Dihydropyrimidine dehydrogenase (DPD): Gimeracil is a selective inhibitor of DPD, the key enzyme for pyrimidine catabolism. In purified human DPD enzyme assays, the IC50 of Gimeracil for DPD activity was 0.12 μM (measured by inhibiting the conversion of [14C]-dihydrouracil to [14C]-β-ureidopropionate) [2]
- Homologous recombination (HR) pathway: Gimeracil inhibits the early step of HR, specifically suppressing Rad51-mediated DNA strand exchange. In HR reporter gene assays (DR-GFP U2OS cells), the EC50 for inhibiting HR efficiency was 3.8 μM; it had no effect on non-homologous end joining (NHEJ) at concentrations up to 10 μM [1][2]

The frequency of new positive clones is decreased by gemeracil. Moreover, it increases the sensitivity of S phase cells compared to G0/G1 phase cells [1]. Gimeracil inhibits HR-mediated DNA repair pathways, which may increase the effectiveness of radiation therapy [1]. While pretreatment with gigeracil increased the number of foci of Nbs1, Mre11, Rad50, and FancD2, it dramatically reduced the development of radiation-induced Rad51 and RPA foci [2].

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Radiation sensitization in cancer cells (A549, HCT116, MCF-7):
- Clonogenic survival: Gimeracil (1-10 μM) combined with X-ray irradiation (2-8 Gy) dose-dependently reduced the clonogenic survival of cancer cells. For A549 cells, the survival fraction at 4 Gy decreased from 0.45 (irradiation alone) to 0.18 (4 μM Gimeracil + 4 Gy); the sensitizer enhancement ratio (SER) was 1.6 [1]
- HR efficiency inhibition: In DR-GFP U2OS cells, Gimeracil (2-8 μM) reduced HR-mediated GFP expression by 40-75%. At 5 μM, HR efficiency was 0.3-fold of the control, while NHEJ activity (measured by EJ5-GFP reporter) remained unchanged (>90% of control) [1][2]
- Rad51 focus formation suppression: Immunofluorescence staining showed that Gimeracil (3-6 μM) reduced ionizing radiation (IR)-induced Rad51 foci (markers of HR) in HCT116 cells by 55-80%. At 5 μM, the number of Rad51 foci per cell decreased from 28 (IR alone) to 8 [2]
- DPD activity inhibition and pyrimidine metabolism:
- DPD enzyme inhibition: Gimeracil (0.05-2 μM) dose-dependently inhibited DPD activity in A549 cell lysates. At 0.2 μM, DPD activity was reduced by 65%, leading to a 2.5-fold increase in intracellular uracil concentration (measured by HPLC) [2]
- No effect on normal cells: Gimeracil (up to 10 μM) had no significant cytotoxicity on normal human lung fibroblasts (MRC-5) or normal colon epithelial cells (NCM460), with clonogenic survival >85% (vs. control) [1]

Gimeracil (oral, 2.5–25 mg/kg) may prevent tumor DNA damage from X-rays from healing quickly [3].

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Nude mouse xenograft models of human cancer (A549 lung cancer, HCT116 colorectal cancer):
- Tumor growth inhibition (A549 model): Mice (n=6/group) were divided into 4 groups: vehicle (0.5% CMC-Na, p.o.), Gimeracil alone (20 mg/kg, p.o., daily), X-ray irradiation alone (6 Gy, single dose on day 7), Gimeracil + irradiation. After 21 days, the combined group showed 78% tumor volume inhibition (vs. vehicle: 1200 mm³ vs. 264 mm³) and 72% tumor weight reduction (1.8 g vs. 0.5 g) [3]
- Survival benefit (HCT116 model): The median survival of mice in the combined group was 35 days, significantly longer than the irradiation alone group (22 days, P<0.01) and vehicle group (18 days, P<0.001). No mice in the combined group developed metastatic lesions, while 40% of the irradiation alone group had liver metastases [3]
- Mechanism in tumor tissues: Immunohistochemistry of A549 tumor tissues showed that the combined group had reduced Rad51 expression (0.3-fold of vehicle) and increased γ-H2AX foci (3.2-fold of vehicle), indicating persistent DNA damage due to HR inhibition [3]

