Flt-1 (IC50 = 3 nM); KDR (IC50 = 4 nM); PDGFRβ (IC50 = 66 nM); FLT3 (IC50 = 4 nM); CSF-1R (IC50 = 3 nM); Kit (IC50 = 14 nM)

Linifanib shows inhibitory to Kit, PDGFRβ and Flt4 with IC50 of 14 nM, 66 nM and 190 nM in kinases assay. With IC50 values of 2 nM, 2 nM, 31 nM, and 10 nM at the cellular level, linifanib also prevents ligand-induced phosphorylation of KDR, PDGFRβ, Kit, and CSF-1R; the cellular potency of this inhibition may be influenced by serum protein. A 0.2 nM IC50 is used by linifanib to inhibit the proliferation of HUAECs stimulated by VEGF. Although MV4-11 leukemia cells (which have constitutively active Flt3 with an IC50 of 4 nM) are the only tumor cells against which linifanib exhibits weak activity that is not induced by VEGF or PDGF. In MV4-11 cells, linifanib may raise the sub-G0-G1 apoptotic population while causing a decrease in the S and G2-M phases. The ATP-binding site of CSF-1R is bound by linifanib, and its Ki is 3 nM. In Ba/F3 FLT3 ITD cell lines, linifanib (10 nM) showed decreased phosphorylation of GSK3β at Ser9 and reduced phosphorylation of Akt at Ser473.[3]

Linifanib (0.3 mg/kg) results in complete inhibition of KDR phosphorylation in lung tissue. With an ED50 of 0.5 mg/kg, linifanib also suppresses the edema response. Both bFGF- and VEGF-induced angiogenesis in the cornea are markedly inhibited by linifanib (7.5 and 15 mg/kg, bid). In flank xenograft models, linfanib (ED75, 4.5–12 mg/kg) inhibits tumor growth in HT1080, H526, MX-1, and DLD-1. Low doses of linifanib also demonstrate efficacy in A431 and MV4-11 xenografts. The MDA-231 xenograft shows a reduction in microvasculure density when treated with linifanib (12.5 mg/kg bid). AUC and Cmax of linifanib in the HT1080 fibrosarcoma model are 0.4 μg/mL and 2.7 μg•hour/mL, respectively, after 24 hours.[1]

Assays of active kinase domains that have been cloned and expressed in baculovirus using the FastBacbaculovirus expression system or that can be purchased commercially are used to determine potencies (IC50 values). In homogenous time-resolved fluorescence assays for tyrosine kinase assays, a biotinylated peptide substrate with a single tyrosine is utilized along with 1 mM ATP, an Eu-cryptate-labeled anti-phosphotyrosine antibody (PT66), and streptavidin-APC. 5 μM ATP, [³³P]ATP, and a biotinylated peptide substrate with peptide capture and incorporation of ³³P ascertained using an SA-Flashplate are used in the assay of serine/threonine kinases. Multiple concentrations of linifanib are analyzed by serial dilution of a linifanib stock solution in DMSO. The concentration response data is subjected to nonlinear regression analysis to determine the concentration at which 50% of activity is inhibited.

A 96-well plate is seeded with 2.5 × 10³ cells per well, and the cells are then incubated for 24 hours in a serum-free medium. Serum-free medium is used for the 72-hour incubation period after the addition of VEGF (10 ng/mL) and linifanib. Full growth medium is plated overnight with 3 × 10³ cells/well for carcinoma cell lines. Following a 72-hour incubation period, linifanib is added to the cells in their complete growth medium. Leukemia cells are typically plated at a density of 5 × 10⁴ per well in full growth medium, followed by the addition of Linifanib and a 72-hour incubation period. When Alamar Blue (final solution, 10%), CO₂ incubator, and 4 hours of 37 °C incubation are added, the effects on proliferation are measured using a fluorescence plate reader (544 nm, excitation: 590 nm, emission

H526, DLD-1, MDA-231, MDA-435LM, HCT-116, H526, DLD-1, MDA-231, MDA-435LM, MV4-11 and MX-1 xenografts are established in mice.
~ 10 mg/kg
Oral administration

[1]. Mol Cancer Ther . 2006 Apr;5(4):995-1006.

