Sunitinib metabolite; VEGFR; PDGFRβ; KIT

Moreover, sunitinib efficiently inhibits FLT-3 and Kit[1]. With a Ki of 8 nM for PDGFRβ and 9 nM for VEGFR2 (Flk1), sunitinib is a strong ATP competitive inhibitor. Compared to FGFR-1, EGFR, Cdk2, Met, and IGFR, it is ten times more selective for VEGFR2 and PDGFR. above. 1. Src and Abl. Sunitinib suppresses VEGF-dependent VEGFR2 phosphorylation and PDGF-dependent PDGFRβ phosphorylation with IC50 values of 10 nM and 10 nM, respectively, in serum-starved NIH-3T3 cells expressing VEGFR2 or PDGFRβ. With an IC50 of 40 nM for VEGF, sunitinib prevents serum-starved HUVEC from proliferating, and with an IC50 of 39 nM and 69 nM for PDGF, it prevents NIH-3T3 cells overexpressing PDGFRβ or PDGFRβ from proliferating [2]. For wild-type FLT3, FLT3-ITD, and FLT3-Asp835, sunitinib inhibits phosphorylation with IC50 values of 250 nM, 50 nM, and 30 nM, respectively. Sunitinib causes apoptosis in MV4;11 and OC1-AML5 cells in a dose-dependent manner and suppresses their growth with IC50 values of 8 nM and 14 nM, respectively [3].

Across a variety of tumor xenograft models, such as HT-29, A431, Colo205, H-460, SF763T, C6, A375, or MDA-dependent antitumor activity of MB-435, sunitinib (20–80 mg/kg/day) demonstrates a broad and effective dosing range that is consistent with a significant and selective inhibition of VEGFR2 or PDGFR phosphorylation and signaling in vivo. In six out of eight mice, sunitinib (80 mg/kg/day) for 21 days led to total tumor shrinkage; no tumor regrowth occurred over the 110-day observation period following the conclusion of treatment. For tumours that did not fully regress during the first round of treatment, a second round of sunitinib remains effective. Treatment with sunitinib greatly decreased tumor MVD, with SF763T glioma tumors showing a 40% reduction. Although there was no decrease in tumor size, SU11248 therapy totally prevented further tumor growth in luciferase-expressing PC-3M xenografts [2]. In the FLT3-ITD bone marrow transplant paradigm, sunitinib therapy (20 mg/kg/day) effectively decreased the growth of subcutaneous MV4;11 (FLT3-ITD) xenografts and extended survival [3].

IC50 values for Sunitinib against VEGFR2 (Flk-1) and PDGFRβ are determined using glutathione S-transferase fusion proteins containing the complete cytoplasmic domain of the RTK. Biochemical tyrosine kinase assays to quantitate the trans-phosphorylation activity of VEGFR2 (Flk-1) and PDGFRβ are performed in 96-well microtiter plates precoated (20 μg/well in PBS; incubated overnight at 4°C) with the peptide substrate poly-Glu,Tyr (4:1). Excess protein binding sites are blocked with the addition of 1-5% (w/v) BSA in PBS. Purified GST-fusion proteins are produced in baculovirus-infected insect cells. GST-VEGFR2 and GST-PDGFRβ are then added to the microtiter wells in 2× concentration kinase dilution buffer consisting of 100 mM HEPES, 50 mM NaCl, 40 μM NaVO4, and 0.02% (w/v) BSA. The final enzyme concentration for GST-VEGFR2 or GST-PDGFRβ is 50 ng/mL. Twenty-five μL of diluted sunitinib are subsequently added to each reaction well to produce a range of inhibitor concentrations appropriate for each enzyme. The kinase reaction is initiated by the addition of different concentrations of ATP in a solution of MnCl2 so that the final ATP concentrations spanned the Km for the enzyme, and the final concentration of MnCl2 is 10 mM. The plates are incubated for 5-15 minutes at room temperature before stopping the reaction with the addition of EDTA. The plates are then washed three times with TBST. Rabbit polyclonal antiphosphotyrosine antisera are added to the wells at a 1:10,000 dilution in TBST containing 0.5% (w/v) BSA, 0.025% (w/v) nonfat dry milk, and 100 μM NaVO4 and incubated for 1 hour at 37°C. The plates are then washed three times with TBST, followed by the addition of goat antirabbit antisera conjugated with horseradish peroxidase (1:10,000 dilution in TBST). The plates are incubated for 1 hour at 37°C and then washed three times with TBST. The amount of phosphotyrosine in each well is quantitated after the addition of 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate] as substrate.[1]

Cells are starved overnight in medium containing 0.1% FBS prior to addition of Sunitinib and FL (50 ng/mL; FLT3-WT cells only). Proliferation is measured after 48 hours of culture using the Alamar Blue assay or trypan blue cell viability assays. Apoptosis is measured 24 hours after Sunitinib addition by Western blotting to detect cleavage of poly (ADP-ribose) polymerase (PARP) or levels of caspase-3.[3]

To predict the target SU11248 exposure required to achieve antitumor activity in mouse xenograft models, we directly measured target phosphorylation in tumor xenografts before and after SU11248 treatment and correlated this with plasma inhibitor levels. In target modulation studies in vivo, SU11248 selectively inhibited Flk-1/KDR (VEGF receptor 2) and PDGF receptor beta phosphorylation (in a time- and dose-dependent manner) when plasma concentrations of inhibitor reached or exceeded 50-100 ng/ml. Similar results were obtained in a functional assay of VEGF-induced vascular permeability in vivo. Constant inhibition of VEGFR2 and PDGF receptor beta phosphorylation was not required for efficacy; at highly efficacious doses, inhibition was sustained for 12 h of a 24-h dosing interval. The pharmacokinetic/pharmacodynamic relationship established for SU11248 in these preclinical studies[2].