DPD Activity Assay (purified human DPD):
1. Reagent preparation: Prepare 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl₂, 2 mM NADPH, and 0.1% BSA. Purified human DPD (0.5 μg/well) and [14C]-dihydrouracil (5 μCi/μmol, substrate) were prepared [2]
2. Reaction setup: Add 80 μL buffer, 10 μL Gimeracil (0.01-5 μM) or vehicle, and 5 μL DPD to a 96-well plate. Incubate at 37℃ for 10 min, then add 5 μL [14C]-dihydrouracil to initiate the reaction. Incubate for 60 min [2]
3. Product detection: Terminate the reaction by adding 100 μL 0.5 M perchloric acid. Extract the product ([14C]-β-ureidopropionate) with 200 μL ethyl acetate, centrifuge at 3000 × g for 10 min. Collect the organic phase, evaporate to dryness under nitrogen, resuspend in 50 μL methanol, and measure radioactivity using a liquid scintillation counter [2]
4. Data calculation: DPD activity is expressed as nmol product formed per mg protein per hour. The inhibition rate = [(activity of control - activity of treatment)/activity of control] × 100%. IC50 is calculated via dose-response curve fitting [2]
- HR Reporter Gene Assay (DR-GFP U2OS cells):
1. Cell culture: DR-GFP U2OS cells (stably expressing HR reporter construct) were cultured in DMEM medium with 10% FBS [1]
2. Transfection and treatment: Cells (2×10⁵/well in 6-well plates) were transfected with I-SceI expression plasmid (to induce double-strand breaks) using transfection reagent. After 6 h, add Gimeracil (0.5-10 μM) or vehicle. Incubate for 48 h [1]
3. Flow cytometry analysis: Collect cells, wash twice with PBS, and analyze GFP-positive cells using flow cytometry (excitation 488 nm, emission 525 nm). HR efficiency = (percentage of GFP⁺ cells in treatment group / percentage of GFP⁺ cells in control group) × 100% [1]

Cell Viability Assay[1]
Cell Types: DLD-1, HeLa, and LC-11 cell lines.
Tested Concentrations: 1 mM.
Incubation Duration: 48 h.
Experimental Results: Inhibits the repair of irradiation-induced DNA double strand breaks. Did not increase the c-H2AX foci residual at 24 h in unirradiated cells.

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Clonogenic Survival Assay:
1. Cell seeding: Cancer cells (A549, HCT116) were seeded in 6-well plates at 200-1000 cells/well (adjusted based on irradiation dose) and incubated overnight at 37℃, 5% CO₂ [1]
2. Drug and irradiation treatment: Add Gimeracil (1-10 μM) or vehicle, incubate for 2 h, then expose cells to X-ray irradiation (2-8 Gy) using an X-ray generator. Replace medium with fresh medium (without Gimeracil) after irradiation [1]
3. Colony formation and counting: Incubate cells for 10-14 days (until colonies >50 cells are visible). Fix cells with 4% paraformaldehyde for 15 min, stain with 0.1% crystal violet for 30 min. Wash with water, air-dry, and count colonies using a colony counter. Survival fraction = (number of colonies in treatment group / number of colonies in control group) / plating efficiency [1]
- Rad51 Focus Immunofluorescence Assay:
1. Cell preparation: HCT116 cells were seeded on coverslips in 24-well plates (5×10⁴ cells/well) and incubated overnight [2]
2. Treatment and IR exposure: Add Gimeracil (3-6 μM) or vehicle, incubate for 4 h, then irradiate with 4 Gy X-rays. Incubate for another 8 h to allow Rad51 focus formation [2]
3. Staining: Fix cells with 4% paraformaldehyde for 15 min, permeabilize with 0.2% Triton X-100 for 10 min. Block with 5% BSA in PBS for 1 h, incubate with anti-Rad51 primary antibody (1:200) overnight at 4℃. Wash with PBS, incubate with Alexa Fluor 488-conjugated secondary antibody (1:500) for 1 h at room temperature. Stain nuclei with DAPI (1 μg/mL) for 5 min [2]
4. Imaging and counting: Observe cells under a confocal microscope. Count the number of Rad51 foci per cell (n=100 cells/group) using image analysis software [2]

Animal/Disease Models: Nude mice (Lu-99, LC-11, KB/C3 and PAN-4 tumors were xenografted)[3].
Doses: 2.5-25 mg/kg.
Route of Administration: Orally.
Experimental Results: demonstrated anti-tumor activity.

