VEGFR1 (IC50 = 2 nM); VEGFR2 (IC50 = 3 nM); VEGFR3 (IC50 = 6 nM)
Motesanib exhibits >1000 selectivity against EGFR, Src, and p38 kinase in addition to having broad activity against the human VEGFR family. With an IC50 of 10 nM, motesanib dramatically suppresses HUVECs' VEGF-induced cellular proliferation, but has minimal effect on bFGF-induced proliferation (IC50 of >3,000 nM). With an IC50 of 207 nM for PDGF-induced proliferation and 37 nM for SCF-induced c-kit phosphorylation, respectively, motesanib also potently inhibits these processes[1]. However, it is ineffective against EGF-induced EGFR phosphorylation and A431 cell viability. Despite having minimal effect on HUVECs' ability to proliferate, motesanib treatment greatly increases the cells' sensitivity to fractionated radiation[2].

Motesanib exhibits >1000 selectivity against EGFR, Src, and p38 kinase in addition to having broad activity against the human VEGFR family. With an IC50 of 10 nM, motesanib dramatically suppresses HUVECs' VEGF-induced cellular proliferation, but has minimal effect on bFGF-induced proliferation (IC50 of >3,000 nM). With an IC50 of 207 nM for PDGF-induced proliferation and 37 nM for SCF-induced c-kit phosphorylation, respectively, motesanib also potently inhibits these processes[1]. However, it is ineffective against EGF-induced EGFR phosphorylation and A431 cell viability. Despite having minimal effect on HUVECs' ability to proliferate, motesanib treatment greatly increases the cells' sensitivity to fractionated radiation[2].

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AMG 706 potently inhibited VEGF-induced proliferation of human umbilical vascular endothelial cells (HUVECs) with an IC50 of 10 nM, but did not inhibit bFGF-induced HUVEC proliferation (IC50 > 3,000 nM). [1]
AMG 706 inhibited PDGF-induced proliferation of normal human dermal fibroblasts (NHDFs) with an IC50 of 207 nM. [1]
AMG 706 inhibited SCF-induced Kit receptor phosphorylation in M07e human megakaryocytic leukemia cells with an IC50 of 37 nM. [1]
AMG 706 displayed no activity against EGF-induced EGFR phosphorylation in A431 human epidermoid carcinoma cells (IC50 > 25 µM). [1]
Incubation of A431 tumor cells with up to 25 µM AMG 706 for 3 days did not affect cell viability. [1]

Motesanib (100 mg/kg) significantly inhibits VEGF-induced vascular permeability in a time-dependent manner. Using the rat corneal model, oral administration of Motesanib twice daily or once daily potently inhibits VEGF-induced angiogenesis in a dose-dependent manner (ED50 of 2.1 mg/kg and 4.9 mg/kg, respectively). By specifically focusing on neovascularization in tumor cells, motesanib causes a dose-dependent tumor regression in well-established A431 xenografts[1]. In xenograft models of head and neck squamous cell carcinoma (HNSCC), motesanib in combination with radiation exhibits strong anti-tumor activity[2]. When used in conjunction with docetaxel or tamoxifen, motesanib treatment also significantly reduces the tumor growth and blood vessel density of MCF-7, MDA-MB-231, or Cal-51 xenografts in a dose-dependent manner[3].

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A single oral dose of AMG 706 (100 mg/kg) significantly inhibited VEGF-induced vascular permeability in a mouse model in a time-dependent manner, with significant inhibition observed at 2 and 4 hours post-dose. [1]
Oral administration of AMG 706 significantly inhibited VEGF-induced angiogenesis in a rat corneal model in a dose-dependent manner. Estimated ED50 values were 2.1 mg/kg (b.i.d. dosing) and 4.9 mg/kg (q.d. dosing). [1]
Oral administration of AMG 706 (10, 30, or 100 mg/kg/dose, b.i.d.) caused dose-dependent inhibition and regression of established A431 human epidermoid tumor xenografts in athymic nude mice. Treatment with 100 mg/kg/dose b.i.d. induced statistically significant tumor regression after 7 days, and continued treatment led to complete regression in some animals. [1]
Treatment of mice bearing large established A431 tumors (>400 mm³) with AMG 706 (100 mg/kg/dose, b.i.d.) resulted in rapid tumor regression. [1]
Mechanistic studies in A431 xenografts showed that treatment with AMG 706 (75 mg/kg/dose, b.i.d.) induced a significant increase in endothelial cell apoptosis and a decrease in blood vessel area within 24 hours, followed by a significant increase in tumor cell apoptosis at 48 hours. [1]

