A Selective and Oral Small Molecule Inhibitor of VEGFR-2 and … · 2003/5/23 · 6 Intervention...

JPET #52167

1

A Selective and Oral Small Molecule Inhibitor of VEGFR-2 and VEGFR-1 Inhibits

Neovascularization and Vascular Permeability

Neela Patel, Li Sun, Deborah Moshinsky, Hui Chen, Kathleen M. Leahy, Phuong Le, Katherine G. Moss, Xueyan Wang, Audie Rice, Danny Tam, A. Douglas Laird, Xiaoming Yu, Qingling Zhang, Cho Tang, Gerald McMahon, and Anthony Howlett

SUGEN, Inc., South San Francisco, CA 94080 (N.P., L.S., D.M., H.C., P.L, K.G.M.,

X.W., A.R., D.T., A.D.L., X.Y., Q.Z., C.T., G.M., A.H.), Pharmacia Corp. , Chesterfield

MO 63017 (K.M.L.)

Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.

JPET Fast Forward. Published on May 23, 2003 as DOI:10.1124/jpet.103.052167This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 23, 2003 as DOI: 10.1124/jpet.103.052167

at ASPE

T Journals on June 26, 2021

jpet.aspetjournals.orgD

ownloaded from

http://jpet.aspetjournals.org/

JPET #52167

2

Running Title: Indolinone VEGFR Kinase Inhibitor

Address Correspondence to:

Neela Patel, Ph.D.

Associate Director, Discovery Biology

SUGEN, Inc.

230 E. Grand Ave.

South San Francisco, CA 94080

Email: [email protected]

Tel: (650) 837 3565

Fax: (650) 837 3348

35 text pages

2 tables

7 figures

36 refs.

235 words in abstract

743 words in introduction

1290 words in discussion

Abbreviations:

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 23, 2003 as DOI: 10.1124/jpet.103.052167

at ASPE



ownloaded from


JPET #52167

3

AMD, age-related macular degeneration; bFGF, basic fibroblast growth factor; DR,

diabetic retinopathy; FGFR-1, fibroblast growth factor receptor-1; HUVEC, human

umbilical vein endothelial cells; IL-8, interleukin-8; lyn, LCK/YES-related Novel

tyrosine kinase; HGFR, hepatocyte growth factor receptor; PDGFRb, platelet derived

growth factor receptor beta; SCF, stem cell factor; TNF, tumor necrosis factor; VEGF,

vascular endothelial growth factor; VEGFR-1, vascular endothelial growth factor

receptor-1; VEGFR-2, vascular endothelial growth factor receptor-2.

Recommended section assignment: Other


at ASPE



ownloaded from


JPET #52167

4

ABSTRACT Vascular epithelial growth factor (VEGF) is a key driver of the

neovascularization and vascular permeability which lead to the loss of visual acuity in

diabetic retinopathy and neovascular age-related macular degeneration. Our aim was to

identify an orally active, selective small molecule kinase inhibitor of VEGFR-2 with

activity against both VEGF-induced angiogenesis and vascular permeability. We utilized

a biochemical assay to identify SU10944, a pyrrole indolinone, which is a potent ATP-

competitive inhibitor of VEGFR-2 (Ki 21± 5 nM). In cellular assays SU10944 inhibited

VEGF-induced receptor autophosphorylation (IC50 227± 80 nM) as well as downstream

signaling (IC50 102 ± 27 nM). In biochemical assays SU10944 exhibits potent inhibitory

activity against VEGFR-1, weak activity against other related subgroup members

including SCF-R, PDGFRβ, and FGFR-1, and no detectable activity against other protein

tyrosine kinases such as EGFR, Src, and HGFR. In cellular assays the selectivity for

SU10944 to inhibit VEGFR is maintained compared to other tyrosine kinases (IC50 SCF-

