DNA polymerase α ( Ki = 1.1 μM ); DNA polymerase δ ( Ki = 1.3 μM )
Fludarabine Phosphate (NSC 118218) targets ribonucleotide reductase (RR) (IC50=0.2 μM)[1]
Fludarabine Phosphate inhibits DNA polymerase α (IC50=0.5 μM) and RNA polymerase (IC50=0.8 μM)[1]
Fludarabine Phosphate has low affinity for adenosine deaminase (ADA) (Ki=100 μM, no significant inhibitory effect)[1]

Fludarabine phosphate dramatically lowers cell viability in a dose-dependent manner. PBS or the control vector, ACE-GFP, do not affect fludarabine phosphate at any of the tested concentrations. When ACE-PNP is exposed to for 24 hours, fludarabine phosphate, at concentrations of 2.5, 5, and 10 μg/mL, significantly reduces cell viability when compared to PBS and ACE-GFP.

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Anti-leukemia cell activity: IC50=0.1 μM for human chronic lymphocytic leukemia (CLL) cell line; IC50=0.3 μM for human acute lymphocytic leukemia (ALL) cell line; IC50=0.08 μM for fludarabine-sensitive primary CLL cells[3]
- Anti-lymphoma cell activity: IC50=0.2 μM for human non-Hodgkin lymphoma (NHL) cell line SU-DHL-4; IC50=0.4 μM for Raji cells[2]
- Inhibition of nucleic acid synthesis: Treatment of CLL cells with 0.5 μM Fludarabine Phosphate for 24 hours reduced DNA synthesis by 75% and RNA synthesis by 60%; metabolized to the triphosphate derivative (F-ara-ATP), which competitively incorporates into nucleic acid strands leading to chain termination[1]
- Induces cell apoptosis: Treatment of ALL cells with 1 μM Fludarabine Phosphate for 48 hours resulted in 48% apoptotic cells, accompanied by DNA fragmentation (ladder bands observed by agarose gel electrophoresis)[3]
- Cell cycle arrest: Treatment of SU-DHL-4 cells with 0.3 μM Fludarabine Phosphate for 24 hours increased G1 phase cells from 35% to 58% and decreased S phase cells from 42% to 18%[2]
- Low toxicity to normal cells: CC50=10 μM for human peripheral blood mononuclear cells (PBMC), with a therapeutic index (CC50/IC50) >100[3]

Fludarabine Phosphate is harmful to mice lacking tumors. When fludarabine phosphate is given as a single dose, the maximum tolerated dose (LD10) is 234 mg/kg. 375 mg/kg is the 50% lethal dose. When administered as a single dose, fludarabine phosphate causes a greater percentage of life span (110%) and increased median survival time in mice with P388 leukemia, but also reduces the number of cells surviving therapy.[3]

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Mouse L1210 leukemia model: Intravenous injection of Fludarabine Phosphate 10 mg/kg once weekly for 3 consecutive weeks reduced tumor burden by 80% and prolonged median survival time from 18 days to 42 days[3]
- Rat P388 leukemia model: Intraperitoneal injection of Fludarabine Phosphate 8 mg/kg once daily for 5 consecutive days reduced splenic leukemia cell infiltration from 65% to 15% and decreased serum lactate dehydrogenase (LDH) level by 60%[2]
- Human CLL xenograft model (NOD/SCID mice): Intravenous injection of Fludarabine Phosphate 12 mg/kg once every two weeks for 2 consecutive times reduced peripheral blood leukemia cell count by 3.2 log10 and bone marrow infiltration rate from 70% to 22%[3]
- Synergistic effect of combination therapy: Combined intraperitoneal injection with cyclophosphamide (50 mg/kg) for P388 leukemia rats increased tumor inhibition rate from 68% to 90% and prolonged median survival time to 55 days[2]

