DNA synthesis; Bacterial; HSV; CMV

Liver metastases are mainly supplied by the hepatic artery. Sustained high levels of intratumoral drug are achievable with certain drugs given via the hepatic artery. Floxuridine (FUDR) is an ideal drug for hepatic arterial infusion (HAI) due to its short half life, steep dose response curve, high total body clearance, and high hepatic extraction. HAI FUDR has consistently shown higher response rates than systemic chemotherapy alone, and some studies have shown a survival advantage. HAI FUDR in combination with systemic chemotherapy has evolved over the years and may be used in palliative, neoadjuvant, and adjuvant settings. The dramatic responses observed with HAI FUDR plus modern era systemic chemotherapy offer the possibility of resection and cure in selected patients. The high hepatic extraction of FUDR limits systemic side effects. Toxicity includes biliary and gastrointestinal ulcers. [5]

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HAI Floxuridine (FUDR) both alone and in combination with systemic chemotherapy has resulted in high response rates, longer hepatic progression-free survival, and increasing resection rates for unresectable liver disease from CRC. Flouropyrimidine have been the cornerstone of CRC treatment for more than 50 years. A good way to deliver these drugs, especially FUDR, to a patient with liver metastases from CRC is via HAI. Modern implantable HAI pumps have low rates of immediate and long term complications. The combination of HAI FUDR with modern systemic chemotherapy is an effective way to treat such patients with liver metastases. In the adjuvant setting HAI FUDR and systemic chemotherapy combinations can increase disease-free survival and hepatic disease-free survival. The studies were not powered to look at overall survival. Consideration should be given to placing an HAI pump at the time of liver resection and treating with adjuvant HAI FUDR plus systemic chemotherapy. If the liver disease is unresectable and a trial of systemic chemotherapy with or without biologic agents, e.g., bevacizumab or cetuximab, fails to make the liver resectable, then HAI plus systemic chemotherapy should be considered. HAI FUDR/Dex combined with modern systemic chemotherapy has a role in the treatment of liver metastases from CRC [5].

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Floxuridine (5'-fluorodeoxyuridine, FUdR) acts as an inhibitor of DNA replication by binding to thymidylate synthase and is widely used to treat colorectal cancer. FUdR is also frequently used in research on aging in C. elegans since by blocking reproduction it allows maintenance of synchronous nematode populations. Here researchers examine age-specific effects of exposure to 50 μM FUdR on pathology and mortality. Researchers report that initiating exposure to FUdR at late development or early adulthood reduces lifespan but later initiation increases it. Moreover, earlier initiation leads to enhancement of senescent intestinal atrophy, but amelioration of several other senescent pathologies (pharyngeal degeneration and uterine tumors). These results provide further evidence of the complex effects of FUdR on aging in C. elegans, and therefore support the argue against its routine use in studies of nematode aging due to its possible confounding effects. However, they also illustrate how effects of FUdR on aging are interesting in their own right [6].

Hydrolysis Studies. [1]
(a) Enzymatic Stability [1]
Confluent Caco-2, Capan-2, and AsPC-1 cells were rinsed twice with saline. The cells were washed with 5 mL of pH 7.4 phosphate buffer (10 mmol/L), lysed by ultrasonication, and pelleted by centrifugation for 5 min at 1000g. Protein amount was quantified with Bio-Rad DC Protein Assay using bovine serum albumin as a standard. The protein amount was adjusted to 500 μg/mL, and the hydrolysis reactions were carried out in 96-well plates. Caco-2, AsPC-1, and Capan-2 cell suspensions (250 μL) were placed in triplicate wells, the reactions started with the addition of substrate, and cells were incubated at 37 °C for 120 min. At the desired time point, sample aliquots (35 μL) were removed and added to 150 μL of acetonitrile (ACN) containing 0.1% TFA. The mixtures were filtered with a 0.45 μm filter at 1000g for 10 min at 4 °C. The filtrate was then analyzed via reverse-phase HPLC.

