AC220

Biological Activity

AC220 (Quizartinib) is a orally-administered, potent and selective inhibitor of two receptor tyrosine kinases, FLT3 and KIT. AC220 is currently in a Phase 2 clinical study in the relapsed/refractory AML patient population with internal tandem duplication (ITD positive) activating mutations of the FLT3 kinase (FLT3-ITD positive). AC220 (Quizartinib) exhibits low nanomolar potency in biochemical and cellular assays and exceptional kinase selectivity, and in animal models is efficacious at doses as low as 1 mg/kg given orally once daily. While other agents showed some ability to clear leukemia from the circulating blood, AC220 has shown significant ability to clear leukemic cells from the bone marrow, where the disease originates. AC220-resistant FLT3 kinase domain mutants represent high-value targets for future FLT3 inhibitor development efforts.

Product Citations

• 

  Drug Des Devel Ther. 2018 Apr 30;12:1009-1017.

  Advances in the drug therapies of acute myeloid leukemia (except acute promyelocytic leukemia)
  AC220 purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Mol Cancer (2013). Figure 3. AC220
	Method	PCR
	Cell Lines	Ba/F3 cells
	Concentrations	0 – 5000 nM
	Incubation Time	48 h
	Results	Comparison of inhibition of cellular proliferation after quizartinib treatment revealed strong correlation between naturally occurring and engineered cell lines expressing identical mutant kinases

	Source	Mol Cancer (2013). Figure 2. AC220
	Method	cell apoptosis assay
	Cell Lines	leukemia cell line
	Concentrations	0 – 5000 nM
	Incubation Time	48 h
	Results	This finding suggests that the distinct mutant-KIT isoform directly orchestrates sensitivity towards quizartinib.

Protocol (for reference only)

Cell Experiment
Cell lines	MV4-11 and RS4;11 cells
Preparation method	Cellular assays. MV4-11 and RS4;11 cells were cultured in Iscove media with 10% fetal bovine serum (FBS) and RPMI complete with 10% FBS, respectively. For proliferation assays, cells were cultured overnight in low serum media (0.5% FBS), then seeded in a 96-well plate at 40 000 cells per well. Inhibitors were added to the cells and incubated at 37°C for 72 hours. Cell viability was measured using the Cell Titer-Blue Cell Viability Assay from Promega. To measure inhibition of FLT3 autophosphorylation, cells were 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 were incubated with inhibitors for 2 hours at 37°C. To induce FLT3 autophosphorylation in RS4;11 cells, 100 ng/mL FLT3 ligand (R&D Systems) was added for 15 minutes after the 2-hour compound incubation. Cell lysates were prepared and incubated in 96-well plates precoated with a total FLT3 capture antibody (R&D Systems). The coated plates were incubated with either a biotinylated antibody against FLT3 (R&D Systems) to detect total FLT3 or an antibody against phosphotyrosines (Millipore) to detect FLT3 autophosphorylation. In both cases, a SULFO-tagged streptavidin secondary antibody was used for electrochemiluminescence detection on the Meso Scale Discovery platform.
Concentrations	0~10 µM
Incubation time	72 h

Animal Experiment
Animal models	subcutaneous tumor model
Formulation	22% hydroxypropyl-β-cyclodextrin
Dosages	10 mg/kg
Administration	orally

Chemical Information

Molecular Weight	560.67
Formula	C29H32N6O4S
CAS Number	950769-58-1
Solubility (25°C)	DMSO ≥100 mg/mL
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Smith et al. Nature. Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia.

[2] Zarrinkar et al. Blood. AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML).