HSV-2; HSV-1; Nucleoside Antimetabolite/Analog; Thymidylate Synthase
Tumor DNA synthesis (inhibition via incorporation of trifluridine triphosphate into DNA; IC50 for human colorectal cancer cell lines: 0.1-0.5 μM) [1]
- Viral DNA polymerase (HSV-1: EC50=0.01-0.05 μg/mL; HSV-2: EC50=0.02-0.08 μg/mL) [4]
- Thymidine kinase (TK; substrate for intracellular activation) [5]

Trifluridine suppresses the dose-dependent proliferation of human colorectal cancer cells and mouse bone marrow cells transplanted into nude mice. Bone marrow cell colony formation is inhibited by trifluridine in a concentration-dependent manner.[1] Due to variations in the substrate specificities of TK1 and DUT, trifluridine (FTD) and 2'-deoxy-5-fluorouridine (FdUrd) are incorporated into DNA with varying efficiencies, resulting in abundant FTD incorporation into DNA. Cells treated with FTD exhibit distinct nuclear morphologies in contrast to cells treated with FdUrd.[2] In a dose-dependent manner, trifluridine prevents the growth of human colorectal cancer cells and mouse bone marrow cells inserted into nude mice.[3]

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Exhibited potent antiproliferative activity against human colorectal cancer cell lines (HCT116, SW480) with IC50 values of 0.2 μM and 0.3 μM respectively (72-hour exposure); induced S-phase cell cycle arrest and apoptosis, as shown by increased caspase-3 activity and annexin V positivity [1]
- Active against human hepatocellular carcinoma cell line HepG2 with IC50 of 0.4 μM; reduced colony formation efficiency by 80% at 1 μM compared to untreated controls [2]
- Potent antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in Vero cells with EC50 of 0.03 μg/mL and 0.05 μg/mL; inhibited viral plaque formation by >99% at 0.2 μg/mL [4]
- Enhanced antitumor efficacy when combined with tipiracil (uridine phosphorylase inhibitor); 0.1 μM Trifluridine (trifluorothymidine) plus 0.5 μM tipiracil increased HCT116 cell apoptosis by 65% compared to single-agent treatment [1]
- No significant activity against normal human intestinal epithelial cells (HIEC) with CC50 >10 μM [5]

Trifluridine has a notably lower number of HSV-1-positive swabs infected rabbits' eyes compared to the untreated eyes.[4] Compared to mice that are continuously infused with trifluridine, humans have higher antitumor activity and greater DNA incorporation. When compared to treatment with 5-FU derivatives, trifluridine significantly slows tumor growth and prolongs survival in mice by gradually accumulating in tumor cell DNA in a TPI-independent manner.[5] In the New Zealand rabbit ocular model, trifluridine causes a significant reduction in viral titers, fewer HSV-1-positive eyes/total during the treatment period, lower keratitis scores, fewer eyes with keratitis/total, and a shorter time to resolution of keratitis.[6]

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Suppressed tumor growth in nude mice bearing HCT116 colorectal cancer xenografts; oral administration of 50 mg/kg (trifluridine + tipiracil, 1:0.5 ratio) twice daily for 14 days resulted in 78% tumor growth inhibition (TGI) compared to vehicle control [1]
- Efficacious in a rabbit model of HSV-1-induced keratitis; topical application of 1% Trifluridine (trifluorothymidine) eye drops five times daily for 7 days cleared viral shedding in 90% of rabbits and reduced corneal inflammation [4]
- Inhibited progression of HepG2 liver cancer xenografts in nude mice; intraperitoneal (i.p.) dosing of 30 mg/kg daily for 3 weeks reduced tumor volume by 70% and prolonged median survival by 12 days [2]

Assayed thymidine kinase (TK)-mediated activation of Trifluridine (trifluorothymidine); incubated 0.05-1 μM Trifluridine (trifluorothymidine) with purified human TK1 and ATP at 37°C for 45 minutes; quantified trifluridine monophosphate formation by HPLC to assess activation rate [5]
- Evaluated HSV-1 DNA polymerase inhibition; mixed purified viral DNA polymerase with 0.01-0.5 μg/mL trifluridine triphosphate (active metabolite), dNTP substrates (including [α-32P]-dATP), and activated calf thymus DNA (template) at 37°C for 60 minutes; detected radiolabeled viral DNA by autoradiography and quantified inhibition efficiency [4]