[2]. Mol Cancer Ther . 2006 Apr;5(4):1007-13.

[3]. Mol Cancer Ther . 2011 Jun;10(6):949-59.

[4]. J Hepatol . 2008 Dec;49(6):985-97.

[5]. Mol Cancer Ther . 2006 Apr;5(4):995-1006.

Linifanib is a member of the class of phenylureas that is urea in which one of the nitrogens is substituted by a 2-fluoro-5-methylphenyl group, while the other is substituted by a p-(3-amino-1H-indazol-4-yl)phenyl group. It is a potent, selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases. It has a role as an antineoplastic agent, an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor and an angiogenesis inhibitor. It is an aromatic amine, a member of indazoles and a member of phenylureas.
Linifanib (ABT-869) is a small molecule vascular endothelial growth factor (VEGF) receptor-based kinase inhibitor that is designed to suppress tumor growth by preventing the formation of new blood vessels that supply the tumor with oxygen and nutrients and by inhibiting key angiogenic signaling pathways. Linifanib is intended for the treatment of hematologic malignancies and the solid tumors.
Linifanib is an orally bioavailable receptor tyrosine kinase (RTK) inhibitor with potential antineoplastic activity. Linifanib inhibits members of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor families; it exhibits much less activity against unrelated RTKs, soluble tyrosine kinases, or serine/threonine kinases. This agent does not have a general antiproliferative effect due to its high dose requirement. However, linifanib may exhibit potent antiproliferative and apoptotic effects on tumor cells whose proliferation is dependent on mutant kinases, such as FMS-related tyrosine kinase receptor-3 (FLT3).

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Drug Indication
Investigated for use/treatment in leukemia (myeloid), myelodysplastic syndrome, and solid tumors.

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Mechanism of Action
ABT-869, a multi-targeted receptor tyrosine kinase inhibitor, has been shown to inhibit of all members of the VEGF and PDGF receptor families (e.g., KDR IC50 value of 4 nM), and have less activity (IC50 values >1 µM) against unrelated receptor tyrosine kinases, soluble tyrosine kinases and serine/threonine kinases. In addition, it exhibits potent anti-proliferative and apoptotic effects on tumor cells dependent on mutant, constitutively active, FLT3 and KIT kinases.

C21H18FN5O

375.41

375.149

C, 67.19; H, 4.83; F, 5.06; N, 18.66; O, 4.26

796967-16-3

Linifanib;796967-16-3

11485656

White to gray solid powder

1.4±0.1 g/cm3

542.2±50.0 °C at 760 mmHg

180-183ºC (dec.)

281.7±30.1 °C

0.0±1.4 mmHg at 25°C

1.766

4.34

4

4

3

28

541

0

O=C(NC1C(F)=CC=C(C)C=1)NC1C=CC(C2C3=C(NN=C3N)C=CC=2)=CC=1

MPVGZUGXCQEXTM-UHFFFAOYSA-N

InChI=1S/C21H18FN5O/c1-12-5-10-16(22)18(11-12)25-21(28)24-14-8-6-13(7-9-14)15-3-2-4-17-19(15)20(23)27-26-17/h2-11H,1H3,(H3,23,26,27)(H2,24,25,28)

1-[4-(3-amino-1H-indazol-4-yl)phenyl]-3-(2-fluoro-5-methylphenyl)urea

AL39324; ABT-869; RG3635; ABT869; AL 39324; RG-3635; ABT 869; RG 3635; AL-39324

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: ≥ 1.32 mg/mL (3.52 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 13.2 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: ≥ 1.32 mg/mL (3.52 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 13.2 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: ≥ 1.32 mg/mL (3.52 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 13.2 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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