thymidine phosphorylase
The primary molecular target of tipiracil is thymidine phosphorylase (TPase), an enzyme involved in pyrimidine metabolism that catalyzes the phosphorolysis of thymidine to thymine. Tipiracil acts as a potent and competitive inhibitor of TPase, with an IC50 of 35 nM for human placental TPase. The compound shows high selectivity for TPase over other pyrimidine-metabolizing enzymes, including uridine phosphorylase (UPase), orotate phosphoribosyltransferase (OPRTase), thymidine kinase (TK), and dihydropyrimidine dehydrogenase (DPDase), with IC50 values >1 mM for these enzymes. By inhibiting TPase, tipiracil prevents the catabolism of trifluridine, a thymidine analog that is incorporated into DNA and causes DNA dysfunction. This inhibition increases the plasma concentration and exposure of trifluridine, enhancing its anticancer activity.

Thymidine phosphorylase is inhibited by the drug tipiracil. Tipiracil increases trifluridine exposure by preventing thymidine phosphorylase from metabolizing the drug. A brand-new oral treatment for metastatic colorectal cancer combines trifluridine and piracil[2]. As a thymidine phosphorylase inhibitor, tipiracil has a first-pass effect that prevents FTD from degrading[3].
In vitro studies demonstrate that tipiracil potently inhibits thymidine phosphorylase activity. The compound shows an IC50 of 35 nM for human placental TPase and is selective for TPase over other pyrimidine-metabolizing enzymes. In enzyme assays, tipiracil inhibits the phosphorolysis of thymidine to thymine, preventing the degradation of thymidine and its analogs. In combination with trifluridine, tipiracil significantly increases the half-life and exposure of trifluridine in cellular assays. The combination of trifluridine and tipiracil (TAS-102) shows synergistic anticancer activity in cancer cell lines, with trifluridine being incorporated into DNA and causing DNA damage, while tipiracil ensures sustained trifluridine concentrations. The combination is effective against a range of cancer cell lines, including those resistant to fluoropyrimidines.

In vivo studies of tipiracil are conducted in the context of TAS-102 (trifluridine/tipiracil combination). In mouse xenograft models of colorectal and gastric cancer, TAS-102 demonstrates significant antitumor activity, including tumor growth inhibition and, in some cases, tumor regression. The combination is effective in tumors that are resistant to other chemotherapeutic agents, including fluorouracil, oxaliplatin, and irinotecan. In vivo, tipiracil prevents the rapid degradation of trifluridine, maintaining its therapeutic concentrations and enhancing its anticancer efficacy. The combination is typically administered orally once or twice daily. Pharmacodynamic endpoints include measurement of trifluridine incorporation into DNA, tumor growth inhibition, and survival prolongation. TAS-102 is FDA-approved for clinical use in metastatic colorectal cancer and gastric cancer.

For TPase inhibition assays, the enzymatic activity of thymidine phosphorylase is measured using a spectrophotometric or HPLC-based method. Purified human placental TPase is incubated with varying concentrations of tipiracil (typically 0.001-100 µM) in assay buffer (50 mM potassium phosphate, pH 7.4, containing 1 mM EDTA and 1 mM DTT) at 37°C for 10 minutes. The reaction is initiated by the addition of thymidine (1-5 mM) and incubated for 30-60 minutes. The reaction is terminated by heat inactivation or acidification. Thymine production is measured by HPLC or by following the decrease in absorbance at 300 nm (for thymidine) or increase at 290 nm (for thymine). IC50 values are calculated from dose-response curves. For selectivity assays, tipiracil is tested against other pyrimidine-metabolizing enzymes, including uridine phosphorylase (using uridine as substrate), orotate phosphoribosyltransferase, thymidine kinase, and dihydropyrimidine dehydrogenase, using similar assay conditions with appropriate substrates.

HeLa cells are plated in triplicate in 96-well plates at a density of 500 cells/180 μL/well and pre-cultured for 24 hours before receiving 20 μL of each drug solution for 24 or 72 hours. Following the 24 h treatment, cells are washed with phosphate-buffered saline (PBS) before being added to each well with drug-free medium and being incubated for an additional 48 h. A Cell Counting Kit-8 is used to assess the inhibition of cell growth. SAS[3] is used to determine the 50% inhibitory concentration (IC50) values from the absorbance data.
For in vitro combination studies, cancer cells (e.g., colorectal, gastric, or pancreatic cancer cell lines) are cultured in appropriate medium (RPMI-1640 or DMEM) with 10% FBS and antibiotics. Cells are seeded in 96-well plates at densities of 5,000-10,000 cells per well. Trifluridine and tipiracil are dissolved in DMSO and diluted in culture medium to achieve desired concentrations. Trifluridine is tested alone and in combination with tipiracil (at a fixed ratio of 1:0.5, mimicking the clinical formulation) at varying concentrations (0.001-100 µM). After 48-96 hours of treatment, cell viability is assessed by MTT, CCK-8, or CellTiter-Glo assays. Combination index values are calculated using the Chou-Talalay method to assess synergy. For mechanistic studies, DNA damage (γ-H2AX), cell cycle distribution, and apoptosis are assessed by Western blot and flow cytometry. Trifluridine incorporation into DNA is measured by mass spectrometry or using radiolabeled trifluridine.