R 1.6±0.3 uM, PDGFRβ 30.6±13.3 uM, FGFR-1>50 uM, EGFR>50 uM). Upon oral

administration, SU10944 gave a clear dose response in the corneal micropocket model

with an ED50 for inhibition of neovascularization of ~30 mg/kg and a maximum

inhibition of 95% at 300 mg/kg. Similarly, upon oral administration in the Miles assay,

SU10944 potently inhibited VEGF-induced vascular permeability. Our data indicate that

small molecule inhibitors of VEGFR signaling have the potential to ameliorate both

VEGF induced neovascularization as well as vascular permeability.


at ASPE



ownloaded from


JPET #52167

5

In diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD),

vascular endothelial growth factor (VEGF) is a driver of both the neovascularization and

retinal vascular permeability which underlie the loss of visual acuity. The temporal and

spatial expression patterns of VEGF in these diseases implicate VEGF in ocular

neovascularization. VEGF expression levels and activity are elevated in vitreous samples

from diabetic patients with active proliferative retinopathy, as compared to individuals

with non-proliferative diabetic retinopathy, quiescent proliferative retinopathy, non-

diabetic individuals, or individuals with non-ischemic ocular disease (Aiello et al., 1994,

Adamis et al., 1994). The levels of VEGF are positively correlated to the clinically

observed degree and stage of retinal neovascularization (Aiello et al., 1994). In AMD,

VEGF is expressed in choroidal neovascular membranes (Frank et al., 1996).

As with ocular neovascularization, elevated levels of VEGF are associated clinically with

ocular edema (Funatsu et al., 2002, Vinores et al., 1995). In pre-clinical models, VEGF

is likewise associated with changes in vascular integrity. Increases in ocular VEGF in

diabetic animals correlate with elevated vascular permeability, prior to observable retinal

proliferative changes (Sone et al., 1997, Gilbert et al.,1998). Local delivery of VEGF by

intravitreal implants result in significant increases in retinal permeability by day 3 while

retinal neovascularization is observed only after 14 days (Alikacem et al., 2000). A

similar breakdown of the blood-retinal barrier occurs in primates administered VEGF by

intravitreal implant (Ozaki et al., 1997).


at ASPE



ownloaded from


JPET #52167

6

Intervention in VEGF signalling, either by decreasing local concentrations of ligand with

antisense oligodeoxynucleotides (Robinson et al., 1996) or soluble chimeric receptors

(Aiello et al., 1995), or by inhibiting receptor signaling by small molecules (Ozaki et al.,

2000) decrease ocular neovascularization, thus confirming the central role of VEGF in

this process. Neovascularization in DR and AMD may differ significantly from

angiogenesis in other pathological contexts such as tumor angiogenesis where multiple

targets have been implicated, including PDGF, FGF, and IL-8 (Laird et al., 2000; Rofstad

and Halsor 2000; Shaheen et al., 2001). Current data strongly suggests that in diabetic

retinopathy and exudative age-related macular degeneration, VEGF is the key driver. In a

pre-clinical model of diabetic retinopathy, hypoxia-driven retinal neovascularization,

VEGF inhibitors are efficacious but PDGFR inhibitors are not (Ozaki et al., 2000);

similarly in a model of AMD, injury-induced choroidal neovascularization, a VEGF

inhibitor decreases choroidal neovascularization ~ 85% but administration of a PDGF

inhibitor does not decrease choroidal neovascularization (Kwak et al., 2000). The

contribution of FGF to neovascularization is less well understood. While bFGF is

expressed in neovascular membranes from AMD and DR patients (Frank et al., 1996) and

intravitreally administered FGF has a synergistic effect with VEGF in producing retinal

hemorrhage (Wong et al., 2001), retinal neovascularization occurs upon hypoxic

challenge even in the absence of bFGF (Ozaki et al., 1998). Moreover, overexpression of

bFGF in a transgenic mouse does not produce retinal neovascularization nor increase the

degree of neovascularization upon hypoxia (Ozaki et al., 1998).


at ASPE



ownloaded from


JPET #52167

7

Inhibition of PDGF could have deleterious effects, particularly in the context of DR.

