Fludarabine

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Biological Activity

Description	Fludarabine is a STAT1 activation inhibitor and a DNA synthesis inhibitor.
Targets
IC50
In vitro	Fludarabine efficiently inhibits the proliferation of RPMI 8226 cells with IC50 of 1.54 μg/mL. The IC50 of Fludarabine against MM.1S and MM.1R cells is 13.48 μg/mL and 33.79 μg/mL, respectively. In contrast, U266 cells are resistant to Fludarabine with IC50 of 222.2 μg/mL. Fludarabine treatment results in increased number of cells in the G1 phase of cell cycle, accompanied with a concomitant reduction of cells at the S phase of cell cycle in a time-dependent manner. Fludarabine induces a cell cycle block and triggers apoptosis in MM cells. Fludarabine triggers time-dependent cleavage of caspase-8, -9, and -3, -7, followed by PARP cleavage. Fludarabine increases expression of Bax in a time-dependent fashion, while the expression of Bak doesn’t change. After exposure to Fludarabine for 12 hours, RPMI 8226 cells shows a loss of membrane potential with 61.05% of the cells expressing low fluorescence of rhodamine 123 compared with 8.62% of cells in untreated control. [1] To enhance solubility, Fludarabine is formulated as the monophosphate (F-ara-AMP, fudarabine), which is instantaneously and quantitatively dephosphorylated to the parent nucleoside upon intravenous infusion. Inside the cells rephosphorylation occurs which leads to fuoroadenine arabinoside triphosphate (F-ara-ATP), the major cytotoxic metabolite of F-ara-A. [2] Fludarabine can also induce pro-inflammatory stimulation of monocytic cells, as evaluated by increased expression of ICAM-1 and IL-8 release. [3]Fludarabine does not affect the growth of ovarian cancer cell lines, whereas it induces marked and dose-dependent inhibition of proliferation in melanoma cell lines. [4] Fludarabine induces significant reduction of STAT-1 phosphorylation, whereas it does not change JAK2 activation. Interestingly, Fludarabine does not significantly affect the phosphorylation of these three STAT proteins. Fludarabine (1.5 mg) significantly prevents STAT-1 phosphorylation and also reduces the increased amount of this protein. No significant changes are demonstrated in JAK2 phosphorylation at 2 days, but Fludarabine inhibits JAK2-increased expression at 7 days. Fludarabine specifically inhibits STAT-1 activation without affecting other STAT proteins and consequently diminishes VSMC proliferation. [5]
In vivo	Tumors treated with PBS grow rapidly to approx-imately 10-fold their initial volume in 25 day, whereas, the tumors in the Fludarabine at 40 mg/kg increase less than 5-fold. A significant antitumor effect of 40 mg/kg Fludarabine on RPMI8226 tumor growth is demonstrated. RPMI8226 tumors treated with 40 mg/kg Fludarabine at day 10 increase apoptotic nuclei. Fludarabine is effective in suppressing RPMI8226 myeloma xenografts in SCID mice. [1]
Features

Protocol(Only for Reference)

Cell Assay: ^[1]

Cell lines	Dexamethasone-sensitive (MM.1S) and -resistant (MM.1R) human MM cell lines, RPMI8226 and U266 cell lines
Concentrations	2 μg/mL
Incubation Time	24 hours
Method	After treated with Fludarabine or control, dexamethasone-sensitive (MM.1S) and -resistant (MM.1R) human MM cell lines, RPMI8226 and U266 cell lines (5 × 105 cells) are washed twice in phosphate-buffered saline (PBS) and fixed with 70% ice-cold ethanol, then centrifuged and suspended in PBS containing 100 μg/mL RNase A. After incubated for 30 minutes at 37 ºC, samples are resuspended in 25 μg/mL propidium iodide. Flow cytometry is performed on a FACSCalibur automated system. Apoptosis is determined by Annexin V-FITC apoptosis detection kit, according to the manufacturer’s instructions. For TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) assay, cells are analyzed by flow cytometry using the in situ cell death detection kit.

Animal Study: ^[1]

Animal Models	Severe combined immunodeficient (SCID) mice bearing RPMI 8226 cells
Dosages	40 mg/kg
Administration	Administered via i.p.
Solubility	30% propylene glycol, 5% Tween 80, 65% D5W, 30 mg/mL

1

References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT01652014	Suspended	Accelerated Phase Chronic Myelogenous Leukemia|Acute Myeloid Leukemia With Multilineage Dysplasia Following Myelodysplastic Syndrome|Adult Acute Lymphoblastic Leukemia in Remission|Adult Acute Myeloid	Rutgers, The State University of New Jersey|University of Medicine and Dentistry New Jersey|National Cancer Institute (NCI)	2014-01	Phase 2
NCT01962636	Not yet recruiting	Acute Myeloid Leukemia (AML)|Acute Lymphocytic Leukemia (ALL)|Chronic Myelogenous Leukemia|Plasma Cell Leukemia|Myelofibrosis|Myelodysplasia|Chronic Lymphocytic Leukemia|Small Lymphocytic Lymphoma|Mar	Masonic Cancer Center, University of Minnesota	2014-01
NCT01163201	Not yet recruiting	Hematologic Malignancy|Acute Myeloid Leukemia|Acute Lymphocytic Leukemia|Chronic Myelogenous Leukemia in Blast Crisis|Anemia, Refractory, With Excess of Blasts|Chronic Myeloproliferative Disease|Chron	Masonic Cancer Center, University of Minnesota	2014-01	Phase 1|Phase 2
NCT01904136	Not yet recruiting	Leukemia|Myeloproliferative Diseases	M.D. Anderson Cancer Center	2014-02	Phase 1|Phase 2
NCT01787474	Not yet recruiting	Leukemia	M.D. Anderson Cancer Center	2014-05	Phase 1|Phase 2

Chemical Information

Download Fludarabine SDF

Chemical Name	(2R,3S,4S,5R)-2-(6-amino-2-fluoro-9H-purin-9-yl)-5-(hydroxymethyl)-tetrahydrofuran-3,4-diol

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.28523	2.8523	5.7046	8.5569