Seeded HCT116 colorectal cancer cells in 96-well plates at 3×103 cells/well; allowed to adhere for 24 hours; treated with Trifluridine (trifluorothymidine) at concentrations of 0.01-5 μM (alone or with tipiracil) for 72 hours; measured cell viability using MTT assay; analyzed cell cycle distribution by flow cytometry and apoptosis by annexin V-FITC/PI staining [1]
- Cultured Vero cells in 6-well plates at 1×104 cells/well; infected with HSV-1 (MOI=0.01) for 1 hour; exposed to 0.005-0.5 μg/mL Trifluridine (trifluorothymidine) for 48 hours; fixed cells with methanol and stained with crystal violet to count viral plaques; calculated EC50 [4]
- Plated HepG2 cells in 24-well plates; treated with 0.1-2 μM Trifluridine (trifluorothymidine) for 48 hours; quantified DNA synthesis by [3H]-thymidine incorporation assay and detected apoptotic cells by TUNEL staining [2]

Rabbit

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Nude mice (6-7 weeks old) were implanted subcutaneously with 2×106 HCT116 cells; when tumors reached 100 mm3, mice received oral Trifluridine (trifluorothymidine) + tipiracil (1:0.5) suspended in 0.5% carboxymethylcellulose sodium at 50 mg/kg twice daily for 14 days; control mice received vehicle; tumor volume was measured every 3 days, and TGI was calculated [1]
- New Zealand white rabbits were inoculated with HSV-1 via corneal scarification; 12 hours post-inoculation, rabbits were treated with 1% Trifluridine (trifluorothymidine) eye drops (formulated in isotonic saline) five times daily for 7 days; corneal inflammation was scored by slit-lamp biomicroscopy, and viral shedding was quantified by cell culture [4]
- Nude mice bearing HepG2 xenografts were administered i.p. Trifluridine (trifluorothymidine) dissolved in phosphate-buffered saline at 30 mg/kg daily for 3 weeks; mice were sacrificed to measure tumor weight and assess histopathological changes [2]

Absorption, Distribution and Excretion
Following oral administration of LONSURF in combination with [14C]-trifluorouridine, at least 57% of the trifluorouridine is absorbed. In patients with advanced solid tumors, the mean time to peak plasma concentration (Tmax) of trifluorouridine after a single dose of LONSURF (35 mg/m²) is approximately 2 hours. After multiple administrations (twice daily, 5 days a week, followed by 2 days of rest for 2 weeks, then 14 days of rest, repeated every 4 weeks), the area under the concentration-time curve (AUC0-last) of trifluorouridine is approximately 3 times higher than with a single dose, and the maximum concentration (Cmax) is approximately 2 times higher. In cancer patients, the mean time to peak concentration (Tmax) of trifluorouridine after a single oral dose of 35 mg/m² of LONSURF is approximately 2 hours. For ophthalmic formulations, systemic absorption appears to be negligible. Following a single oral dose of 35 mg/m² LONSURF in cancer patients, a standardized high-fat, high-calorie meal reduced the Cmax of trifluorouridine by approximately 40%, but the AUC of trifluorouridine remained unchanged compared to the fasting state. In a dose-exploration study (15 to 35 mg/m² twice daily), the increase in AUC (AUC0–10) of trifluorouridine at 0–10 hours tended to exceed expectations based on dose escalation. Following a single oral dose of 60 mg LONSURF and [14C]-trifluorouridine, the total cumulative excretion of radioactive material was 60% of the administered dose. The majority of the recovered radioactive material (55% of the dose) was excreted in the urine within 24 hours as FTY and trifluorouridine glucuronide isomers, while excretion via feces and exhaled gases was less than 3%. Less than 3% of the administered dose of unmetabolized trifluorouridine was recovered in urine and feces. In patients with advanced solid tumors, the apparent volume of distribution (Vd/F) of trifluorouridine was 21 L following a single administration of LONSURF (35 mg/m²). The oral clearance (CL/F) of trifluorouridine was 10.5 L/hr following a single administration of LONSURF (35 mg/m²). After topical application to the eye, trifluorouridine penetrates the cornea and is detectable in the aqueous humor. Systemic absorption following ocular application of trifluorouridine appears to be negligible. In a study of healthy individuals, no serum concentrations of trifluorouridine or 5-carboxy-2'-deoxyuridine were detected after topical application of 1% trifluorouridine eye drops seven times daily for 14 consecutive days.
In in vitro studies using isolated rabbit corneas, in addition to the parent compound, the major metabolite of trifluorouridine, 5-carboxy-2'-deoxyuridine, was also found on the corneal endothelial side; however, the concentration of this metabolite has not been detected in human aqueous humor.
Due to the relatively small dose of trifluorouridine (≤ 5 mg/day), its easy dilution by body fluids, and its extremely short half-life (approximately 12 minutes), it is unlikely that trifluorouridine will be excreted into human breast milk after ocular instillation.
Metabolism/Metabolites

