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Eniluracil (also known as 5-Ethynyluracil; GW776C85) is a novel and potent uracil analog and also a mechanism-based irreversible inhibitor of dihydropyrimidine dehydrogenase (DPD), which increases the oral bioavailability of 5-fluorouracil (5-FU) to 100%, facilitating uniform absorption and predictable toxicity.
Physicochemical Properties
| Molecular Formula | C6H4N2O2 |
| Molecular Weight | 136.1082 |
| Exact Mass | 136.027 |
| CAS # | 59989-18-3 |
| PubChem CID | 43157 |
| Appearance | Typically exists as solid at room temperature |
| Density | 1.39g/cm3 |
| Melting Point | 320 °C(dec.) |
| Index of Refraction | 1.589 |
| LogP | -0.7 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 2 |
| Rotatable Bond Count | 1 |
| Heavy Atom Count | 10 |
| Complexity | 269 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C1C(C#C[H])=C([H])N([H])C(N1[H])=O |
| InChi Key | JOZGNYDSEBIJDH-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C6H4N2O2/c1-2-4-3-7-6(10)8-5(4)9/h1,3H,(H2,7,8,9,10) |
| Chemical Name | 5-ethynyl-1H-pyrimidine-2,4-dione |
| Synonyms | 5-Ethynyluracil; GW-776C85; GW 776C85; GW776C85 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
Eniluracil is a mechanism-based irreversible inhibitor of dihydropyrimidine dehydrogenase (DPD). It binds to the enzyme with a Km of 1.6 µM and inactivates it with a first-order rate constant of 20 min⁻¹ [2] |
| ln Vitro |
Eniluracil enhances the cytotoxicity of 5-FU in human tumor cell lines expressing high levels of DPD, producing a 1- to 5-fold enhancement compared to 5-FU alone. The degree of enhancement correlates with pretreatment DPD activity [2] Eniluracil itself is not cytotoxic in cell lines [2] |
| ln Vivo |
Rats treated with Eniluracil (5-ethynyluracil) at a dose of 1 mg/kg intraperitoneally once a day for three days experience a significant improvement in FUra (5-fluorouracil) response to treatment, without any side effects or inherent antitumor activity[1]. In rats with colorectal tumors, pretreatment with Eniluracil followed by 5-FU yields a 100% complete and sustained response rate, compared to 13% with 5-FU alone. Eniluracil potentiates the antitumor efficacy and improves the therapeutic index of 5-FU six-fold [2] Eniluracil is neither toxic nor active as a single agent in animals [2] |
| Enzyme Assay |
DPD inactivation by Eniluracil proceeds via a two-step mechanism: initial reversible binding followed by irreversible inactivation. The enzyme is inactivated with a first-order rate constant of 20 min⁻¹ [2] In rat liver extracts, Eniluracil inhibits more than 99% of DPD activity within minutes of dosing. New DPD is resynthesized with a half-life of 63 hours [2] |
| Cell Assay |
A panel of human tumor cell lines was treated with Eniluracil and 5-FU. Cytotoxicity was assessed and compared to 5-FU alone. Enhancement was observed only in cell lines with high DPD activity [2] |
| Animal Protocol |
Animal/Disease Models: Female Fischer 344/HSD rats (6 to 7 weeks old; 150-200 g) [1]. Doses: 1 mg/kg Route of Administration: intraperitoneal (ip) injection; one time/day for 3 days (1 hour before FUra (5-fluorouracil) and an additional 2 days after FUra treatment) Experimental Results: When combined with 3.5 mg/kg FUra, tumor Complete regression persisted for at least 90 days after treatment, but 35 mg/kg FUra alone produced a partial response in 75% of treated animals (tumor regrowth in all of these animals). Rats bearing colorectal tumors were pretreated with Eniluracil followed by 5-FU. Tumor response was evaluated [2] In dogs, co-administration of Eniluracil with 5-FU prevented neurotoxicity associated with high-dose 5-FU [2] In a Phase I trial in human subjects, Eniluracil was administered orally at 3.7 mg/m² on days 1–2, followed by oral or intravenous 5-FU on day 2. Escalating doses of 5-FU (10–25 mg/m²/day) were given on days 2–6 with Eniluracil 3.7 mg/m² on days 1–7 every 28 days [2] |
