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

二氢叶酸还原酶 (DHFR) 将二氢叶酸转化为四氢叶酸 (THF)，它会受到甲氧苄啶的抑制，从而停止叶酸的代谢[1]。甲氧苄啶 (3 μg/mL；1 小时) 在大肠杆菌中诱导热休克蛋白 (Hsps) 和蛋白质聚集。大肠杆菌细胞，表明蛋白质错误折叠是由硫酸甲氧苄啶的存在引起的[1]。当用甲氧苄啶（1.5–3 μg/mL；1 小时）处理时，DnaK、DnaJ、GroEL、ClpB 和 IbpA/B Hsp 在 E 中均可诱导。大肠杆菌细胞受到热应激和叶酸的影响[1]。

甲氧苄啶（10 mg/kg；静脉注射；每 12 小时一次；3 天）在受感染的小鼠中表现出对流感嗜血杆菌、肺炎链球菌、大肠杆菌和脑膜炎奈瑟氏球菌的抗菌作用[2]。甲氧苄啶对大肠杆菌的 MIC 值约为 1 μM，可与硫代麦芽糖 (TM-TMP) 连接。它还表现出稳定性，在全血清中的半衰期约为 1 小时[2]。甲氧苄啶（10 mg/mL；0.5 mL；甲氧苄啶-锌混合悬液注射）可提高鸡胚胎的存活率，同时降低病毒滴度[4]。

从病人身上分离出流感病毒并在鸡蛋中繁殖。我们确定了感染50%鸡蛋的病毒载量(50%鸡蛋致死剂量，ELD50)。在受精卵中引入10个ELD50，用100个ELD50重复实验。用氧化锌(Zn)和甲氧苄氨嘧啶(TMP)的混合物(体重比为0.01 ~ 0.3,Zn/TMP递增0.1)对感染的胚胎存活率进行试验(每比值12个，3个重复)。每天用蜡烛点燃卵子，连续7天测定胚胎存活率。对照组以锌、TMP、生理盐水或恢复期血清为对照。用鸡红细胞进行血凝抑制(HAI)试验，评价了Tri-Z对病毒与其细胞表面受体结合的影响。制备Tri-Z至TMP为10 mg / ml, Zn为1.8 mg / ml，进行连续稀释。HAI效应以分数表示，其中++++ =无影响;0 =完全的HAI效果。 结果:TMP、Zn和生理盐水对胚胎存活无影响，感染流感病毒的胚胎无一存活。用恢复期血清处理的胚胎全部存活。在0.15 ~ 0.2(最佳比例0.18)Zn/TMP范围内，Tri-Z使胚胎在病毒载量增加的情况下存活(最佳比例> 80%)。在最佳配比浓度为15µg/ml时，Tri-Z具有完全HAI效应(0分)，而在临床浓度为5µg/ml时，Tri-Z具有部分HAI效应(+ +)。 结论:适当比例的Tri-Z作用于宿主细胞，可降低甲型流感病毒对鸡胚的致死性。Tri-Z具有HAI效果。这些发现表明，甲氧苄氨嘧啶和锌以最佳比例联合使用可用于治疗流感和其他呼吸道RNA病毒感染。[1]

甲氧苄啶(TMP)是一种二氢叶酸还原酶抑制剂，可降低四氢叶酸的水平，四氢叶酸为核苷酸、蛋白质和泛酸的生物合成提供一碳单位。我们首次证明了TMP在大肠杆菌细胞中的作用之一是蛋白质聚集和诱导热休克蛋白(Hsps)。TMP诱导DnaK、DnaJ、GroEL、ClpB和IbpA/B Hsps。在这些热休克蛋白中，TMP诱导的IbpA/B蛋白最有效，并与不溶性蛋白共聚。在叶酸胁迫下，δ ibpA/B操纵子的缺失导致蛋白质聚集增加，但不影响细胞活力。[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 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.

Route of Elimination
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.

Volume of Distribution
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.

Clearance
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.

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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). Mengelers MF, et al; Vet Res Commun 25 (6): 461-481. 2001.

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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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Interactions
Concomitant admin of trimethoprim or trimethoprim/sulfamethoxazole with methotrexate may incr bone marrow suppression, probably as an additive antifolate effect.

