DHFR/Dihydrofolate reductase; Influenza A virus

通过阻止二氢叶酸还原酶 (DHFR) 发挥作用，甲氧苄啶会破坏叶酸的代谢，并将二氢叶酸转化为四氢叶酸 (THF) [1]。在大肠杆菌中，甲氧苄啶（3 μg/mL；1 小时）会导致显着的热休克蛋白 (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引起的[1]。用甲氧苄啶（1.5–3 μg/mL；1 小时）处理时，大肠杆菌会产生 DnaK、DnaJ、GroEL、ClpB 和 IbpA/B Hsp。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脑膜炎奈瑟菌、大肠杆菌、流感嗜血杆菌和肺炎链球菌的抗菌活性[2]。甲氧苄啶在全血清中的半衰期约为 1 小时，针对大肠杆菌的 MIC 值约为 1 μM。它可以与硫代麦芽糖（TM-TMP）结合并表现出稳定性。大肠杆菌[2]。注射甲氧苄啶-锌混合悬液（10 mg/mL；0.5 mL）可降低病毒滴度并提高鸡胚胎的存活率[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 (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.

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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
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. 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 (has active moiety).

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

Typically exists as solid at room temperature

526ºC at 760 mmHg

271.9ºC

1.919

106.97

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

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。