For in vivo efficacy studies, 6-8 week old female immunodeficient mice (e.g., nude or SCID) are used. Mice are subcutaneously implanted with 5 × 10⁶ cancer cells (colorectal or gastric cancer lines) in the flank. When tumors reach approximately 100-200 mm³, animals are randomized into treatment groups (n = 6-10 per group). TAS-102 (trifluridine/tipiracil combination) is formulated as a suspension in 0.5% methylcellulose or other suitable vehicle and administered orally once or twice daily at doses of 50-200 mg/kg/day (based on the combination). Treatment is given for 2-4 weeks. Tumor volumes are measured twice weekly using calipers. Body weights are monitored for toxicity. At study termination, tumors are excised, weighed, and processed for histopathology, trifluridine incorporation analysis, and biomarker assessment. Blood samples may be collected for pharmacokinetic analysis.

Absorption, Distribution and Excretion
Tilpirimidine is primarily absorbed via the gastrointestinal tract. Following a single dose of 35 mg/m² of TAS-102 (containing tilpirimidine and trifluorouridine), the area under the absorption curve (AUC) of tilpirimidine was 301 ng·h/ml, the maximum plasma concentration (Cmax) was 69 ng/ml, and the time to peak concentration (Tmax) was 3 hours. Consumption of high-fat, high-calorie foods reduced Cmax and AUC by 40%. A standardized high-fat, high-calorie diet resulted in approximately a 40% reduction in Cmax and AUC of tilpirimidine in cancer patients after a single dose of LONSURF 35 mg. Following a single oral dose of LONSURF (60 mg) with [14C]-tilpirimidine hydrochloride, 77% of the radioactivity was recovered, of which 27% was excreted in the urine and 50% in the feces. Tipiracil is the main component in urine and feces, with 6-hydroxymethylaminomethane (6-HMU) being the major metabolite. In patients with advanced solid tumors, the apparent volume of distribution (Vd/F) of tilpyrimidine hydrochloride after a single dose of LONSURF (35 mg/m²) was 333 L. The oral clearance (CL/F) of tilpyrimidine hydrochloride after a single dose of LONSURF (35 mg/m²) was 109 L/hr. Metabolism/Metabolites Tipiracil undergoes minimal first-pass metabolism. It is not metabolized by the liver or hepatocytes, nor by cytochrome P450 enzymes. The only tilpyrimidine metabolite present in extremely low amounts in human plasma, urine, or feces is 6-hydroxymethyluracil (6-HMU), but this metabolite is not specific to tilpyrimidine. It is presumed to be produced by Enterobacteriaceae. In plasma, the proportions of these two metabolites were: tepyrimidine 53.1% and 6-HMU 30.9%.
Biological half-life
After administration of LONSURF 35 mg/m², the mean elimination half-life and steady-state half-life (t_1/2) of tepyrimidine were 2.1 hours and 2.4 hours, respectively.
Pharmacokinetic data for tipiracil are well-established from clinical studies of TAS-102. At least 27% of tipiracil is absorbed from the gut, and in cancer patients, highest blood plasma concentrations are reached after approximately 3 hours. Tipiracil has a half-life of 2.1 hours in patients. The compound has a volume of distribution of 333 L and a clearance of 109 L/h. Tipiracil is metabolized and cleared via renal excretion. Its primary function is to inhibit thymidine phosphorylase, thereby increasing the bioavailability and exposure of trifluridine. The combination of trifluridine and tipiracil has been developed as an oral treatment for metastatic colorectal cancer.

Protein Binding
Tipiracil has a plasma protein binding rate of less than 8%.
Toxicological data for tipiracil are derived from clinical studies of TAS-102. The combination is generally well-tolerated, with the most common adverse effects being myelosuppression (neutropenia, thrombocytopenia, anemia), gastrointestinal effects (nausea, vomiting, diarrhea, decreased appetite), and fatigue. These toxicities are primarily attributed to trifluridine, the cytotoxic component of the combination, rather than to tipiracil. Tipiracil itself has a favorable safety profile, as it is a non-cytotoxic enzyme inhibitor. The combination is FDA-approved for use in patients with metastatic colorectal cancer and gastric/gastroesophageal junction adenocarcinoma. The safety and tolerability of TAS-102 have been established in multiple clinical trials, and it is now a standard treatment option for refractory metastatic colorectal cancer.