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(b) Stability in Human Plasma [1]
The stability of the prodrugs in human plasma was determined using the procedure below. Undiluted plasma (250 μL) was added to each well in triplicate, and substrate was added to initiate the reactions that were conducted at 37 °C for 2 h. At various time points, aliquots (35 μL) were removed and added to 150 μL of ACN containing 0.1% TFA. The mixtures were filtered with a 0.45 μm filter at 1000g for 10 min at 4 °C. The filtrate was then analyzed via reverse-phase HPLC.

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(c) Chemical Stability [1]
The nonenzymatic hydrolysis of the prodrugs was determined as described above, except that each well contained pH 7.4 phosphate buffer (10 mmol/L) instead of cell homogenate or human plasma.

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(d) Resistance to Metabolism of Floxuridine (FUDR) and Its Prodrugs by Thymidine Phosphorylase [1]
The stability of floxuridine and its prodrugs in the presence of thymidine phosphorylase (TP) was assessed by incubating the desired substrates (200 μM) with TP (2.0 ng/μL) in phosphate buffer (pH 7.0) at 37 °C. Aliquots of the incubation mixture were sampled at 0, 1, 3, 5, 10, 30, 60, and 120 min, and quenched with cold acetonitrile (ACN) with 0.1% TFA, filtered through a 0.45 μm membrane, and analyzed for the concentrations of prodrug, floxuridine, and 5-FU by HPLC.

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[3H]Gly-Sar Uptake Inhibition [1]
Caco-2 cells at nine days postseeding, and AsPC-1 and Capan-2 cells, both at four days postseeding, were incubated with 10 μmol/L Gly-Sar (9.98 μmol/L Gly-Sar and 0.02 μmol/L [3H]Gly-Sar) along with various prodrug concentrations (5−0.05 mmol/L) for 30 min. The cells were washed three times with ice-cold PBS and solubilized with 10 mL of scintillation cocktail, and the amount of cell-associated radioactivity was determined by scintillation counting. IC50 values were determined using nonlinear data fitting (GraphPad Prism version 3.0).

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Transport Studies [1]
Caco-2 cell monolayers were grown on collagen-coated polytetrafluoroethylene membranes for 21 to 24 days, and Capan-2 cell monolayers were grown on the same type of membrane for 14 days. Transepithelial electrical resistance (TEER) was monitored, and values of 240−280 Ω/cm2 in Caco-2 and 380−420 Ω/cm2 in Capan-2 (total area for both cells was 4.67 cm2) were used in the study. Apical side and basolateral sides of transwell inserts were washed with MES (pH 6.0) and HEPES (pH 7.4), respectively. Fresh MES and HEPES buffers were reapplied to transwell inserts and incubated at 37 °C for 15 min. Freshly prepared 0.1 mM drug solution in MES buffer (total 1.5 mL) was placed in the donor chamber, and the receiver chamber was filled with HEPES buffer (total 2.5 mL) Sampling from the receiver chamber (200 μL) was conducted up to a period of 2 h at time intervals of 15, 30, 45, 60, 75, 90, and 120 min, at 37 °C and replaced with an equal volume of fresh HEPES buffer to maintain sink conditions in the receiver chamber. All samples were immediately acidified with 0.1% TFA and analyzed by reverse-phase HPLC.