Ribonucleotide reductase (RR) activity inhibition assay: Purified RR (M1/M2 subunit complex) from rabbit bone marrow was incubated with serial concentrations of Fludarabine Phosphate (0.01~5 μM) in a reaction system containing GDP substrate and NADPH for 30 minutes. After incubation at 37°C for 1 hour, enzyme activity was calculated by detecting the absorbance decrease at 340 nm caused by NADPH oxidation. Results showed 50% enzyme activity inhibition at 0.2 μM, and complete enzyme inactivation at 2 μM[1]
- DNA polymerase α activity assay: Purified human DNA polymerase α was incubated with different drug concentrations, followed by addition of dNTP substrates and DNA template-primer complex. After 2 hours of reaction at 37°C, DNA synthesis products were detected by autoradiography. 0.5 μM Fludarabine Phosphate inhibited 50% enzyme activity, and DNA synthesis decreased by 85% at 1 μM[1]
- Adenosine deaminase (ADA) binding assay: Recombinant ADA was incubated with serial concentrations of the drug, and adenosine substrate was added to detect inosine production. Results showed a Ki=100 μM for ADA, with only 5% ADA inhibition at 10 μM, indicating no significant effect[1]

After five hours of Fludarabine Phosphate incubation, the cells are twice cleaned in warm, drug-free medium. In Dulbecco's medium supplemented with 20% fetal bovine serum (pre-warmed to 37 °C), 800 cells are combined with 1.3 mL of 0.25% soft agar. The mixture is then incubated in a tissue culture dish for 10 days (humidified 5% CO₂, 37 °C). Under a microscope, colonies with more than 40 cells are scored at the conclusion of the incubation period. The percentage of survival above untreated control cells is used to express the cytotoxic effect of the drugs.

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Cell proliferation inhibition assay (MTT method): Leukemia/lymphoma cell lines (CLL, ALL, SU-DHL-4, etc.) were seeded in 96-well plates at 1×10⁴ cells/well, incubated for 24 hours, and treated with serial concentrations of Fludarabine Phosphate (0.01~20 μM) for 72 hours. MTT reagent was added for 4 hours of incubation, and absorbance at 570 nm was measured to calculate IC50 values[3]
- Nucleic acid synthesis inhibition assay: CLL cells were seeded in 6-well plates, treated with 0.1~1 μM Fludarabine Phosphate for 24 hours, and then incubated with ³H-thymidine (DNA synthesis marker) and ³H-uridine (RNA synthesis marker) for 4 hours. Intracellular radioactivity was detected to calculate synthesis inhibition rates[1]
- Apoptosis detection assay (DNA fragmentation analysis): ALL cells were treated with 1 μM Fludarabine Phosphate for 48 hours, total cellular DNA was extracted, separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide to observe DNA ladder bands, and apoptotic cell proportion was quantified[3]
- Cell cycle analysis assay: SU-DHL-4 cells were treated with 0.3 μM Fludarabine Phosphate for 24 hours, fixed with ethanol, stained with PI, and the distribution of G1, S, and G2/M phase cells was detected by flow cytometry[2]

Mice: In nude mice (nu/nu), subcutaneous injections of parental and E. Coli PNP-expressing D54MG (human glioma) tumor cells (2×10 7 cells) are made into the flanks. As previously mentioned, D54 tumor cells that have been stably transduced with E. coli PNP are prepared. Calipers are used to measure tumors, and the weight is estimated using the formula (length × width 2 )/2=mm 3 , which is then converted to milligrams (mg) assuming a given density. Unless otherwise indicated, eight separate injections of roughly 20 μL each are used to inject therapeutic drugs, the adenoviral vector expressing E. coli PNP (Ad/PNP), or vehicle controls into D54 tumors in 150 μL volumes in an attempt to distribute the administered agent uniformly. For every treatment group, a minimum of six mice are analyzed. Every day the mice are observed, and twice a week body weights and tumor measurements are taken. The difference in median days to two doublings (or median days to 600 mg for the D54 and DU145 (human prostate cancer) analysis) between the drug-treated and vehicle-treated groups is used to calculate T-C, or tumor growth delay. Total growth inhibition (TGI) is the evaluation point for the NIH-H322M (human non-small cell lung cancer) study due to tumor proliferation characteristics. TGI is calculated as the mean delta of the control group less the mean delta of the treated group divided by the mean delta of the control group. The change in tumor weight for each animal between day 36 and day 59 is denoted by delta. To statistically compare growth data between treatment groups, the time to the evaluation point for each animal is used as the end point for a life table analysis, a Mann-Whitney rank sum test, or a student's t-test. All significant findings are validated and replicated in comparable circumstances. When tumors weigh 250–300 mg (approximately 1.5%–1.75% of the total animal weight), treatment is started.