Homogeneous time-resolved fluorescence (HTRF) assays are used to determine suitable enzyme, ATP, and substrate (gastrin peptide) concentrations for each enzyme. Using a two-thirds Km ATP concentration for each enzyme, motesanib is tested in a 10-point dose-response curve. An enzyme is combined with kinase reaction buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 5 mM MnCl₂, 100 mM NaCl, 1.5 mM EGTA) in the majority of assays. Prior to each experiment, a final concentration of 20 μg/mL BSA, 0.2 mM NaVO₄, and 1 mM DTT is added. The HTRF reaction is preceded in all assays by the addition of 0.1125 nM Eu-PT66 and 5.75 mg/mL streptavidin-allophycocyanin. Using a Discovery instrument, plates are read after 30 minutes of room temperature incubation. Levenberg-Marquardt algorithm is used to calculate IC50 values, which are then entered into a four-parameter logistic equation.

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In vitro kinase inhibition assays were performed using homogeneous time-resolved fluorescence (HTRF) methods. Recombinant kinases were mixed with a kinase reaction buffer containing Tris-HCl, MgCl2, MnCl2, NaCl, and EGTA. DTT, sodium orthovanadate, and BSA were added prior to the assay. For most assays, ATP concentration was set at two-thirds of the Km for each enzyme. AMG 706 was tested in a 10-point dose-response curve. After incubation, streptavidin-allophycocyanin and a europium-labeled antibody were added for the HTRF reaction. Plates were incubated and read on a suitable instrument. IC50 values were calculated using a four-parameter logistic equation. [1]
A modified kinase reaction buffer with adjusted MnCl2 concentration was used for the Src kinase assay. [1]

After exposing the cells to either 50 ng/mL VEGF or 20 ng/mL bFGF for an extra 72 hours, the cells are preincubated for two hours at varying concentrations of motesanib. Plates are frozen for 24 hours at -70°C after cells are twice cleaned with DPBS. Plates are read using a Victor 1420 workstation, and proliferation is measured by adding CyQuant dye. The four-parameter logistic equation is derived from the IC50 data using the Levenberg-Marquardt algorithm.

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For the HUVEC proliferation assay, cells were seeded in 96-well plates and pre-incubated with serial dilutions of AMG 706 for 2 hours before stimulation with VEGF or bFGF. After 72 hours, cell proliferation was assessed using a fluorescent DNA-binding dye. [1]
For the PDGF proliferation assay, NHDFs were serum-starved, treated with AMG 706 for 2 hours, then stimulated with PDGF. DNA synthesis was measured by [14C]thymidine incorporation after 24 hours. [1]
For the EGFR phosphorylation assay in A431 cells, serum-starved cells were treated with AMG 706 for 2 hours, stimulated with EGF, and lysed. Phosphorylated EGFR was quantified using a bead-based immunoassay with a biotinylated anti-EGFR antibody and an anti-phosphotyrosine antibody. [1]
For the Kit phosphorylation assay in M07e cells, serum-starved cells were treated with AMG 706 for 2 hours, stimulated with SCF, and lysed. Kit was immunoprecipitated, followed by Western blotting using an anti-phosphotyrosine antibody and a Kit antibody for detection and quantitation. [1]

Penicillin, streptomycin, and glutamine are added to 10% FBS-containing DMEM (low glucose) medium for the cultivation of A431 cells. Trypsinization is used to extract the cells, which are then cleaned and concentrated to 5×107/mL in serum-free medium. Animals are given 1x107 cells in 0.2 mL over their left flank as a challenge. A little over ten days later, mice are treated with either vehicle (Ora-Plus) or motesanib, randomly assigned according to initial tumor volume measurements. Body weights and tumor volumes are noted once a week, as well as on the day of sacrifice. The Pro-Max electronic digital caliper is used to measure the tumor volume, which is then computed using the formula length (mm)×width (mm)×height (mm) and expressed in mm3. The data are presented as mean±SE. Repeated actions ANOVA is used to assess the statistical significance of observed differences, with Scheffe post hoc testing for multiple comparisons used afterwards.