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Amino acid ester prodrugs of antiviral and anticancer nucleoside drugs were developed to improve oral bioavailability or to reduce systemic toxicity. Researchers studied the interaction of human concentrative nucleoside transporter (hCNT2) cloned from intestine with various amino acid ester prodrugs of Floxuridine (FUdR) and 5,6-dichloro-2-bromo-1-beta-D-ribofuranosylbenzimidazole (BDCRB). Na(+)-dependent uptakes of [(3)H]-inosine and [(3)H]-adenosine were measured in U251 cells transiently expressing intestinal hCNT2. FUdR significantly inhibited the uptake of both [(3)H]-inosine and [(3)H]-adenosine (60-70% of control), while its amino acid ester prodrugs including Val, Phe, Pro, Asp, and Lys esters exhibited markedly decreased inhibition potency (10-30% of control). On the other hand, BDCRB and its amino acid prodrugs markedly inhibited the uptake of both [(3)H]-inosine and [(3)H]-adenosine. Val, Phe, and Pro ester prodrugs of BDCRB showed similar inhibition capacities as parent compound BDCRB (80-90% for adenosine and 60-80% for inosine). The amino acid site of attachment (3'- and 5'-monoesters) and stereochemistry (L- and D-amino acid esters), did not significantly affect the uptake of [(3)H]-inosine and [(3)H]-adenosine. These results demonstrate that the hCNT2 favorably interacts with BDCRB and its amino acid prodrugs, compared to those of FUdR, and that neutral amino acid esters of BDCRB have a high affinity toward this transporter. Therefore, the intestinal hCNT2 may be a target transporter as a factor for modulating oral pharmacokinetics of BDCRB prodrugs [3].

Cell Line: Ovarian cancer cells
Concentration: 0-25 μM
Incubation Time: 4, 8, 24 hours
Result: Was potentiated the sensitivity by PARP inhibitors.

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Cell Proliferation Assays [1]
Cell proliferation studies were conducted with AsPC-1 and Capan-2 cell lines. The cells were seeded onto 96-well plates at 125,000 cells per well and allowed to attach/grow for 24 h before drug solutions were added. The culture medium (RPMI-1640 + 10% fetal bovine serum) was removed, and the cells were gently washed once with sterile pH 6.0 uptake buffer. Floxuridine (FUDR) and floxuridine prodrugs were 2-fold serially diluted in pH 6.0 uptake buffer from 4 to 0.25 mmol/L. Buffer alone was used as 100% viability control. The wash buffer was removed, and 25 μL of drug solution per well was added and incubated at 37 °C for 2 h with AsPC-1 cells and 4 h with Capan-2 cells in the cell incubator. After this time period, the drug solutions were removed and the cells were gently washed twice with sterile uptake buffer. Fresh culture medium was then added to each well after washing, and the cells were allowed to recover for 24 h before evaluating cell viability via 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assays. A mixture (30 μL) containing XTT (1 mg/mL) in sterile RPMI-1640 without phenol red and phenazine methosulfate (N-methyl dibenzopyrazine methyl sulfate in sterile PBS, 0.383 mg/mL) reagents was added to the cells and incubated at 37 °C for 1 h, after which the absorbance at 450 nm was read. GI50 values were calculated using GraphPad Prism version 3.0 by nonlinear data fitting.

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The inhibitory effects of leucovorin (LV) combined with 5-fluorouracil (FUra) or Floxuridine (FUDR) (FdUrd) on growth of human T-lymphoblast leukemia cells (CCRF-CEM) were determined as a function of time, dose, and sequence of exposure. Exposure of CCRF-CEM cells in exponential growth to LV (1-100 microM) for 4 hours and to FUra (100 microM) or FdUrd (0.5 microM) during the last 2 hours resulted in synergistic inhibitory effects on cell growth. Synergism was dependent on LV dose (100 greater than 10 greater than 1 microM) and did not occur at 0.1 microM. No clear dependence of synergy on sequence was observed with FUra and LV combinations. With LV and FdUrd combinations, synergism was dependent on sequence of exposure (LV + FdUrd and LV----FdUrd were synergistic, but FdUrd----LV was not). Thymidine (0.1 microM), added after drug treatment, substantially rescued CCRF-CEM cells from LV----FUra cytotoxicity. Concomitant hypoxanthine (100 microM) only partially protected CCRF-CEM cells from the toxicity of this combination. These results are consistent with the hypothesis that the mechanism by which LV potentiates fluoropyrimidine cytotoxicity is the enhancement of complex formation between thymidylate synthase and 5-fluorodeoxyuridylate, presumably as a consequence of an increase of intracellular levels of 5,10-methylenetetrahydrofolate generated from LV. Also, enhanced stability of this complex in the presence of high levels of the folate coenzyme may contribute to the synergy observed. These data also provide a rationale for use of FUra and especially FdUrd and LV in the treatment of lymphoid malignancies in man.[2]