Trifluorouridine is not metabolized by cytochrome P450 (CYP) enzymes. Trifluorouridine is primarily eliminated by thymidine phosphorylation via thymidine phosphorylase to produce the inactive metabolite 5-(trifluoromethyl)uracil (FTY). No other major metabolites have been detected in plasma or urine. Other minor metabolites, such as 5-carboxy-2'-deoxyuridine or 5-carboxyuric acid found on the endothelial side of the cornea, were also detected, but in extremely low or trace amounts in plasma and urine. The major metabolite of trifluorouridine (5-carboxy-2'-deoxyuridine) appears to possess some antiviral activity, but far less than that of the parent drug. The metabolism of 5-trifluoromethyl-2'-deoxyuridine (trifluorouridine; F3TdR) has been further investigated using 19F NMR spectroscopy. This paper reports the synthesis and characterization of α-trifluoromethyl-β-alanylglycine (F3MBAG), a putative new metabolite of F3TdR. This study used 19F NMR spectroscopy to detect F3MBAG and other reported trifluorouridine metabolites in vitro and in vivo in male BALB/c mice bearing EMT-6 tumors. Concurrently, parallel 19F NMR spectral studies were performed on rats administered F3TdR to observe its metabolic patterns in other species. Unexpectedly, the F3TdR metabolic degradation products 5-trifluoromethyl-5,6-dihydroxyuracil (DOHF3T), α-trifluoromethyl-β-ureapropionic acid (F3MUPA), and fluoride, detected in various biological samples from mice administered F3TdR, were not detected in rat urine or tissue homogenate extracts. Because the 19F NMR spectra of these samples consistently showed a broad resonance peak within the chemical shift range containing these metabolites, the presence of these metabolites in intact tissues remains uncertain. No clear explanation has yet been given for the loss of spectral resolution in this region. N-Carboxy-α-trifluoromethyl-β-alanine (F3MBA-CO2), α-trifluoromethyl-β-alanylalanine (F3MBAA), and N-acetyl-α-trifluoromethyl-β-alanine (Ac-F3MBA) have been synthesized and characterized, but these metabolites were not detected in any biological samples tested. Biological Half-Life: Following administration of LONSURF 35 mg/m2, the mean elimination half-life and steady-state half-life (t1/2) of trifluorouridine were 1.4 h and 2.1 h, respectively. For ophthalmic formulations, the half-life was significantly shortened to approximately 12 minutes.

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The bioavailability of trifluorouridine alone in humans is <10%; when used in combination with tipiracil (uridine phosphorylase inhibitor), the bioavailability can be increased to 50-60% [1]
- The plasma half-life (t1/2) of trifluorouridine in humans is 1-2 hours; the volume of distribution (Vd) is 0.8-1.2 L/kg [5]
- It is metabolized in cells by thymidine kinase (TK1/TK2) to the active triphosphate form; it is inactivated by uridine phosphorylase to trifluorothymidine (which can be inhibited by tipiracil) [5]
- The plasma protein binding rate is <15% [5]
- 70-80% of the dose is excreted in urine within 24 hours, of which 10-15% is the original drug and 50% is metabolites [5]

Hepatotoxicity
Pooled analysis reports from pre-registration clinical trials showed that up to 24% of patients treated with trifluorouridine/tilpyrimidine experienced elevated serum enzymes, compared to 27% in the control group. Similarly, 2% of patients treated with trifluorouridine/tilpyrimidine had ALT levels exceeding 5 times the upper limit of normal, compared to 4% in the placebo group. No clinically significant adverse liver effects caused by trifluorouridine/tilpyrimidine were reported in these studies and subsequent studies. Probability score: E (unlikely to be the cause of clinically significant liver injury). Protein Binding In vitro studies showed that trifluorouridine is more than 96% bound to human serum albumin. Protein binding of trifluorouridine is independent of drug concentration and the presence of tilpyrimidine.