| ADME/Pharmacokinetics |
Eniluracil achieves 100% oral bioavailability of 5-FU in animals and humans [2] The half-life of Eniluracil in plasma is 4 hours [2] Eniluracil prolongs the half-life of 5-FU from 8–22 minutes to 4.4–4.5 hours and reduces its clearance 20- to 22-fold [2] Renal excretion becomes the primary elimination pathway for 5-FU when co-administered with Eniluracil, with 77% of 5-FU excreted unchanged in urine [2] Systemic clearance of 5-FU correlates with creatinine clearance in patients treated with Eniluracil [2] |
| Toxicity/Toxicokinetics |
In Phase I trials, the dose-limiting toxicity for the 5-day schedule is myelosuppression (neutropenia), and for the 28-day schedule, it is diarrhea [2] Eniluracil irreversibly inhibits DPD; DPD activity recovers to normal levels about 2 weeks after cessation, but severe toxicity may occur if 5-FU is given within 4 weeks. It is recommended to wait at least 8 weeks before administering another fluoropyrimidine [2] Patients with renal impairment are at higher risk of toxicity due to reduced clearance of 5-FU [2] |
| References |
[1]. 5-Ethynyluracil (776C85): modulation of 5-fluorouracil efficacy and therapeutic index in rats bearing advanced colorectal carcinoma. Cancer Res. 1994 Mar 15;54(6):1507-10. [2]. Eniluracil: an irreversible inhibitor of dihydropyrimidine dehydrogenase. Expert Opin Investig Drugs. 2000 Jul;9(7):1635-49. |
| Additional Infomation |
Eniluracil is a pyrimidone. Eniluracil, which was previously under development by GlaxoSmithKline (GSK), is being developed by Adherex to enhance the therapeutic value and effectiveness of 5-fluorouracil (5-FU), one of the world’s most widely-used oncology agents. 5-FU is widely used in the U.S. and is often first or second line therapy for a variety of cancers including colorectal, breast, gastric, head and neck, ovarian and basal cell cancer of the skin. Eniluracil could improve 5-FU by increasing its effectiveness, reducing its side effects and/or making it orally available. Eniluracil has received Orphan Drug status from the FDA for the treatment of hepatocellular cancer in combination with fluoropyrimidines (including 5-FU). Eniluracil is an orally-active fluoropyrimidine analogue. Eniluracil inhibits dihydropyrimidine dehydrogenase, the rate-limiting enzyme that catabolizes and inactivates 5-fluorouracil (5-FU) in the liver. Co-administration of ethynyluracil permits the oral administration of 5-FU. Drug Indication For the treatment of cancer in combination with 5-fluorouracil. Mechanism of Action Normally, 5-FU is rapidly broken down in the body by an enzyme known as dihydropyrimidine dehydrogenase (DPD). Eniluracil irreversibly inhibits DPD, thereby substantially slowing the breakdown of 5-FU and prolonging exposure of the tumor cells to the drug. Eniluracil is developed to overcome erratic oral bioavailability of 5-FU caused by variable DPD activity [2] It may overcome 5-FU resistance mediated by DPD overexpression [2] Phase II trials in colorectal, breast, liver, and pancreatic cancers have been completed; Phase III trials in colorectal and pancreatic cancer are pending analysis [2] Eniluracil is considered a promising oral chemotherapy option due to its predictable pharmacokinetics and favorable toxicity profile compared to continuous IV infusion [2] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~16.67 mg/mL (~122.47 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (15.28 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (15.28 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 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (15.28 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 7.3470 mL | 36.7350 mL | 73.4700 mL | |
| 5 mM | 1.4694 mL | 7.3470 mL | 14.6940 mL | |
| 10 mM | 0.7347 mL | 3.6735 mL | 7.3470 mL |