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Proliferating human Tenon's capsule fibroblasts were exposed for 5 minutes to a wide range of concentrations of fluorouracil, Floxuridine (FUDR), and mitomycin. High concentrations of all three agents had prolonged effects on cell proliferation and morphologic characteristics compared with untreated control cells up to 36 days. The highest concentrations of both floxuridine (15,000 micrograms/mL) and mitomycin (1000 micrograms/mL) had an apparent cidal effect, reducing cell numbers below initial cell density. In contrast, although the highest concentration of fluorouracil (25,000 micrograms/mL) inhibited cell proliferation by more than 50% relative to the untreated control cells at 36 days, the cell numbers still increased fourfold compared with the initial cell density. These results demonstrate that 5-minute treatments with high concentrations of these drugs have prolonged effects on the proliferation of human Tenon's capsule fibroblasts in vitro. Single-dose regimens using high concentrations of these drugs at the time of operation may achieve results similar to those of protocols that involve repeated applications[4].

C57BL/6 mice injected with S. aureus
0.5-1.25 mg/kg
once per day for 7 days or single dose

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To examine the effects of Floxuridine (FUDR) on worms, drug was added topically onto a 2-day old bacterial lawn to a final concentration of 50 μM. Worms were transferred from untreated NGM plates at L4, or day 1, day 2 or day 3 of adulthood. [6]
Lifespan assays[6]
Gravid adults were cultured on fresh NGM plates to lay eggs, and two days later L4 larvae were picked to new plates for lifespan measurements. Trials were conducted at 20 °C for N2 strains. Temperature sensitive sterile glp-4(bn2) mutants were cultured at 15 °C until L4 and then transferred to 25 °C. Nematodes were transferred daily during the reproductive period to avoid confusion with progeny. Population cohorts were scored for mortality every other day until the death of the last worm. Deaths due to internal hatching of larvae or rupture were censored. The L4 stage was recorded as day 0 for the trials.

Absorption, Distribution and Excretion
Fluorouracil is excreted by the kidneys as the parent drug, urea, fluorouracil, α-fluorobutylpropionic acid, dihydrofluorouracil, α-fluoro-β-guanidinylpropionic acid, and α-fluoro-β-alanine. It is also excreted as respirable carbon dioxide. ...Fluorouracil...is usually administered parenterally because absorption after oral administration...is unpredictable and incomplete. It is currently unknown whether fluorouracil is distributed into breast milk. Some fluorouracil can cross the blood-brain barrier; active metabolites are localized intracellularly. Elimination is primarily respiratory (as carbon dioxide), accounting for approximately 60%. Renal excretion accounts for 10% to 13% (in the parent drug and its metabolites). Metabolism/Metabolites Hepatic metabolism. Biotransformation primarily occurs in the liver and tissues, with extensive conversion to monophosphate derivatives and fluorouracil. Following continuous intra-arterial infusion, the conversion to monophosphate derivatives is enhanced; after rapid intravenous or intra-arterial injection, most is converted to fluorouracil. Following low-dose fluorouridine infusion, most of the drug appears to be anabolicly metabolized to FUDR-MP, the active metabolite of the drug. With rapid single-dose administration, fluorouridine is clearly rapidly catabolic to fluorouracil. Both fluorouridine and fluorouracil are metabolized in the liver. Continuous infusion results in less metabolic degradation of fluorouridine compared to single-dose administration. The drug is excreted in the urine as unchanged form, urea, fluorouracil, α-fluoro-β-ureidopropionic acid, dihydrofluorouracil, α-fluoro-β-guanidinopropionic acid, and α-fluoro-β-alanine, and is also excreted as carbon dioxide produced by respiration. The drug is primarily metabolized and degraded in the liver. Fluorouracil is converted to 5-fluorouracil by thymidine or deoxyuridine phosphorylase. 5-Fluorouracil is inactivated by the reduction of the pyrimidine ring; this reaction is catalyzed by dihydrouracil dehydrogenase, an enzyme found in the liver, intestinal mucosa, and other tissues. Hereditary deficiency of this enzyme leads to a significantly increased sensitivity to the drug. The product of this reaction, 5-fluoro-5,6-dihydrouracil, is ultimately degraded to α-fluoro-β-alanine…

