Background
Quizartinib (AC220) is a 2nd-generation FLT3 inhibitor for Flt3(ITD/WT) with IC50 value of 1.1 nM/4.2 nM, and it is ten-fold more selective for Flt3 than PDGFRα, PDGFRβ, KIT, RET and CSF-1R [1].
Quizartinib inhibits FLT3 with low nanomolar potency in cellular assays and shows high selectivity when screened against most of the human protein kinome. In addition, the combination of high potency and selectivity exhibited by quizartinib is unique compared with CEP-701, PKC-412, MLN-518, sunitinib, and sorafenib.
Quizartinib (AC220) was identified to be the most potent and selective FLT3 inhibitor with good pharmaceutical properties and superior efficacy in tumor xenograft models. A single dose of 10 mg/kg was administered to mice by oral gavage and plasma levels were measured over a 24-hour period. Quizartinib was well absorbed, achieving a maximum plasma level (Cmax) of 3.8 μM (2100 ng/mL) within 2 hours of dosing. To determine the effect of FLT3-ITD inhibition on cell growth,. These results establish that AC220 has strong activity against FLT3 in biochemical and cellular assays in MV4-11 cell proliferation in the presence of 1.1nM quizartinib [1].
As a FLT3 inhibitor for the treatment of acute myeloid leukemia (AML), when at doses as low as 1 mg/kg orally once a day, quizartinib inhibits FLT3 activity in vivo extending survival significantly. And this eradicates tumors in a FLT3-dependent mouse xenograft model, and potently inhibits FLT3 activity in primary patient cells at a dose of 10 mg/kg. In addition, quizartinib has been demonstrated a desirable safety and PK profile in humans. The emergence of resistant mutations is a common mechanism of resistance to FLT3 inhibitors used clinically, with a mutation emerging in at least 20% of the patients. This shows that the survival of AML blasts depends to a great extent on FLT3 signaling in these cases [2, 3].
参考文献:
[1].? Zarrinkar PP, Gunawardane RN, Cramer MD, et al. AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML). Blood, 2009, 114(14): 2984-2992.
[2].?Chao Q, Sprankle KG, Grotzfeld RM, et al. Identification of N-(5-tert-Butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea Dihydrochloride (AC220), a Uniquely Potent, Selective, and Efficacious FMS-Like Tyrosine Kinase-3 (FLT3) Inhibitor. Journal of Medicinal Chemistry, 2009, 52(23): 7808-7816.
[3].? Alvarado Y , Kantarjian HM, Luthra R, et al. Treatment With FLT3 Inhibitor in Patients With FLT3-Mutated Acute Myeloid Leukemia Is Associated With Development of Secondary FLT3-Tyrosine Kinase Domain Mutations. Cancer, 2014, 120(14): 2142-2149.
Protocol
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Kinase experiment [1]:
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Inhibition of FLT3 autophosphorylation
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To measure inhibition of FLT3 autophosphorylation, MV4-11 or RS4;11 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 different concentrations of AC220 for 2 hrs at 37 °C. To induce FLT3 autophosphorylation in RS4;11 cells, 100 ng/mL FLT3 ligand was added for 15 mins after the 2-hr AC220 incubation. Cell lysates were prepared and incubated in 96-well plates precoated with a total FLT3 capture antibody. The coated plates were 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 was used for electrochemiluminescence detection on the Meso Scale Discovery platform. The concentration of AC220 that inhibited FLT3-ITD or TLT3-WT autophosphorylation by 50% represented the IC50 value.
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Cell experiment [1]:
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Cell lines
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MV4-11 and RS4;11 cells
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Preparation method
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The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.
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Reaction Conditions
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20 μM; 72 hrs
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Applications
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In MV4-11 and RS4;11 cells, AC220 inhibited the autophosphorylation of FLT3, with the IC50 values of 1.1 nM and 4.2 nM, respectively.
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Animal experiment [1]:
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Animal models
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Mice bearing MV4-11 tumors
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Dosage form
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10 mg/kg; p.o.
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Applications
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In mice bearing MV4-11 tumors, AC220 inhibited FLT3 autophosphorylation in a time-dependent manner.
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Other notes
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Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.
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参考文献:
[1]. Zarrinkar PP, Gunawardane RN, Cramer MD, et al. AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML). Blood, 2009, 114(14): 2984-2992.
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