AG-490

AG-490 inhibits HER-2 driven cell proliferation with IC50 of 3.5 μM. ^[1] Corresponding to the specific dose-dependent inhibition of constitutively activated JAK2 in pre-B acute leukemia (ALL) cells, AG-490 (5 μM) almost completely blocks the growth of all ALL cells by inducing programmed cell death, with no deleterious effect on normal hematopoiesis. AG-490 does not inhibit the activities of Lck, Lyn, Btk, Syk, and Src. ^[2] AG-490 (60-100 μM) blocks the constitutive activation of Stat3sm, and inhibits spontaneous as well as interleukin 2-induced growth of mycosis fungoides (MF) tumor cells with IC50 values of 75 μM and 20 μM, respectively. ^[3] AG-490 potently inhibits IL-2-mediated human T cell growth with an IC50 of 25 μM by blocking the activities of JAK3 and STAT5a/b. ^[4] AG-490 significantly inhibits the constitutive activation of Stat3 in MOPC, MPC11, and S194 cells, leading to dramatic dose-dependent apoptosis. ^[6] AG-490 (100 μM) inhibits Akt phosphorylation, inhibits the activation of nuclear factor-κB, and causes the activation of GSK-3β, leading to the reduction of c-Myc. AG-490 (50 μM) can induce apoptosis of BaF3 cells expressing T315I and E255K mutants of Bcr-Abl. ^[7] AG-490 at 30 μM inhibits not only Epo-induced phosphorylation of wild-type JAK2 but also constitutive phosphorylation of the JAK2 V617F mutant. AG-490 also potently inhibits cytokine-independent cell growth induced by the JAK2 V617F mutant in BaF3 cells. ^[8]

Administration of AG-490 drastically reduces the numbers of CD45⁺ and HLA-DR⁺ cells from 48 % and 46% in bone marrow of untreated mice, as well as 38% and 22% in the spleen of untreated mice to undetectale levels. ^[2] In vivo administration of AG-490 causes murine myeloma tumor cell apoptosis but does not inhibit IL-12-mediated macrophage activation and IFN-γ production by lymphocytes. ^[6] Consistent with the in vitro blocking of JAK2 V617F mutant activity, AG-490 treatment at 0.5 mg/day for 10 days effectively inhibits JAK2 V617F mutant-induced tumorigenesis and tumor cell invasion in nude mice. ^[8] Combined therapy with AG-490 and IL-12 induces greater antitumor effects than either agent alone in a murine myeloma tumor model. ^[6]

• In vitro kinase autophosphorylation

  AG-490 is dissolved in DMSO 10%-H₂O-ethanol 45%. Crude membrane extracts (0.125 μg/mL) are preactivated with EGF (20 nM) in 50 mM HEPES buffer, pH 7.6, and 125 mM NaCl, for 15 minutes at 4 °C. Autophosphorylation activity of EGFR or ErbB2 kinase is assayed at 4 °C for 30 seconds in V-shaped 96-well plates. Membrane extracts (8 μL) are added to each well containing reaction mixture (12 μL, 50mM, HEPES, pH 7.4,125 mM NaCl, 12 mM M₈Ac₂, 2 mM MnCl₂, 1 mM NaVO₃, 1 μM ATP, and 1 μCi[γ-³²P]ATP, final concentrations) and increasing concentrations of AG-490 (4 μL). After termination by addition of hot sample buffer, the samples are run on a 6% SDS-polyacrylamide gel electrophoresis minigel, the gels dried, and autoradiography performed during the linear exposure time period. The receptor bands are scanned densitrometrically, and the results analyzed by the Ez-Fit program. For the analysis of autophosphorylation of JAK2, JAK2 is immunoprecipitated by using anti-JAK2 antibody from lysates of G2 cells pretreated for 16 hours with increasing concentrations of AG-490 (0-50 μM). Immune complexes are then immunoblotted with anti-phosphotyrosine antibody. A dose-dependent inhibition of in vitro kinase activity is demonstrated by assessing JAK2 autophosphorylation.

• Cell lines

  Pre-B ALL

• Concentrations

  Dissolved in DMSO, final concentrations ~ 50 μM

• Incubation Time

  16 hours

• Method

  Cells are exposed to different concentrations of AG-490 for 16 hours. For the determination of cell proliferation, [³H]tymidine (1 μCi) is added 6 hours or more before the cultures are terminated. Cells are then collected and samples counted in a liquid scintillation counter.

• Animal Models

  SCID mice intravenously injected with ALL cells

• Dosages

  0.85 mg + 0.5 mg daily

• Administration

  Continuous pump infusion supplemented with daily intraperitoneal injections