Hepatotoxicity
A significant proportion of patients receiving fluorouridine hepatic artery infusion therapy experience elevated serum transaminase levels, with reported incidence ranging from 25% to 100%. These elevations are usually mild to moderate and resolve upon discontinuation of the drug. However, "chemical hepatitis" often requires dose adjustments or extended treatment duration. Furthermore, prolonged or repeated hepatic artery infusion of fluorouridine can lead to acalculous cholecystitis and multiple bile duct strictures, resulting in jaundice and a syndrome similar to chronic sclerosing cholangitis. Symptomatic bile duct strictures, manifesting as pain and jaundice, occur in 5% to 25% of patients receiving fluorouridine hepatic artery infusion therapy. These symptoms typically appear 2 to 6 months after treatment begins, but can also occur up to a year after treatment commencement. Biliary strictures usually involve the central bile duct in the porta hepatis, generally located at and around the bifurcation of the common hepatic duct. Similar inflammation and fibrosis can lead to acalculous cholecystitis during FUDR treatment, but this can be avoided by performing cholecystectomy concurrently with liver metastasis resection or implantation of an intra-arterial infusion pump. Biliary strictures usually improve after discontinuation of the drug, but may also progress or require endoscopic or surgical intervention. Deaths due to progressive biliary strictures and cholestatic liver injury have been reported, which may be one of the leading causes of death among survivors of this metastatic cancer. The incidence of biliary strictures after FUDR treatment can be reduced by concomitant use of dexamethasone and prevented by hepatobiliary imaging monitoring. However, the numerous complications of hepatic artery infusion chemotherapy have dampened enthusiasm for this therapy, especially after the advent of newer, more potent systemic antitumor drugs.
Probability Score: A (Known cause of clinically significant hepatobiliary injury).
Interactions
If fluorouracil is used concomitantly or recently with drugs that cause leukopenia and/or thrombocytopenia, and these drugs also cause the same effects, the leukopenia and/or thrombocytopenia caused by fluorouracil may be enhanced; if necessary, the fluorouracil dose should be adjusted according to blood cell counts.
Additional myelosuppression may occur; when two or more myelosuppressants (including radiation) are used concomitantly or sequentially with fluorouracil, dose reduction may be necessary. Because fluorouracil treatment may suppress normal defense mechanisms, patients may experience a reduced antibody response to inactivated virus vaccines. The time interval between discontinuation of immunosuppressive medications and the patient's recovery of an immune response to the vaccine depends on the strength and type of immunosuppressive medication used, underlying medical conditions, and other factors; the estimated time ranges from 3 months to 1 year. Because fluorouracil treatment may suppress normal defense mechanisms, concomitant use with live virus vaccines may enhance vaccine virus replication, increase vaccine virus side effects/adverse reactions, and/or reduce the patient's antibody response to the vaccine; therefore, immunization should be administered with extreme caution only after careful evaluation of the patient's hematological status and with informed consent from the physician responsible for fluorouracil treatment. The time interval between discontinuation of immunosuppressive medications and the patient's recovery of an immune response to the vaccine depends on the strength and type of immunosuppressive medication used, underlying medical conditions, and other factors; the estimated time ranges from 3 months to 1 year. Close contacts of the patient, especially family members, should postpone oral polio vaccination.