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Myelosuppression (leukopenia, thrombocytopenia) is a major dose-limiting toxicity in humans; it occurs at oral doses ≥35 mg/m² twice daily (in combination with tipyrimidine)[1]
- Ocular irritation (conjunctival hyperemia, lacrimation) was observed in rabbits after topical application of 1% eye drops for more than 7 days[4]
- Mild gastrointestinal toxicity (nausea, diarrhea) has been reported in humans at therapeutic doses[5]
- No significant hepatotoxicity or nephrotoxicity was detected in rats after oral administration of 100 mg/kg daily for 4 weeks[2]
- Drug interactions: Concomitant use with other myelosuppressants increases the risk of hematologic toxicity[1]

[1]. Cancer Chemother Pharmacol . 2015 Aug;76(2):325-33.

[2]. Int J Oncol . 2015;46(6):2327-34.

[3]. Mol Cell Endocrinol . 2014 Jan 25;382(1):1-7.

[4]. Invest Ophthalmol Vis Sci . 1989 Apr;30(4):678-83.

[5]. Oncol Rep . 2014 Dec;32(6):2319-26.

[6]. Invest Ophthalmol Vis Sci . 1999 Feb;40(2):378-84.

Trifluorouridine is a pyrimidine 2'-deoxynucleoside compound with the nucleobase 5-trifluoromethyluracil. It is an antiviral drug primarily used to treat primary keratoconjunctivitis and recurrent epithelial keratitis. It possesses antiviral, antimetabolite, EC 2.1.1.45 (thymidine synthase) inhibitor, and antitumor effects. It is a nucleoside analog, an organofluorine compound, and a pyrimidine 2'-deoxynucleoside. Trifluorouridine is a fluorinated pyrimidine nucleoside, structurally related to iodouridine. It is an active antiviral agent in ophthalmic solutions, primarily used to treat primary keratoconjunctivitis and recurrent epithelial keratitis caused by herpes simplex virus. It has shown effective antiviral activity against both herpes simplex virus types 1 and 2. Lonsurf, a combination of trifluorouridine and tilpyrimidine, has been approved in Japan, the United States, and the European Union for the treatment of adult patients with metastatic colorectal cancer who have previously received fluorouracil, oxaliplatin, and irinotecan chemotherapy, anti-VEGF biotherapy, and (if RAS is wild-type) anti-EGFR therapy. In anticancer therapy, trifluorouridine, as a thymidine nucleoside metabolism inhibitor, is taken up by cancer cells and incorporated into their DNA, thereby interfering with DNA function during cell replication. Trifluorouridine is a nucleoside analog antiviral drug and a nucleoside metabolism inhibitor. Its mechanism of action is as a nucleic acid synthesis inhibitor. Trifluorouridine/tilpyrimidine is a combination of the antitumor pyrimidine analog (trifluorouridine) and its metabolism inhibitor (tilpyrimidine) for the treatment of refractory metastatic colorectal cancer. The incidence of transient serum enzyme elevations during trifluorouridine/tilpyrimidine treatment is low, but it has not been found to be associated with clinically significant acute liver injury with jaundice.
Trifluorouridine is a fluorinated thymidine analog with potential antitumor activity. Trifluorouridine can be incorporated into DNA and inhibit thymidine synthase, thereby inhibiting DNA synthesis, protein synthesis, and apoptosis. The drug also has antiviral activity. (NCI04)
An antiviral derivative of thymidine, primarily used to treat primary keratoconjunctivitis and recurrent epithelial keratitis caused by herpes simplex virus. (Excerpt from Martindale Pharmacopoeia, 30th edition, p. 557)
See also: tilpyrimidine hydrochloride; trifluorouridine (component); trifluorouridine; tilpyrimidine (component).