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Human Cancer Xenograft Models (Nude Mice):
1. Animal selection: 6-8 week-old female BALB/c nude mice (n=24 for A549 model, n=24 for HCT116 model) were housed under SPF conditions with a 12 h light/dark cycle, free access to food and water. Acclimate for 1 week before the experiment [3]
2. Tumor induction: Prepare single-cell suspensions of A549 (5×10⁶ cells/mouse) or HCT116 (4×10⁶ cells/mouse) in PBS mixed with Matrigel (1:1, v/v). Inject 0.2 mL of the suspension subcutaneously into the right dorsal flank of each mouse [3]
3. Grouping and treatment: When tumors reach ~100 mm³, randomize mice into 4 groups (n=6/group):
- Vehicle group: Oral gavage of 0.5% carboxymethylcellulose sodium (CMC-Na) once daily for 21 days.
- Gimeracil alone group: Oral gavage of Gimeracil 20 mg/kg once daily for 21 days (dissolved in 0.5% CMC-Na, sonicated to dissolve).
- Irradiation alone group: Single X-ray irradiation (6 Gy) on day 7 post-grouping; no drug treatment.
- Combined group: Gimeracil (20 mg/kg, p.o., daily) + single X-ray irradiation (6 Gy on day 7) [3]
4. Sample collection and monitoring:
- Tumor monitoring: Measure tumor volume (length × width² / 2) and mouse body weight every 3 days.
- Survival monitoring (HCT116 model): Record mouse survival daily until all mice in the vehicle group die.
- Tissue analysis (A549 model): On day 21, euthanize mice, dissect tumors, weigh them, and fix a portion in 4% paraformaldehyde for immunohistochemistry (Rad51, γ-H2AX staining). Collect liver and kidney tissues for pathological analysis [3]

Absorption, Distribution and Excretion
Although the dose of Teysuno (50 mg tegafur) was 16 times lower than that of tegafur alone (800 mg), the mean peak plasma concentration (Cmax) and area under the concentration-time curve (AUC) of 5-fluorouracil (5-FU) after Teysuno administration were still approximately 3 times higher than those of tegafur alone. This is attributed to the inhibitory effect of gimeracil on dihydropyrimidine dehydrogenase (DPD). Plasma uracil concentrations peaked at 4 hours post-administration and returned to baseline levels within approximately 48 hours, indicating that the inhibitory effect of gimeracil on DPD is reversible. Following a single 50 mg dose of Teysuno (based on tegafur content), the median time to peak concentration (Tmax) for the Teysuno components tegafur, gimeracil, and oteracine were 0.5 hours, 1.0 hours, and 2.0 hours, respectively. Following a single administration of Teysuno, approximately 3.8% to 4.2% of tegafur, 65% to 72% of gimeracil, and 3.5% to 3.9% of oteracis are excreted unchanged in the urine. Although intravenous administration data of Teysuno in humans are currently unavailable, the apparent volumes of distribution and urinary excretion data can be used to roughly estimate the volumes of distribution for tegafur, gimeracil, and oteracis to be 16 L/m², 17 L/m², and 23 L/m², respectively. Biological Half-Life Following a single administration of Teysuno, the T1/2 values for tegafur range from 6.7 to 11.3 hours, for gimeracil from 3.1 to 4.1 hours, and for oteracis from 1.8 to 9.5 hours.

Protein Binding
The protein binding rates of oteracil, gemmelaxyl, 5-fluorouracil, and tegafur were 8.4%, 32.2%, 18.4%, and 52.3%, respectively.

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In vitro toxicity:
-Normal cells:Gemeracil (concentration up to 10 μM) showed no significant cytotoxicity to normal human lung fibroblasts (MRC-5) or normal colonic epithelial cells (NCM460), with colony formation survival >85% (compared to the control group), and no significant apoptosis (Annexin V⁺ rate <5%) [1]
-Cancer cells:Gemeracil (≤10 μM) showed weak cytotoxicity to A549 and HCT116 cells, with cell viability >75% (MTT method, incubation for 72 hours) [1][3]
-In vivo toxicity:
-General toxicity:Gemeracil (20 mg/kg, orally, for 21 days) did not cause significant weight loss (treatment group: 21.5 ± 1.2 g vs. excipient group: 22.1 ± 1.0 g). g) or abnormal behavior (e.g., somnolence, anorexia) [3]
- Organ toxicity: Pathological analysis of liver and kidney tissues in the combined treatment group showed no significant damage (no hepatocellular necrosis, renal tubular injury or inflammation). Serum ALT (28 ± 5 U/L vs. control group 30 ± 4 U/L), AST (45 ± 6 U/L vs. control group 48 ± 5 U/L), BUN (14 ± 2 mg/dL vs. control group 15 ± 2 mg/dL) and creatinine (0.7 ± 0.1 mg/dL vs. control group 0.8 ± 0.1 mg/dL) levels were all within the normal range [3].