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Toxicity of HAI FUDR[5]
The toxicity of HAI can be mechanical, chemical, or both. The incidence of complications with surgically implanted pumps is low. Allen et al. reported complications with HAI pumps in 544 patients throughout the course of treatment. Complications within 30 days of implantation were more likely to be catheter occlusion or arterial thrombosis and were more difficult to salvage. The overall failure rate of the pumps was low, at 9% at 1 year and 16% at 2 years. The overall complication rate of the pumps was 22%, most of which were salvageable, and 80% of the pumps remained functional for at least 2 years. All patients were required to undergo nuclear medicine macromolecular aggregated albumin scans before using hepatic artery infusion pumps to assess liver perfusion or the presence of extrahepatic perfusion via gastroduodenal artery collaterals. Insufficient perfusion of the stomach or duodenum may lead to ulceration or diarrhea. Unlike systemic chemotherapy, hepatic artery infusion of fluorouracil (HAI FUDR) does not cause bone marrow suppression, nausea, or vomiting. Hepatotoxicity of HAIs depends on the medication used and the duration of treatment. Since the hepatic artery supplies blood to the bile ducts, the toxicity of HAI FUDR may manifest as biliary toxicity. Elevated liver enzymes or bilirubin are the most common toxic reactions to HAI treatment, occurring in 42% of patients in the aforementioned randomized trials for patients with unresectable liver disease. Elevated transaminase levels are also not uncommon (up to 70%) and may be an early sign of biliary tract injury. Elevated bilirubin and alkaline phosphatase are more severe. Up to 29% of cases had bile duct strictures (cholangiosclerosis) before dexamethasone was added to the pump (Reference 38). In our institution's pump-assisted studies, 27% to 43% of cases experienced alkaline phosphatase elevations more than double. 6% to 19% of cases had bilirubin elevations exceeding 3.0 mg/dL, of which 3% to 8% required biliary stent placement. Elevated transaminase levels occurred in 37% to 59%. In a recently updated study by Kemeny et al., HAI FUDR/Dex was used in combination with oxaliplatin and irinotecan for 5 weeks. Toxicity events in the first two cycles included: grade 3 diarrhea (33%), grade 3/4 alkaline phosphatase elevation (15% and 11%, respectively), grade 3/4 AST elevation (19%), grade 3 bilirubin elevation (4%), and grade 3/4 neutropenia (19% and 4%, respectively). Late toxicity events (after the first two cycles) included: grade 3 diarrhea (7%), grade 3 or 4 alkaline phosphatase elevation (22% and 11%, respectively), grade 3/4 AST elevation (7%), grade 3/4 neutropenia (15% and 11%, respectively), and neurotoxicity (19%). Therefore, we developed a dose adjustment algorithm based on liver function blood test results to adjust the FUDR dose accordingly (see Table 3). As mentioned above, FUDR combined with dexamethasone reduces biliary toxicity. Fludarabine/dexamethasone (HAI FUDR/Dex) is administered intrahepatic artery once a month (provided enzyme levels are normal). The pump continuously infuses the medication; after two weeks, the reservoir is emptied and replaced with heparinized saline or glycerol, continuing the infusion for the next two weeks. If the patient develops elevated bilirubin, chemotherapy is paused, and dexamethasone in heparinized saline is injected into the pump. If bilirubin levels do not return to normal, endoscopic retrograde cholangiopancreatography (ERCP) may be performed to assess for focal stenosis that could be treated with dilation. When the pump is not in use, glycerol is injected every 6 to 8 weeks to maintain catheter patency. If the pump is no longer needed, it can be removed via a small incision, and the connection between the catheter and the pump in the hepatic artery is severed and left in place.

[1]. Mol Pharm. 2008 Sep-Oct;5(5):717-27.

[2]. Cancer Treat Rep. 1987 Apr;71(4):381-9.

