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Research Area
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Description
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Solid tumours, Acute myeloid leukaemia |
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Biological Activity
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Description
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AC220 (Quizartinib) is a potent and selective inhibitor of Flt3ITD and Fltwt with IC50 of 1.1 nM and 4.2 nM, respectively. |
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Targets
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Flt3ITD |
Fltwt |
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IC50 |
1.1 nM [1] |
4.2 nM [1] |
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In Vitro
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AC220, a unique, potent and selective inhibitor of FLT3, has high affinity for FLT3 with a Kd value of 1.6 nM. AC220 inhibits the autophosphorylation of FLT3 in the human leukemia cell lines MV4-11 which harbor a homozygous FLT3-ITD mutation and is FLT3 dependent, and RS4;11 which expresses wild-type FLT3 with IC50 values of 1.1 nM and 4.2 nM, respectively. AC220 is the most potent cellular FLT3-ITD inhibitor, leading to the most significant inhibition of MV4-11 cell proliferation with IC50 of 0.56 nM compared to all other FLT3 inhibitors whose IC50 values range from 0.87 nM to 64 nM. AC220 has no inhibitory activity against the proliferation of A375 cells which harbor an activating mutation in BRAF and are not FLT3 dependent, indicating a large window between FLT3 inhibition and general cytotoxic effects. [1] |
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In Vivo
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Oral administration of AC220 (10 mg/kg) induces time-dependent inhibition of FLT3 autophosphorylation in the FLT3-ITD–dependent MV4-11 tumor xenograft mouse model; the inhibition being 90% at 2 hours and 40% at 24 hours. AC220 significantly extends survival in a mouse model of FLT3-ITD AML with doses as low as 1 mg/kg given orally once a day. Treatment with AC220 at 10 mg/kg for 28 days results in rapid and complete regression of tumors in all mice with no tumor regrowth during the 60-day posttreatment period. AC220 displays more significant efficacy compared to sunitinib treatment which causes tumors to shrink slowly and resume growth immediately upon discontinuation of treatment in all but one of the mice. [1] |
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Clinical Trials
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A Phase I study of AC220 in patients with relapsed/refractory acute myeloid leukemia, regardless of FLT3 status, has been completed. |
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Features
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AC220 is the most potent cellular FLT3-ITD inhibitor. |
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Protocol
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Kinase Assay
[1]
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| Inhibition of FLT3 autophosphorylation |
To measure inhibition of FLT3 autophosphorylation, MV4-11 or RS4;11 cells are cultured in low serum media (0.5% FBS) overnight and seeded at a density of 400 000 cells per well in a 96-well plate the following day. The cells are incubated with different concentrations of AC220 for 2 hours at 37 °C. To induce FLT3 autophosphorylation in RS4;11 cells, 100 ng/mL FLT3 ligand is added for 15 minutes after the 2-hour AC220 incubation. Cell lysates are prepared and incubated in 96-well plates precoated with a total FLT3 capture antibody. The coated plates are incubated with either a biotinylated antibody against FLT3 to detect total FLT3 or an antibody against phosphotyrosines to detect FLT3 autophosphorylation. In both cases, a SULFO-tagged streptavidin secondary antibody is used for electrochemiluminescence detection on the Meso Scale Discovery platform. The concentration of AC220 that inhibits FLT3-ITD or TLT3-WT autophosphorylation by 50% represents IC50 values. |
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Cell Assay
[1]
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| Cell Lines |
MV4-11 and RS4;11 cells |
| Concentrations |
Dissolved in DMSO, final concentration ~20 μM |
| Incubation Time |
72 hours |
| Methods |
Cells are cultured overnight in low serum media (0.5% FBS), seeded in a 96-well plate at 40 000 cells per well and exposed to AC220 for 72 hours at 37 °C. Cell viability is measured using the Cell Titer-Blue Cell Viability Assay. |
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Animal Study
[1]
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| Animal Models |
Female NU/NU or severe combined immunodeficient mice implanted with MV4-11 cells |
| Formulation |
Formulated in 22% hydroxypropyl-β-cyclodextrin |
| Doses |
~10 mg/kg |
| Administration |
Oral gavage |
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