacterial cell wall, overproduction of dihydrofolate reductase, or the development of resistant dihydrofolate reductase. In rare cases, trimethoprim can induce hematologic disorders (e.g., thrombocytopenia, leukopenia), which may initially present with symptoms such as sore throat, fever, pallor, and/or purpura—patients should be closely monitored for these symptoms throughout treatment. Due to regional variations in antimicrobial susceptibility patterns, the local antimicrobial spectrum should be consulted before use to ensure adequate coverage against relevant pathogens. 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, allowing it to selectively interfere with bacterial biosynthesis. Trimethoprim is often used in combination with sulfamethoxazole, which inhibits the first step in bacterial protein synthesis—the combination of sulfamethoxazole and trimethoprim inhibits two consecutive steps in bacterial nucleic acid and protein biosynthesis. Trimethoprim is considered a bacteriostatic agent as a monotherapy, but is considered to have bactericidal activity when used in combination with sulfamethoxazole.
Trimethoprim is a bacteriostatic, lipophilic, weak base with a structure related to pyrimethamine. It binds to and reversibly inhibits bacterial 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 bacterial nucleic acid and protein synthesis. Trimethoprim binds to bacterial dihydrofolate reductase with approximately 50,000 to 60,000 times stronger force than it binds to the corresponding mammalian enzyme. To determine the incidence and severity of hyperkalemia during trimethoprim treatment, we studied 30 patients with acquired immunodeficiency syndrome who received consecutive high-dose (20 mg/kg/day) trimethoprim; furthermore, this study investigated the mechanism by which trimethoprim induces hyperkalemia in rats. Despite normal adrenal cortical function and glomerular filtration rate, trimethoprim increased serum potassium concentration by 0.6 mmol/L. Of the 30 patients treated with trimethoprim, 15 developed serum potassium levels >5 mmol/L during treatment. In rats, intravenous administration of trimethoprim inhibited renal potassium excretion by 40% and increased sodium excretion by 46%. The study concluded that trimethoprim blocks sodium channels on the apical membrane of the distal renal tubules in mammals. Therefore, the transepithelial voltage decreases, and potassium secretion is inhibited. Due to the direct effect of trimethoprim on the renal tubules, reduced renal potassium excretion leads to a significant proportion of patients receiving trimethoprim-containing medications developing hyperkalemia.

C17H24N4O6

380.40

380.169

C, 53.68; H, 6.36; N, 14.73; O, 25.23

23256-42-0

Trimethoprim;738-70-5;Trimethoprim-d3;1189923-38-3;Trimethoprim sulfate;56585-33-2;Trimethoprim hydrochloride;60834-30-2;Trimethoprim-13C3;1189970-95-3

3084396

White to off-white solid powder

526ºC at 760mmHg

271.9ºC

3.74E-11mmHg at 25°C

1.871

4

10

6

27

366

0

IIZVTUWSIKTFKO-UHFFFAOYSA-N

InChI=1S/C14H18N4O3.C3H6O3/c1-19-10-5-8(6-11(20-2)12(10)21-3)4-9-7-17-14(16)18-13(9)15;1-2(4)3(5)6/h5-7H,4H2,1-3H3,(H4,15,16,17,18);2,4H,1H3,(H,5,6)

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

TRIMETHOPRIM LACTATE; Trimethoprim lactate salt; 5-(3,4,5-Trimethoxybenzyl)pyrimidine-2,4-diamine 2-hydroxypropanoate; Trimethoprim lactic Acid; Trimethoprim (lactate); MLS000069832; MFCD00171722;

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 : 250 mg/mL (657.20 mM)
H2O : 16.67 mg/mL (43.82 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.47 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 20.8 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: ≥ 2.08 mg/mL (5.47 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 20.8 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: ≥ 2.08 mg/mL (5.47 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2 mg/mL (5.26 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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