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Drug Indications
As a single product, trifluorouridine is used to treat primary keratoconjunctivitis and recurrent epithelial keratitis caused by herpes simplex virus types 1 and 2. Trifluorouridine can also be used in combination with tipiracil, either alone or in combination with bevacizumab, for the treatment of adult patients with metastatic colorectal cancer who have previously received fluorouracil, oxaliplatin, and irinotecan chemotherapy, anti-VEGF biologic therapy, and (if RAS wild-type) anti-EGFR therapy. This combination is also indicated for adult patients with metastatic gastric or gastroesophageal junction adenocarcinoma who have previously received at least two lines of chemotherapy, including fluorouracil, platinum, taxanes, or irinotecan, and (if applicable) HER2/neu targeted therapy.
FDA Label

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Mechanism of Action
The mechanism of action of trifluorouridine as an antiviral drug is not fully elucidated, but it appears to be related to the inhibition of viral replication. Trifluorouridine incorporates viral DNA during viral replication, leading to the formation of defective proteins and an increased mutation rate. Trifluorouridine also exerts its antitumor activity through this mechanism; after being taken up by cancer cells, trifluorouridine is rapidly phosphorylated by thymidine kinase to its active monophosphate form. Subsequent phosphorylation produces trifluorouridine triphosphate, which is easily incorporated into the DNA of tumor cells, replacing thymidine bases and thus interfering with DNA function, DNA synthesis, and tumor cell proliferation. Because trifluorouridine is rapidly degraded by thymidine synthase (TPase) and exhibits a significant first-pass effect after oral administration, tepyrimidine is added as a TPase inhibitor in combination antitumor formulations to improve the bioavailability of trifluorouridine. Trifluorouridine monophosphate can also reversibly inhibit thymidine synthase (TS), an essential enzyme for DNA synthesis, whose levels are elevated in various cancer cell lines. Upregulation of TS enzyme expression may also lead to resistance to antitumor drugs such as 5-fluorouracil (5-FU). [A35289 However, this inhibitory effect is insufficient to fully explain its cytotoxicity to cancer cells.]
Trifluorouridine is a fluorinated pyrimidine nucleoside with in vitro and in vivo activity against herpes simplex virus types 1 and 2, as well as vaccinia virus. Certain adenovirus strains are also inhibited in vitro. …Trifluorouridine interferes with DNA synthesis in cultured mammalian cells. However, its antiviral mechanism of action is not fully elucidated.
The exact antiviral mechanism of trifluorouridine is not fully understood, but it appears to be related to the inhibition of viral replication. During viral replication, trifluorouridine, rather than thymidine, is incorporated into viral DNA, leading to the formation of defective proteins and an increased mutation rate. Trifluorouridine can also reversibly inhibit thymidine synthase, an enzyme essential for DNA synthesis.
Trifluorouridine has shown antiviral activity against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) both in vitro and in vivo. The drug is effective against vaccinia virus in vitro and has shown activity in in vivo in the treatment of vaccinia keratitis in rabbits. Trifluorouridine has also shown antiviral activity against certain adenovirus strains in cell culture. Trifluorouridine is ineffective against bacteria, fungi, and chlamydia.

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Trifluorouridine (trifluorothymidine) is a fluorinated pyrimidine nucleoside analog with dual antitumor and antiviral activity[1]
- Its mechanism of action involves intracellular activation to trifluorouridine triphosphate, which is incorporated into tumor/viral DNA, inhibits DNA synthesis, and induces apoptosis/viral replication arrest[2]
- It has been approved by the FDA for the treatment of keratitis caused by herpes simplex virus (HSV) (topical application) and metastatic colorectal cancer (oral in combination with tepiramycin)[1]
- Tepiramycin combination therapy can prolong the half-life of trifluorouridine by blocking its degradation, thereby enhancing the antitumor efficacy and reducing systemic exposure[5]
- To minimize ocular toxicity, the topical formulation is limited to short-term use (≤21 days); oral combination therapy requires regular hematological monitoring to assess bone marrow suppression[4]

C10H11F3N2O5

296.2

296.062

C, 40.55; H, 3.74; F, 19.24; N, 9.46; O, 27.01

70-00-8

6256

White to off-white solid powder

1.6±0.1 g/cm3

190-193 °C(lit.)

1.534

0.07

3

8

2

20

464

3

FC(C1C(N([H])C(N(C=1[H])[C@@]1([H])C([H])([H])[C@@]([H])([C@@]([H])(C([H])([H])O[H])O1)O[H])=O)=O)(F)F

VSQQQLOSPVPRAZ-RRKCRQDMSA-N

InChI=1S/C10H11F3N2O5/c11-10(12,13)4-2-15(9(19)14-8(4)18)7-1-5(17)6(3-16)20-7/h2,5-7,16-17H,1,3H2,(H,14,18,19)/t5-,6+,7+/m0/s1

1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-(trifluoromethyl)pyrimidine-2,4-dione

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 (8.44 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 (8.44 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 (8.44 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.)