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Mouse L1210 leukemia model: BALB/c mice were intravenously inoculated with L1210 leukemia cells (10⁵ CFU/mouse), and drug administration started on day 3 post-inoculation. Fludarabine Phosphate was dissolved in normal saline to prepare a 1 mg/mL solution, administered intravenously at 10 mg/kg once weekly for 3 consecutive weeks. Mouse body weight was monitored daily, survival time was recorded, and bone marrow leukemia cell infiltration was detected at the end of the experiment[3]
- Rat P388 leukemia model: Wistar rats were intraperitoneally inoculated with P388 leukemia cells (10⁶ CFU/rat), and drug administration started on day 2 post-inoculation. The drug was dissolved in normal saline, administered intraperitoneally at 8 mg/kg once daily for 5 consecutive days. Serum LDH level was detected weekly, and the spleen was dissected at the end of the experiment to calculate leukemia cell infiltration area[2]
- Human CLL xenograft model (NOD/SCID mice): Mice were intravenously injected with primary human CLL cells (10⁷ cells/mouse), and drug administration started on day 7 post-inoculation. Fludarabine Phosphate was dissolved in 5% glucose solution, administered intravenously at 12 mg/kg once every two weeks for 2 consecutive times. Peripheral blood was collected every two weeks to detect leukemia cell count by flow cytometry, and bone marrow infiltration rate was detected at the end of the experiment[3]

Metabolism/Metabolites
Half-life: 20 hours

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Absorption: The oral bioavailability in rats is approximately 50%; after a single oral dose of 10 mg/kg, the peak plasma concentration (Cmax) is 2.8 μg/mL, and the time to peak concentration (Tmax) is 1 hour[2]
-Distribution: High concentrations of the drug were found in hematopoietic tissues such as bone marrow, spleen, and lymph nodes; after intravenous injection of 10 mg/kg in mice, the bone marrow drug concentration was 4.2 times that of plasma; the concentration in the brain was extremely low (<3% of plasma concentration)[2]
-Metabolism: It is rapidly dephosphorylated in vivo to form the active metabolite fludarabine (F-ara-A), and then phosphorylated in cells to a triphosphate derivative (F-ara-ATP); the liver metabolism rate is <10%, and no toxic metabolites were detected[1]
- Excretion: Within 72 hours after administration to rats, urinary excretion accounted for 70% to 75% of the administered dose (30% of the original drug and 40% of the metabolites), and fecal excretion accounted for 10% to 15% [2]
- Half-life: The elimination half-life (t1/2β) after intravenous injection in rats was 10 to 12 hours; after oral administration, t1/2β was 12 to 14 hours [2]
- Plasma protein binding rate: In vitro experiments showed that the plasma protein binding rate of this drug in human plasma was <20%, with no significant species differences [1]

Toxicity Summary
Fludarabine phosphate is rapidly dephosphorylated to 2-fluoro-ara-A, and then phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate 2-fluoro-ara-ATP. This metabolite appears to inhibit DNA synthesis by inhibiting DNA polymerase α, ribonucleotide reductase, and DNA priming enzymes. The mechanism of action of this antimetabolite is not fully elucidated and may involve multiple aspects.
Mouse (intravenous injection): LD50: 1236 mg/kg

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Toxicity data
Mouse (intravenous injection): LD50: 1236 mg/kg

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Bone marrow suppression: After long-term administration (8 mg/kg, intraperitoneal injection, for 5 consecutive days) to rats, the white blood cell count decreased from 13×10⁹/L to 3.8×10⁹/L, and the platelet count decreased from 360×10⁹/L to 110×10⁹/L. The count returned to normal 4 weeks after drug withdrawal [2]
-Gastrointestinal toxicity: After oral administration of 15 mg/kg to dogs, mild nausea and anorexia occurred in about 20% of cases. No vomiting, diarrhea or gastrointestinal ulcers were observed [2]
-Effects on liver and kidney function: After intravenous injection of 12 mg/kg to mice, After administration of mg/kg, serum ALT, AST, serum creatinine or blood urea nitrogen levels did not increase significantly; after long-term administration (once a week for 4 consecutive weeks), no liver and kidney tissue pathological damage was observed [3]
- Median lethal dose (LD50): The LD50 for mice via intravenous injection was 40 mg/kg, the LD50 via intraperitoneal injection was 55 mg/kg, and the LD50 via oral administration was 120 mg/kg [3]
- Immunosuppression: After administration to rats, the proportion of peripheral blood CD4⁺ and CD8⁺ T cells decreased by 30%, and the antibody production capacity decreased by 25%, which recovered 6 weeks after drug withdrawal [2]

[1]. J Biol Chem . 1990 Sep 25;265(27):16617-25.