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For the in vivo vascular permeability assay, HEK 293 cells expressing murine VEGF or vector control were mixed with Matrigel and injected subcutaneously into nude mice. Twenty-four hours later, mice received a single oral dose of AMG 706 (100 mg/kg) or vehicle. At specified times, Evans blue dye was injected intravenously, and dye extravasation in the skin overlying the implant was quantified. [1]
For the rat corneal angiogenesis model, a VEGF-soaked nylon disc was implanted into the corneal stroma of anesthetized rats. Rats were treated orally with AMG 706 (at various doses, either once or twice daily) or vehicle for 7 days. Angiogenesis was assessed by counting blood vessels growing toward the implant. [1]
For tumor xenograft studies, A431 cells were injected subcutaneously into the flank of athymic nude mice. When tumors reached approximately 125 mm³, mice were randomized and treated orally with AMG 706 (10, 30, or 100 mg/kg/dose, twice daily) or vehicle. Tumor volume and body weight were monitored. For mechanistic studies, tumors were harvested at various time points after treatment initiation for histopathological analysis. [1]
For all in vivo studies, AMG 706 was formulated as a suspension in a pH-adjusted oral vehicle. [1]

Following a single oral dose of 100 mg/kg in mice, plasma concentrations of AMG 706 remained above the HUVEC proliferation IC50 (10 nM) for up to 16 hours, consistent with the duration of significant inhibition of VEGF-induced vascular permeability. [1] In a rat model of corneal angiogenesis, the estimated ED50 corresponding to plasma AUC0-24 was 2.2 µg•h/mL and Cmax was 0.28 µg/mL for twice-daily administration (2.1 mg/kg). The estimated ED50 corresponding to AUC0-24 was 5.0 µg•h/mL and Cmax was 2.1 µg/mL for once-daily administration (4.9 mg/kg). [1]

In mice, oral administration of AMG 706 at doses up to 100 mg/kg/dose twice daily for 24 days was well tolerated, and no significant effects on body weight or overall health were observed in the described studies. [1]

[1]. AMG 706, an oral, multikinase inhibitor that selectively targets vascular endothelial growth factor, platelet-derived growth factor, and kit receptors, potently inhibits angiogenesis and induces regression in tumor xenografts.Cancer Res . 2006 Sep 1;66(17):8715-21.

[2]. Augmentation of radiation response by motesanib, a multikinase inhibitor that targets vascular endothelial growth factor receptors. Clin Cancer Res . 2010 Jul 15;16(14):3639-47.

[3]. Broad antitumor activity in breast cancer xenografts by motesanib, a highly selective, oral inhibitor of vascular endothelial growth factor, platelet-derived growth factor, and Kit receptors. Clin Cancer Res, 2009, 15(1), 110-118.

Motesarib is a pyridine carboxamide compound.
Motesarib is an orally bioavailable receptor tyrosine kinase inhibitor with potential antitumor activity. AMG 706 selectively targets and inhibits vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), Kit and Ret receptors, thereby inhibiting angiogenesis and cell proliferation.

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Drug indications
It has been investigated in the treatment of solid tumors.

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AMG 706 (motesarib) is an orally bioavailable ATP-competitive small molecule multi-kinase inhibitor. [1]
Its main antitumor mechanism of action is anti-angiogenesis, achieved by inhibiting VEGFR, thereby leading to endothelial cell apoptosis, tumor angiogenesis reduction, and ultimately tumor cell apoptosis and regression. [1]
At the time of publication, AMG 706 was in the clinical research stage for the treatment of human malignancies, and early phase I clinical trials showed acceptable safety and encouraging efficacy in patients with advanced solid tumors. [1]

C22H23N5O

373.460

373.19

C, 70.76; H, 6.21; N, 18.75; O, 4.28

453562-69-1

Motesanib Diphosphate;857876-30-3

11667893

White to off-white solid powder

1.3±0.1 g/cm3

517.3±50.0 °C at 760 mmHg

266.6±30.1 °C

0.0±1.3 mmHg at 25°C

1.669

4.22

3

5

5

28

533

0

O=C(C1=CC=CN=C1NCC2=CC=NC=C2)NC3=CC(NCC4(C)C)=C4C=C3

RAHBGWKEPAQNFF-UHFFFAOYSA-N

InChI=1S/C22H23N5O/c1-22(2)14-26-19-12-16(5-6-18(19)22)27-21(28)17-4-3-9-24-20(17)25-13-15-7-10-23-11-8-15/h3-12,26H,13-14H2,1-2H3,(H,24,25)(H,27,28)

N-(3,3-dimethyl-1,2-dihydroindol-6-yl)-2-(pyridin-4-ylmethylamino)pyridine-3-carboxamide

motesanib free base; AMG-706; AMG 706; AMG706

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.69 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.69 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)