[1]. Discovery of 5-[5-fluoro-2-oxo-1,2- dihydroindol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase. J Med Chem. 2003 Mar 27;46(7):1116-9.

[2]. In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res. 2003 Jan;9(1):327-37.

[3]. SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood. 2003 May 1;101(9):3597-605.

To enhance the antitumor activity of indoline-2-one and optimize its pharmacological properties, including solubility and protein binding, we designed and synthesized a series of different basic and weakly basic analogs. The results showed that 5-[5-fluoro-2-oxo-1,2-dihydroindole-(3Z)-methylene]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide (12b or SU11248) exhibited the best overall performance in terms of activity, solubility, protein binding and bioavailability of VEGF-R2 and PDGF-Rβ tyrosine kinases at both biochemical and cellular levels. 12b is currently in a Phase I clinical trial for the treatment of cancer. [1]

---

Molecular targeted therapy represents an emerging and promising approach to the treatment of cancer, and a major challenge in its clinical development is how to determine the bioactive dose rather than the maximum tolerated dose. This study aimed to determine the pharmacokinetic/pharmacodynamic relationship that can be used to guide clinical dose selection through preclinical models. SU11248 is a novel small-molecule receptor tyrosine kinase inhibitor that exerts direct antitumor and anti-angiogenic activities by targeting vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), KIT, and FLT3 receptor tyrosine kinases. This study used this drug as the research subject. In a mouse xenograft model, SU11248 exhibited broad and potent antitumor activity, leading to regression, growth arrest, or significant reduction in growth of various established xenografts derived from human or rat tumor cell lines. To predict the SU11248 target exposure required to achieve antitumor activity in a mouse xenograft model, we directly measured the phosphorylation level of the target in xenografts before and after SU11248 treatment and correlated it with plasma inhibitor concentrations. In in vivo target regulation studies, SU11248 selectively inhibited phosphorylation of Flk-1/KDR (VEGF receptor 2) and PDGF receptor β (in a time- and dose-dependent manner) when plasma inhibitor concentrations reached or exceeded 50–100 ng/ml. Similar results were obtained in in vivo VEGF-induced vascular permeability assays. Sustained inhibition of VEGFR2 and PDGF receptor β phosphorylation is not a necessary condition for therapeutic efficacy. At high doses, the inhibitory effect can last for 12 hours of a 24-hour dosing interval. The pharmacokinetic/pharmacodynamic relationships of SU11248 established in these preclinical studies can help design, select and evaluate dosing regimens being tested in human trials. [2]

---

FLT3 (fms-associated tyrosine kinase/Flk2/Stk-2) is a receptor tyrosine kinase (RTK) that is primarily expressed on hematopoietic cells. Two classes of FLT3 activating mutations have been identified in the primitive cells of patients with acute myeloid leukemia (AML): internal tandem repeat (ITD) mutations in the juxtamembrane domain (25%–30% of patients) and point mutations in the kinase domain activation loop (7%–8% of patients). FLT3-ITD mutations are the most common molecular defect in AML and have been proven to be an independent prognostic factor. Therefore, FLT3-ITD is a highly attractive molecular therapeutic target. SU11248 is a recently discovered selective inhibitor selective for splitting kinase domain receptor tyrosine kinases (RTKs), including platelet-derived growth factor receptor, vascular endothelial growth factor receptor, and KIT. We found that SU11248 exhibited potent activity against wild-type FLT3 (FLT3-WT), FLT3-ITD, and FLT3 activation loop (FLT3-Asp835) mutants in phosphorylation assays. SU11248 inhibited FLT3-driven phosphorylation and induced apoptosis in vitro. In addition, SU11248 can also inhibit FLT3-induced VEGF production. This study investigated the in vivo efficacy of SU11248 in two FLT3-ITD models: a subcutaneous tumor xenograft model and a bone marrow transplantation model. The results showed that SU11248 (20 mg/kg/d) significantly inhibited FLT3-ITD tumors in the subcutaneous tumor xenograft model and prolonged survival in the bone marrow transplantation model. Pharmacokinetic and pharmacodynamic analyses of subcutaneous tumors showed that a single effective dose of the drug could strongly inhibit FLT3-ITD phosphorylation, and the inhibitory effect could last for up to 16 hours. These results suggest that further research is needed on the activity of SU11248 in patients with acute myeloid leukemia (AML). [3]

C₂₀H₂₃FN₄O₂

370.42

370.181

356068-97-8

N-Desethyl Sunitinib-d5;1217247-62-5;N-Desethyl Sunitinib hydrochloride

10292573

Light yellow to orange solid powder

3.522

4

4

6

27

597

0

CCNCCNC(=O)C1=C(NC(=C1C)/C=C\2/C3=C(C=CC(=C3)F)NC2=O)C

LIZNIAKSBJKPQC-GDNBJRDFSA-N

InChI=1S/C20H23FN4O2/c1-4-22-7-8-23-20(27)18-11(2)17(24-12(18)3)10-15-14-9-13(21)5-6-16(14)25-19(15)26/h5-6,9-10,22,24H,4,7-8H2,1-3H3,(H,23,27)(H,25,26)/b15-10-

N-[2-(ethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide

SU-11662; SU11662; N-Desethyl Sunitinib; 356068-97-8; N-DesethylSunitinib; SU-12662; N-[2-(ethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide; CHEMBL3542344; 42LJ35612R; UNII-42LJ35612R; SU 11662

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)

DMSO : ~6.25 mg/mL (~16.87 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.75 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

---

Solubility in Formulation 2: ≥ 0.62 mg/mL (1.67 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 6.2 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.

---

View More

Solubility in Formulation 3: 0.62 mg/mL (1.67 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 6.2 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.

---

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