KW-2449 is a multiple-targeted inhibitor, mostly for Flt3 with IC50 of 6.6 nM, modestly potent to FGFR1, Bcr-Abl and Aurora A; little effect on PDGFRβ, IGF-1R, EGFR. Phase 1.
|製品説明||KW-2449 is a multiple-targeted inhibitor, mostly for Flt3 with IC50 of 6.6 nM, modestly potent to FGFR1, Bcr-Abl and Aurora A; little effect on PDGFRβ, IGF-1R, EGFR. Phase 1.|
|特性||Investigated as a FLT3 inhibitor in clinical trials, with others in early development.|
KW-2449, a multikinase inhibitor of FLT3, ABL, ABL-T315I, and Aurora kinase, is under investigation to treat leukemia patients. KW-2449 shows the potent growth inhibitory effects on leukemia cells with FLT3 mutations by inhibition of the FLT3 kinase, resulting in the down-regulation of phosphorylated-FLT3/STAT5, G1 arrest, and apoptosis. Oral administration of KW-2449 shows dose-dependent and significant tumor growth inhibition in FLT3-mutated xenograft model with minimum bone marrow suppression. In FLT3 wild-type human leukemia, KW-2449 induces the reduction of phosphorylated histone H3, G2/M arrest, and apoptosis. In Imatinib-resistant leukemia, KW-2449 contributes to release of the resistance by the simultaneous down-regulation of BCR/ABL and Aurora kinases. The inhibitory activity of KW-2449 is not affected by the presence of human plasma protein, such as α1-acid glycoprotein. KW-2449 has potent growth inhibitory activity against various types of leukemia by several mechanisms of action. KW-2449 has significant activity and warrants clinical study in leukemia patients with FLT3 mutations as well as Imatinib-resistant mutations. Phosphorylation levels of FLT3 and STAT5 are decreased by KW-2449 in a dose-dependent manner. In addition, it potently inhibits ABL-T315I, which is associated with IM resistance, with IC50 of 4 nM. On the other hand, KW-2449 has little effect on PDGFRβ, IGF-1R, EGFR, and various serine/threonine kinases even at a concentration of 1 μM. KW-2449 has the potent growth inhibitory activities against not only FLT3/ITD-expressing leukemia cells but also FLT3/KDM-activated and wild-type FLT3-overexpressing leukemia cells. In accordance with growth inhibitory effect, KW-2449 suppresses the phosphorylations of FLT3 (P-FLT3) and its downstream molecule phospho-STAT5 (P-STAT5) in MOLM-13 cells in a dose-dependent manner. Furthermore, KW-2449 increases the percentage of cells in the G1 phase of the cell cycle and reciprocally reduces the percentage of cells in the S phase, resulting in the increase of apoptotic cell population. KW-2449 can dephosphorylate constitutively active WT-FLT3 kinase but not inhibit the proliferation of leukemia cells if they are not mainly addicted to FLT3 the kinase.  KW-2449 is rapidly absorbed and converted to a major metabolite M1.Preclinical studies reveal that KW-2449 is converted by monoamine oxidase-B (MAO-B) and aldehyde oxidase into its major metabolite M1.KW-2449 mediates cytotoxicity thru inhibition of FLT3/ITD.KW-2449 is a direct inhibitor of FLT3 and induces inhibition of its downstream target STAT5.  KW2449 interacts synergistically with HDACIs to induce apoptosis in Ph+ CML cells in a time- and concentration-dependent manner. KW2449 synergistically enhances the lethality of vorinostat/SNDX275 in CML cells.KW-2449 regimens are active against additional IM-resistant Bcr/Abl+ leukemia cells. KW2449 moderately reduces phosphorylation of histone H3, an indicator of Aurora B activity, in nocodozole-treated K562 cells. 
|体内試験||In the MOLM-13 tumor xenograft model, oral administration of KW-2449 for 14 days shows a potent and significant antitumor effect in a dose-dependent manner. |
FLT3 phosphorylation:Leukemia cells are washed in phosphate-buffered saline (PBS), then lysed by resuspending the cells in lysis buffer (20 mM Tris pH 7.4, 100 mM NaCl, 1% Igepal, 1 mM EDTA, 2 mM NaVO4, plus Complete protease inhibitor KW-2449 for 30 minutes while rocking. The extract is clarified by centrifugation at 1.6 × 104 g and the supernatant is assayed for protein (Bio-Rad). A 50-μg aliquot is removed as a whole-cell lysate for analysis of STAT5, and the remainder is used for immunoprecipitation with anti-FLT3. Anti-FLT3 antibody is added to the extract for overnight incubation, then protein A sepharose is added for 2 additional hours. Separate sodium dodecyl sulfate–polyacrylamide electrophoresis (SDS-PAGE) gels for whole-cell lysate and immunoprecipates are run in parallel. After transfer to Immobilon membranes, immunoblotting is performed with antiphosphotyrosine antibody (4G10) to detect phosphorylated FLT3 or, for the whole-cell lysate gels, with a rat monoclonal antibody against phosphorylated STAT5 (residue Y694) then stripped and reprobed with anti-FLT3 antibody to measure total FLT3. Proteins are visualized using chemiluminescence, exposed on Kodak BioMax XAR film, developed, and scanned using a Bio-Rad GS800 densitometer. The concentration of KW-2449 for which the phosphorylation of FLT3 or STAT5 is inhibited to 50% of its baseline (IC50) is determined using linear regression analysis of the dose response curves. For direct analysis of FLT3 and STAT5 in circulating blasts, peripheral blood is collected in heparinized tubes and promptly chilled on ice. Samples are centrifuged for 10 minutes at 900 g, at 4 °C. The plasma is removed and stored frozen at −80 °C. The buffy coat is carefully transferred to ice-cold PBS, layered onto chilled Ficoll-Hypaque, and centrifuged for 5 minutes at 600 g, at 4 °C. All subsequent steps are carried out at 4 °C. Mononuclear cells are collected and washed rapidly once in red blood cell lysis buffer (0.155 M NH4Cl, 0.01 M KHCO3, 0.1 mM EDTA), then washed once in PBS. Cells are then lysed as described for FLT3 and STAT5 analysis.
|体外||DMSO||67 mg/mL (201.56 mM)|
|Ethanol||67 mg/mL (201.56 mM)|
質量 (g) = 濃度 (mol/L) x 体積 (L) x 分子量 (g/mol)
開始濃度 x 開始体積 = 最終濃度 x 最終体積
この方程式は、一般に略語を使われます：C1V1 = C2V2 ( 入力 出力 )
チップス: 化学式は大文字と小文字の区別ができます。C10H16N2O2 c10h16n2o2
|NCT Number||Recruitment||Conditions||Sponsor/Collaborators||Start Date||Phases|
|NCT00779480||Terminated||Acute Myelogenous Leukemia (AML)||Kyowa Hakko Kirin Pharma Inc.|Kyowa Kirin Pharmaceutical Development Inc.||January 2009||Phase 1|
|NCT00346632||Terminated||Acute Myelogenous Leukemia|Acute Lymphoblastic Leukemia|Myelodysplastic Syndromes|Chronic Myelogenous Leukemia||Kyowa Kirin Pharmaceutical Development Inc.||June 2006||Phase 1|