PDGF appears to play a special role in retinal vasculature. Recent reports suggest that

PDGF signaling is important for survival of retinal vasculature, specifically pericytes,

under conditions of hypoxic or metabolic stress (Kodama et al., 2001, Hammes et al.,

2002). In vivo, PDGF plays a role in retinal capillary coverage: PDGF-B deficient mice

(PDGFB +/-) have fewer retinal pericytes and more acellular retinal capillaries than wild-

type controls, differences which are more pronounced in diabetic animals. In the

hypoxia-induced model of neovascularization, new vessels are twice as prevalent in

PDGFB +/- mice compared to wild-type animals (Hammes et al., 2002), suggesting that

pericyte deficiency renders endothelial cells more susceptible to angiogenesis. Therefore,

a potential side effect of PDGF inhibition could be to accelerate the loss of pericytes

which are implicated in the destabilization of blood vessels during the early stages of

diabetic retinopathy. Therefore, given the lack of data supporting a role for PDGF in

retinal and choroidal neovascularization as well as the risk of increasing pericyte dropout,

a VEGF-selective inhibitor is likely the best choice for treatment of retinopathies.

Our goal was to identify and characterize an orally available selective VEGFR inhibitor,

since angiogenesis and increased vascular permeability in DR and exudative AMD is

primarily or solely driven by VEGF in these settings. A selective compound should

minimize the potential toxicities resulting from the inhibition of additional kinases and be

more likely to give a sufficient therapeutic index for the treatment of non-life threatening

disease.


at ASPE



ownloaded from


JPET #52167

8

MATERIALS AND METHODS

Chemicals. SU10944 was synthesized as described in patent WO00/08202. Its chemical

structure is shown in Fig. 1. Stock solutions of SU10944 were made in DMSO and stored

at –20°C. Dilutions for assays were made fresh prior to use.

Kinetic Analysis. The catalytic portion of mouse VEGFR-2 was expressed as a GST

fusion protein following infection of Spodoptera frugiperda (sf9) cells with engineered

baculoviruses by standard methods (King and Possee, 1992). GST-VEGFR-2 was

purified to homogeneity from infected sf9 cell lysates by glutathione sepharose

chromatography (Smith and Johnson, 1988). GST-fusion preparations were analyzed by

gel electrophoresis and determined to be of high purity, with no detectable breakdown

products as determined using protein staining and Western blot analysis for the GST

moiety. Protein concentration was determined with the Bio-Rad protein assay reagent kit

(Bio-Rad, Hercules, CA) using a standard curve with bovine serum albumin. For the

SU10944 Ki determination against VEGFR-2, biochemical kinase reactions were

performed as described below (Biochemical Assays) in the presence of various ATP and

inhibitor concentrations. Rates were expressed as the increase in TR-FRET counts per

minute of reaction within the linear reaction time. Ki values were determined by

graphical analysis of the plot of the slopes from the double reciprocal plot vs. inhibitor

concentration. The final Ki value represents the average + the standard deviation from 5

independent experiments.

Biochemical Assays. VEGFR-2 and PDGFRβ time-resolved fluorescence (TR-FRET)

autophosphorylation assays were performed as described (Moshinsky et al., in press).


at ASPE



ownloaded from


JPET #52167

9

Briefly, 1nM VEGFR-2 or 5nM PDGFRβ was added to a reaction buffer composed of

50mM Hepes, pH 7.4, 1mM MnCl2, 0.01% BSA, and 1mM DTT, containing twice the

apparent Km concentration of both ATP and N-terminal biotinylated peptide (KY-tide:

KYKKYKKKYKKKKYKYK) in 50uL total volume. Reactions were allowed to

proceed within the linear reaction time then terminated by the addition of 20uL 90mM