[1]. Thymidine phosphorylase influences [(18)F]fluorothymidine uptake in cancer cells and patients with non-small cell lung cancer. Eur J Nucl Med Mol Imaging. 2014 Jul;41(7):1327-35.

[2]. Lonsurf (Trifluridine plus Tipiracil): A New Oral Treatment Approved for Patients with Metastatic Colorectal Cancer. Am Health Drug Benefits. 2016 Mar;9(Spec Feature):97-100.

[3]. Repeated oral dosing of TAS-102 confers high trifluridine incorporation into DNA and sustained antitumor activity in mouse models. Oncol Rep. 2014 Dec;32(6):2319-26.

Tipiracil belongs to the pyrimidinone class of compounds and is a compound in which uracil is substituted at the 5 and 6 positions with chlorine and (2-iminopyrrolidone-1-yl)methyl groups, respectively. It (in hydrochloride form) is used in combination with the nucleoside metabolism inhibitor trifluorouridine for the treatment of advanced/recurrent unresectable colorectal cancer. It is an antitumor drug and also an EC 2.4.2.4 (thymidine phosphorylase) inhibitor. It is a pyrimidinone, organochlorine, carboxymidine, and pyrrolidine compound. Functionally, it is related to uracil. It is the conjugate base of tilpyrimidine (1+). Tipiracil is a thymidine phosphorylase inhibitor. When used in combination with trifluorouridine in a 1:0.5 ratio, it forms TAS-102. The primary action of tilpyrimidine in TAS-102 is to enhance the bioavailability of trifluorouridine by inhibiting its catabolism. TAS-102 is indicated for the treatment of metastatic colorectal cancer in patients who have previously received fluorouracil, oxaliplatin, and irinotecan chemotherapy, or anti-VEGF or anti-EGFR therapy. Tipiracil is a thymidine phosphorylase inhibitor. Its mechanism of action is as a thymidine phosphorylase inhibitor.

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Drug Indications
Tipiracil can also be used in combination with [trifluoperidine], 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 biotherapy, and (if RAS wild-type) anti-EGFR therapy. This combination therapy is also indicated for the treatment of 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, where appropriate, HER2/neu targeted therapy).
FDA Label

Mechanism of Action

Tipiramycin is a thymidine phosphorylase inhibitor. Its function is to prevent the breakdown of the active ingredient of trifluorouridine, thereby increasing the bioavailability of trifluorouridine and enhancing its systemic concentration. Furthermore, thymidine phosphorylase is reported to be an angiogenic factor, typically overexpressed in solid tumors. Thymidine phosphorylase is directly associated with poor prognosis; tumors with elevated enzyme expression tend to have increased angiogenesis and are therefore more malignant. Therefore, studies have shown that tilpyrimidine plays an additional role by downregulating tumor angiogenesis.
Pharmacodynamics

Tipiramycin prevents the conversion of trifluorouridine to 5-trifluoromethyl-2,4(1H,3H)-pyrimidinidone (an inactive major metabolite) by inhibiting thymidine phosphorylase. Therefore, tilpyrimidine can improve the bioavailability of trifluorouridine. On the other hand, thymidine phosphorylase is a known platelet-derived endothelial growth factor; inhibiting this enzyme can produce an indirect anti-angiogenic effect.

Tipiracil is a potent and selective inhibitor of thymidine phosphorylase (TPase) with an IC50 of 35 nM for human placental TPase. It is used in combination with trifluridine in a 1:0.5 molar ratio to form TAS-102 (Lonsurf). The primary function of tipiracil is to increase trifluridine bioavailability by inhibiting its catabolism, thereby preventing rapid degradation of trifluridine. TAS-102 is FDA-approved for the treatment of metastatic colorectal cancer in patients who have previously received fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy, or anti-VEGF/anti-EGFR therapy. It is also approved for gastric/gastroesophageal junction adenocarcinoma. Tipiracil shows high selectivity for TPase over other pyrimidine-metabolizing enzymes. The combination represents an important therapeutic option for refractory metastatic colorectal cancer.

C9H11N4O2CL

242.66224

242.057

183204-74-2

Trifluridine/tipiracil hydrochloride mixture;733030-01-8;Tipiracil hydrochloride;183204-72-0

6323266

White to off-white solid

1.7±0.1 g/cm3

245ºC (decomposition)

1.743

-1.37

3

3

2

16

404

0

O=C1NC(C(Cl)=C(CN2C(CCC2)=N)N1)=O

QQHMKNYGKVVGCZ-UHFFFAOYSA-N

InChI=1S/C9H11ClN4O2/c10-7-5(12-9(16)13-8(7)15)4-14-3-1-2-6(14)11/h11H,1-4H2,(H2,12,13,15,16)

5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]-1H-pyrimidine-2,4-dione

Tipiracil

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)

H2O: ~1 mg/mL (~4.1 mM)
DMSO: <1 mg/mL

Solubility in Formulation 1: 2 mg/mL (8.24 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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