Concurrent use with trimethoprim or use of trimethoprim between courses of other folic acid antagonists, such as methotrexate or pyrimethamine, is not recommended because of the possibility of an increased incidence of megaloblastic anemia.

Trimethoprim may inhibit the metab of phenytoin, increasing the half-life of phenytoin by up to 50% & decreasing its clearance by 30%

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Non-Human Toxicity Values
LD50 Mice oral 7000 mg/kg

LD50 Rat oral 200 mg/kg

LD50 Mouse oral 3960 mg/kg

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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 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 an odorless white powder. Bitter taste. National Toxicology Program, Institute of Environmental Health Sciences, National Institutes of Health (NTP). 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 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.

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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.
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.

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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.

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Therapeutic Uses
Anti-Infective Agents, Urinary; Antimalarials; Antimetabolites; Folic Acid Antagonists

Trimethoprim given alone has also been effective for urinary tract infections, but the development of resistant organisms limits the usefulness of this treatment.

Trimethoprim is indicated in the treatment of initial, uncomplicated urinary tract infections caused by susceptible strains of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Enterobacter species, & coagulase-negative Staphylococcus species, including Staphylococcus saprophyticus. /Included in US product labeling/

trimethoprim is used in the prophylaxis of bacterial urinary tract infections.

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Drug Warnings
Because trimethoprim may interfere with folic acid metabolism, the drug should be used with caution in nursing women.

Adverse GI reactions occur in approximately 6% of patients receiving trimethoprim and may include epigastric discomfort, nausea, vomiting, glossitis, and abnormal taste sensation. Elevations in serum aminotransferase (transaminase) and bilirubin concentrations have been reported in patients receiving the drug, but the clinical importance of these findings is not known. Cholestatic jaundice has been reported rarely.

The most frequent adverse effects of trimethoprim are rash and pruritus. Mild to moderate rashes appearing 7-14 days after initiation of trimethoprim therapy reportedly occur in 2.9-6.7% of patients receiving 200 mg of the drug daily. Rashes are generally maculopapular, morbilliform, and pruritic. Rashes have been reported to occur in up to 24% of patients receiving 400 mg or more trimethoprim for 14 days. Photosensitivity (e.g., erythematous phototoxic eruptions with subsequent hyperpigmentation of sun-exposed skin) also has occurred. Exfoliative dermatitis, toxic epidermal necrolysis (Lyell's syndrome), erythema multiforme, and Stevens-Johnson syndrome have been reported rarely in patients receiving the drug. Anaphylaxis also has occurred rarely.

Safety and efficacy of trimethoprim in infants younger than 2 months of age and efficacy of the drug when used as single agent in children younger than 12 years of age have not been established. Trimethoprim should be used with caution in children who have the fragile X chromosome associated with mental retardation, because folate depletion may worsen the psychomotor regression associated with the disorder.

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Drug Tolerance
Most gram-negative and gram-positive microorganisms are sensitive to trimethoprim, but resistance can develop when the drug is used alone.

Bacterial resistance to trimethoprim-sulfamethoxazole is a rapidly increasing problem, although resistance is lower than it is to either of the agents alone. Resistance often is due to the acquisition of a plasmid that codes for an altered dihydrofolate reductase. The development of resistance is a problem for treatment of many different bacterial infections.

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Pharmacodynamics
Trimethoprim exerts its antimicrobial effects by inhibiting an essential step in the synthesis of bacterial nucleic acids and proteins. It has shown activity against several species of gram-negative bacteria, as well as coagulase-negative _Staphylococcus_ species. Resistance to trimethoprim may arise via a variety of mechanisms, including alterations to the bacterial cell wall, overproduction of dihydrofolate reductase, or production of resistant dihydrofolate reductase. Rarely, trimethoprim can precipitate the development of blood disorders (e.g. thrombocytopenia, leukopenia, etc.) which may be preceded by symptoms such as sore throat, fever, pallor, and or purpura - patients should be monitored closely for the development of these symptoms throught the course of therapy. As antimicrobial susceptibility patterns are geographically distinct, local antibiograms should be consulted to ensure adequate coverage of relevant pathogens prior to use.

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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.

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

163.04

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.47 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (5.47 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (5.47 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2 mg/mL (5.26 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶 (<60°C).

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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