[2]. Invest New Drugs . 1984;2(3):263-5.

[3]. Cancer Res . 1982 Jul;42(7):2587-91.

Fludarabine phosphate is a purine arabinoside monophosphate with the nucleobase 2-fluoroadenine. As a prodrug, it is rapidly dephosphorylated to 2-fluoro-ara-A, and then phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate 2-fluoro-ara-ATP. Once incorporated into DNA, 2-fluoro-ara-ATP acts as a DNA chain terminator. It is used to treat adult patients with B-cell chronic lymphocytic leukemia (CLL) who have failed or experienced disease progression to at least one standard alkylating agent regimen. It possesses multiple functions, including antimetabolite, antitumor, immunosuppressive, antiviral, prodrug, and DNA synthesis inhibitor. It is an organofluorine compound, nucleoside analog, and purine arabinoside monophosphate. Functionally, it is related to 2-fluoroadenine. Fludarabine phosphate is the phosphate salt of the fluorinated nucleotide antimetabolite analog of the antiviral drug vidarabine (ara-A) and exhibits antitumor activity. Fludarabine phosphate is rapidly dephosphorylated to 2-fluoro-ara-A, and then phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate 2-fluoro-ara-ATP. This metabolite inhibits DNA polymerase α, ribonucleotide reductase, and DNA primase, thereby blocking DNA synthesis and inhibiting tumor cell growth. Fludarabine (marketed under the brand name Fludara, trade name fludarabine phosphate) is a chemotherapeutic drug used to treat hematologic malignancies. See also: Fludarabine (containing the active fraction). Mechanism of action: Fludarabine phosphate is a purine nucleoside analog. It is dephosphorylated in vivo to form fludarabine, which is then phosphorylated into cells to its triphosphate derivative (F-ara-ATP). It can competitively incorporate into DNA/RNA chains, inhibiting nucleic acid synthesis and ribonucleotide reductase activity, thereby reducing the supply of deoxynucleotides and ultimately inducing tumor cell apoptosis [1]
- Indications: Used to treat hematologic malignancies, such as chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) [3]
- Resistance mechanisms: Some leukemia cells develop resistance by upregulating the expression of nucleoside transporter 1 (ENT1) to reduce intracellular drug concentration or by enhancing the activity of DNA repair enzymes (e.g., PARP) [3]
- Route of administration: Can be administered intravenously or orally, with the oral dose being twice that of the intravenous dose (because the oral bioavailability is approximately 50%) [2]

C10H13FN5O7P

365.21

365.053

C, 32.89; H, 3.59; F, 5.20; N, 19.18; O, 30.67; P, 8.48

75607-67-9

30751

White solid powder

2.4±0.1 g/cm3

864.2±75.0 °C at 760 mmHg

203 °C(dec.)

476.4±37.1 °C

0.0±0.3 mmHg at 25°C

1.879

0.41

5

12

4

24

514

4

P(=O)(O[H])(O[H])OC([H])([H])[C@]1([H])[C@]([H])([C@@]([H])([C@]([H])(N2C([H])=NC3=C(N([H])[H])N=C(N=C23)F)O1)O[H])O[H]

GIUYCYHIANZCFB-FJFJXFQQSA-N

InChI=1S/C10H13FN5O7P/c11-10-14-7(12)4-8(15-10)16(2-13-4)9-6(18)5(17)3(23-9)1-22-24(19,20)21/h2-3,5-6,9,17-18H,1H2,(H2,12,14,15)(H2,19,20,21)/t3-,5-,6+,9-/m1/s1

[(2R,3S,4S,5R)-5-(6-amino-2-fluoropurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate

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 (6.85 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 (6.85 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 (6.85 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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Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL

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Solubility in Formulation 5: 18.33 mg/mL (50.19 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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Solubility in Formulation 6: 20 mg/mL (54.76 mM) in phosphate buffer Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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