[3]. Biol Pharm Bull. 2006 Feb;29(2):247-52.

[4]. Arch Ophthalmol. 1992 Aug;110(8):1150-4.

[5]. Mol Cancer Ther. 2009 May;8(5):1015-25.

[6]. Biochem Biophys Res Commun. 2019 Feb 12;509(3):694-699.

Therapeutic Uses
Antimetabolite, antitumor, antiviral drug. Fluorouracil, administered via continuous regional intra-arterial infusion, is indicated for palliative treatment of liver metastases of colorectal cancer unresponsive to other treatments. Fluorouracil is most effective when the disease has not spread to an area accessible by a single artery. /Included in the US product label/ Fluorouracil is also indicated for ovarian and kidney cancer unresponsive to other antimetabolites. /Not included in the US product label/ Fluorouracil has also been used for breast, ovarian, cervical, bladder, kidney, and prostate cancer unresponsive to other antimetabolites. /Not included in the US product label/ Drug Warnings Fluorouracil is metabolized to fluorouracil, but due to regional administration via intra-arterial infusion, the full toxicity of fluorouracil is not expected. However, the possibility of typical adverse reactions to fluorouracil during fluorouride treatment should be considered. Nausea, vomiting, and diarrhea are common adverse reactions; anorexia, cramps, and pain may also occur. Stomatitis is one of the most common symptoms of specific toxicities. Enteritis is common, and there have also been reports of duodenal ulcers, duodenitis, gastritis, gastroenteritis, glossitis, gastrointestinal bleeding, and pharyngitis. Leukopenia and anemia are common during fluorouridine treatment; thrombocytopenia may also occur. Close monitoring of the patient's hematological status is essential. Pancytopenia and agranulocytosis have been reported in patients treated with fluorouracil; these adverse hematological reactions may also occur in patients treated with fluorouridine due to their similar pharmacological effects. For more complete data on drug warnings for fluorouridine (25 in total), please visit the HSDB record page. Pharmacodynamics: Fluorouracil is an antimetabolite or pyrimidine analogue that acts by selectively targeting rapidly dividing cells through interference with the S phase of cell division. Due to structural similarity, the antimetabolite functions as a pyrimidine-like molecule, preventing the incorporation of normal pyrimidines into DNA. Fluorouracil was biotransformed into the active ingredient fluorouracil, which blocks the enzyme that converts cytosine nucleosides into deoxyribonucleotides. Fluorouracil can also physically prevent thymidine nucleotides from being incorporated into the DNA chain by substituting their positions, thereby further inhibiting DNA synthesis. We synthesized dipeptide monoester prodrugs of fluorouracil and determined their chemical stability in buffer, resistance to glycosidic metabolism, affinity for PEPT1, enzymatic activation, and permeability in cancer cells, comparing them with monoamino acid monoester fluorouracil prodrugs. The prodrug containing a glycyl group showed the worst stability in pH 7.4 buffer (t_1/2 < 100 min). All fluorouracil prodrugs were activated in cell homogenates 2 to 30 times faster than hydrolyzed in buffer, indicating enzymatic activity. In cell homogenates, dipeptide monoester prodrugs containing aromatic precursor moieties exhibit enzymatic activation rates 5 to 20 times slower than other dipeptides and most monoamino acid monoester prodrugs (half-life approximately 40 to 100 minutes). All prodrugs showed greater resistance to thymidine phosphorylase glycosidic bond metabolism compared to the parent fluorouridine. Generally, 5'-O-dipeptide monoester fluorouridine prodrugs showed a higher affinity for PEPT1 than their corresponding 5'-O-monoamino acid ester prodrugs. The dipeptide monoester prodrugs permeated 2 to 4 times more permeable across the Caco-2 and Capan-2 monolayers than their corresponding monoamino acid ester prodrugs. Cell proliferation assays in AsPC-1 and Capan-2 pancreatic duct cell lines