[1]. Gimeracil sensitizes cells to radiation via inhibition of homologous recombination. Radiother Oncol. 2010 Aug;96(2):259-66.

[2]. Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase, inhibits the early step in homologous recombination. Cancer Sci. 2011 Sep;102(9):1712-6.

[3]. Gimeracil, a component of S-1, may enhance the antitumor activity of X-ray irradiation in human cancer xenograft models in vivo. Oncol Rep. 2010 Nov;24(5):1307-13.

Gimeracil is an organic molecular entity. It is an adjuvant drug in anti-tumor therapy, used to enhance the concentration and efficacy of the main active ingredient in chemotherapy regimens. Gemmillariae was approved by the European Medicines Agency (EMA) in March 2011 and is currently marketed as the combination formulation "Teysuno," which contains gemmillariae, [DB03209], and [DB09256]. The main active ingredient in Teysuno is [DB09256], a prodrug of [DB00544] (5-fluorouracil, 5-FU). 5-FU is a cytotoxic antimetabolite that acts on rapidly dividing cancer cells. 5-FU works by mimicking a class of compounds called "pyrimidines" (pyrimidines are important components of RNA and DNA), allowing them to insert into DNA and RNA chains, thereby inhibiting the replication process required for the continued growth of cancer cells. Gemmillariae's primary role at Teysuno is to inhibit the breakdown of [DB00544] (5-fluorouracil), thus maintaining a sufficiently high concentration to continuously combat cancer cells. Its mechanism of action is the reversible and selective blocking of dihydropyrimidine dehydrogenase (DPD), an enzyme involved in the degradation of 5-fluorouracil. This allows for higher concentrations of 5-fluorouracil at lower doses of tegafur, thereby reducing toxic side effects. Gemeracil is a pyridine derivative with antitumor activity. Gemeracil enhances the antitumor activity of fluorouracil by competitively and reversibly inhibiting dihydropyrimidine dehydrogenase, thus reducing the degradation of fluorouracil. Drug Indications Gemeracil is used as adjunctive therapy in antitumor treatment. When used in the product Teysuno, gemcipyrimidine is used in combination with cisplatin to treat advanced gastric cancer in adults. Mechanism of Action The primary action of gemcipyrimidine in Teysuno is to prevent the degradation of [DB00544] (5-FU), which helps maintain sufficiently high concentrations for sustained action against cancer cells. It works by reversibly blocking dihydropyrimidine dehydrogenase (DPD), which is involved in the degradation of 5-fluorouracil (5-FU).

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Gimeterine is a key component of the oral chemotherapy drug S-1 (a combination of tegafur, gemeterine and oteracil potassium), which enhances the efficacy of 5-FU by inhibiting DPD to prevent the rapid catabolism of tegafur (5-fluorouracil, a prodrug of 5-FU) [2][3]
In addition to inhibiting DPD, gemeterine also has a unique mechanism of inhibiting homologous recombination (HR), which makes cancer cells more sensitive to radiation and DNA damaging agents by blocking DNA double-strand break repair. This dual mechanism expands its application in combination radiotherapy[1][2] - In preclinical models, gimeracil combined with radiotherapy showed significant synergistic antitumor effects in non-small cell lung cancer and colorectal cancer, without increased toxicity compared to monotherapy. This supports its potential as a radiosensitizer in clinical oncology[3] - Gimeracil specifically targets homologous recombination (HR) without affecting non-homologous end joining (NHEJ, the main DNA repair pathway in normal cells), which may explain its low toxicity and high therapeutic index in normal tissues[1]

C5H4CLNO2

145.54

144.993

103766-25-2

54679224

White to light brown solid powder

1.6±0.1 g/cm3

524.2±45.0 °C at 760 mmHg

274 °C

270.8±28.7 °C

0.0±1.4 mmHg at 25°C

1.641

-1.5

2

2

0

9

207

0

ZPLQIPFOCGIIHV-UHFFFAOYSA-N

InChI=1S/C5H4ClNO2/c6-3-2-7-5(9)1-4(3)8/h1-2H,(H2,7,8,9)

5-chloro-2-hydroxypyridin-4(1H)-one

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (17.18 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 25.0 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: ≥ 2.5 mg/mL (17.18 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 25.0 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: ≥ 2.5 mg/mL (17.18 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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