生物活性

Fluorouracil is a potent antitumor agent that interferes with DNA synthesis via noncompetitive inhibition of thymidylate synthase (TS). The interruption of this enzyme blocks the synthesis of pyrimidine thymidine, a nucleotide required for DNA replication. Fluorouracil Induces apoptosis in human primary and metastatic colon adenocarcinoma cell lines in vitro. It has also been shown to arrest the cell cycle at the G2 phase. Fluorouracil acts as an antimetabolite to uracil. Research in mice has shown that fluorouracil administration may lead to potential degeneration of the CNS by damage to oligodendrocytes responsible for production of myelin sheaths. Metabolized to form fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine (FUTP). FdUMP inhibits thymidylate synthase, causing a reduction in dTMP synthesis. FUTP and FdUTP are misincorporated into RNA and DNA respectively. 5-Fluorouracil (5-FU), a broad spectrumcytotoxic agent, is used clinical treatment for solid tumors and other cancersand initiates apoptosis by targeting thymidylate synthase (TS) and directincorporation of 5-FU metabolites into DNA and RNA.

In vitro cytotoxicity of 5-FU

体外研究

体内研究

PBS

激酶实验

Inhibition of thymidylate synthase[7]

Cells were seeded at densities of 1.5 x 10⁶ in each well of 6-well plates, and after 24hr were treated with concentrations of5-FU (FUra) from 12.5 to 125μM for 1hr at 37 ^oC. Plates were washed with 3 x 3 mL HBSS, and incubatedat 37 ^oC for a further 72 hr. Cells were harvested, as described, andinitial and residual thymidylate synthase activities were determined at varioustimes as described above.

细胞实验

Cellculture[8]

The human breast cancer cell line MCF-7were cultured in RPMI-1640 with 10 % newborn bovine serum, 100 U/mL ofpenicillin, and 100 mg/mL of streptomycin in a humidified atmosphere with 5 %CO₂ at 37 ^oC.

Cellviability assay

Cells were seeded in 96-well microplate for24 h and treated with various concentrations of 5-Fu for 48 h. Then 20μLof MTT solution (5 mg/mL) was added to each well and incubated at 37^oCfor 4 h. The purple crystals were dissolved in 150μL of DMSO.Optical absorbance was determined at 490nm with a microplate reader. Eachtreatment was performed in sextuplicate and each experiment was repeated threetimes. Cell survival ratio was calculated using Atreated/Acontrol 9

100 %, where Atreated and Acontrol were theabsorbance of treated and control cells after 48 h incubation, respectively. Calculatethe half-maximal inhibitory concentration (IC50) of 5-Fu in MCF-7 cells. TheIC50 value was defined as the concentration that caused 50 % inhibition of cellproliferation.

动物实验

Animals[2]

A total number of 140 Swiss mice (female,25–30 g) were housed in cages with free access to food and water. All animalswere kept under a 12 h: 12h light-dark cycle (lights on at 6:00 a.m.).

Assayof Antitumor Activity and Toxicological Study

Ten-day-old Sarcoma 180 ascites tumor cells(2 × 10⁶ cell/500μl) were implanted subcutaneously into the lefthind groin of the mice. One day after inoculation, piplartine or piperine (50mg/ kg) alone or combined with 5-FU (10 mg /kg) was dissolved in 10% DMSO andadministered intraperitoneally for 7 days in mice transplanted with sarcoma 180tumor and in healthy mice.  5-FU (10 mg/kg) was used as a positive control. The negative control was injected with 10%DMSO or 0.9% NaCl. On day 8, the mice were killed. Tumors, livers, spleens andkidneys were extirpated, weighed and fixed in 10% formaldehyde. The tumorinhibition ratio (%) was calculated by the following formula: inhibition ratio(%) = [(A-B)/A] × 100, where A is the mean tumor weight of the negativecontrol, and B is that of the treated group. Body weights were measured at thestart and at the last day of treatment. Peripheral blood was obtained byretro-orbital venous plexus sampling with a heparinized capillary tube fromhealthy mice or mice transplanted with sarcoma 180 tumor and used for hematologicaland biochemical analyses.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Yang L, Wang MJ, Zhang ZJ, et al. Synthesis of novel spin-labeled derivatives of 5-FU as potential antineoplastic agents. Med Chem Res. 2014;23(7):3269-3273.
[2] Bezerra DP, de Castro FO, Alves AP, et al. In vitro and in vivo antitumor effect of 5-FU combined with piplartine and piperine. J Appl Toxicol. 2008;28(2):156-163.
[3] El-Hammadi MM, Delgado AV, Melguizo C, Prados JC, Arias JL. Folic acid-decorated and PEGylated PLGA nanoparticles for improving the antitumour activity of 5-fluorouracil. Int J Pharm. 2017;516(1-2):61-70.
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体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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