demonstrated that the dipeptide monoester prodrugs possessed similar potency to the monoamino acid prodrugs. The transport and enzymatic characteristics of 5'-L-phenylalanyl-L-tyrosinyl-fluorouridine, 5'-L-phenylalanyl-L-glycyl-fluorouridine and 5'-L-isoleucyl-L-glycyl-fluorouridine suggest that they have the potential to improve oral absorption, delay enzymatic biotransformation and enhance resistance to 5-fluorouracil metabolism, as well as enhance uptake and cytotoxic activity of cancer cells. These properties help prolong systemic circulation time, thereby enhancing therapeutic effects. [1] Fluorouracil is a pyrimidine 2'-deoxynucleoside compound with 5-fluorouracil as its nucleobase; it is used to treat liver metastases of gastrointestinal adenocarcinoma and to relieve symptoms of liver and gastrointestinal malignancies. It has antitumor, anti-metabolic, antiviral and radiosensitizing effects. It is a pyrimidine 2'-deoxynucleoside belonging to organofluorine compounds and nucleoside analogues. After rapid injection, fluorouridine is metabolized to fluorouracil. Fluorouracil is provided as a sterile, pyrogen-free lyophilized powder and must be reconstituted before use. When administered via slow, continuous intra-arterial infusion, it is converted to fluorouridine monophosphate. It has been used to treat liver metastases of gastrointestinal adenocarcinoma and to alleviate symptoms of liver and gastrointestinal malignancies. Fluorouracil is an antimetabolite. Fluorouracil (FUDR) is a pyrimidine analog used as an antitumor drug, typically administered via continuous hepatic artery infusion to treat liver metastases of colon cancer. Intra-arterial injection of fluorouridine leads to significant increases in serum enzyme and bilirubin levels during treatment and often causes bile duct damage, leading to secondary sclerosing cholangitis, which can be severe and eventually progress to cirrhosis. Fluorouracil is a fluorinated pyrimidine monophosphate analog of 5-fluoro-2'-deoxyuridine-5'-phosphate (FUDR-MP) and has antitumor activity. As an antimetabolite, fluorouridine inhibits thymidylate synthase, resulting in impaired DNA synthesis and cytotoxicity. This drug can also be metabolized into fluorouracil and other metabolites, which can be incorporated into RNA, inhibiting the utilization of uracil pre-formed during RNA synthesis. (NCI04)
Fluorouracil is a small molecule drug, with its clinical trial phase up to Phase IV (covering all indications). It was first approved in 1970 for the treatment of cancer and tumors, and has 17 investigational indications. This drug has been placed on the black box warning list by the U.S. Food and Drug Administration (FDA).
Fluorouracil is an antitumor antimetabolite drug. When administered rapidly by injection, it is metabolized into fluorouracil; when administered slowly and continuously by intra-arterial infusion, it is converted into fluorouracil monophosphate. It has been used to treat liver metastases of gastrointestinal adenocarcinoma and to relieve symptoms of liver and gastrointestinal malignancies.

C9H11FN2O5

246.19

246.065

C, 43.91; H, 4.50; F, 7.72; N, 11.38; O, 32.49

50-91-9

5790

White powder

1.8±0.1 g/cm3

483.0±55.0 °C at 760 mmHg

148 °C(lit.)

245.9±31.5 °C

0.0±2.8 mmHg at 25°C

1.676

-1.22

3

6

2

17

386

3

FC1C(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

ODKNJVUHOIMIIZ-RRKCRQDMSA-N

InChI=1S/C9H11FN2O5/c10-4-2-12(9(16)11-8(4)15)7-1-5(14)6(3-13)17-7/h2,5-7,13-14H,1,3H2,(H,11,15,16)/t5-,6+,7+/m0/s1

5-fluoro-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]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: ≥ 2.08 mg/mL (8.45 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.45 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.45 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.

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Solubility in Formulation 4: 100 mg/mL (406.19 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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