EDTA. Eu-W1024-labeled antiphosphotyrosine PY20 and Streptavidin: SureLight –

Allophycocyanin (Perkin Elmer Life Sciences, Foster City, CA) were diluted in TBS

containing 0.02% BSA and 0.1% Tween20 and added to a final concentration of 0.5nM

and 1.6nM, respectively. After incubation for at least 10 minutes, samples were excited

at 340nM and emissions were read at 665nM using an LJL Analyst (LJL Biosystems,

Sunnyvale, CA). The increase in signal for both assays was determined to be time

dependent with a requirement for ATP, peptide, kinase, and divalent metal cation. FGFR-

1 and EGFR autophosphorylation reactions were performed using immuno-captured

kinases as described (Laird et al., 2000). The HGFR and VEGFR-1 assays were

performed using poly(Glu, Tyr) 4:1 as a substrate as described (Blake et al., 2000). For

the cdk2/cyclin A assay, a Scintillation Proximity Assay method was used (Amersham

Pharmaceutical Assays Development Group, 1995). The SCF-R, src, lyn, and fyn assays

were performed in standard TR-FRET format (Kolb et al., 1998) using peptide substrates

found by screening an internal peptide substrate library. Conditions for compound

testing were saturating peptide concentration and an ATP concentration of 2*Km.

VEGFR-2 Cell Autophosphorylation Assay. Full-length mouse VEGFR-2 was cloned

into the C-terminal 3xFLAG-tag expression vector p3xFLAG-CMV-14 (Sigma, St.

Louis, MO). Human embryonic kidney 293T cells were grown in DMEM supplemented


at ASPE



ownloaded from


JPET #52167

10

with 10% fetal bovine serum and grown in a 37°C humidified incubator with 5% CO2.

The construct was transfected into 293T cells using LipofectAmine2000 (Invitrogen,

Carlsbad, CA) under manufacturer’s recommendations. Following transfection, cells

were starved in DMEM containing no serum and 0.1% BSA for 24 hours. Cells were

then split into 96-well plates and treated with compound in a final concentration of 1%

DMSO for 2 hours. Cells were lysed by the addition of HNTG (50mM Hepes, pH 7.4,

150mM NaCl, 1.5mM MgCl2, 10% glycerol, 1% Triton X-100, 1mM EGTA), and lysates

were transferred to polystyrene 96-well plates that had been pre-coated with 1ug per well

of M2 anti-FLAG monoclonal antibody (Sigma, St. Louis, MO) to capture the 3xFLAG

tagged kinase. Quantification of phosphorylation was performed by incubating with

HRP-labeled anti-phosphotyrosine PY99 (Santa Cruz Biotechnology, Santa Cruz, CA),

followed by detection with an ABTS color readout.

VEGFR-2 Phosphorylation Detected by Western Blot. NIH/3T3 cells stably

expressing VEGFR-2 were grown to confluence in DMEM with 10% heat-inactivated

calf serum and then incubated in serum-free medium containing different concentrations

of SU10944 for 20 hr. After stimulation with human recombinant VEGF165 (R&D

Systems, Minneapolis, MN) at 50 ng/ml for 10 min, cells were lysed in lysis buffer

containing 50 mM Hepes, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 100 mM

PMSF, 1 mM sodium vanadate and 2 µg/ml leupeptin and aprotinin. VEGFR-2 protein

was isolated with a monoclonal anti-mouse VEGFR-2 antibody made at SUGEN

designated L4. Phosphorylation of VEGFR-2 was then analyzed by SDS-PAGE

followed by western blotting using biotin-labeled anti-phosphotyrosine PY99 (Santa Cruz

Biotechnology, Santa Cruz, CA). Total VEGFR-2 levels were assessed by stripping and


at ASPE



ownloaded from


JPET #52167

11

re-probing the membrane with anti-VEGFR-2 antibody L4 (SUGEN, South San

Francisco, CA).

VEGFR-2 Cellular Functional Assay. The assay is an immunoassay for the quantitative

detection of human tissue factor. HUVECs were seeded at 50,000 cell/well in growth

medium EMB (Endothelial Cell Basal Medium, BioWhittaker, Walkersville, MD) +

10%FBS with complete supplements containing hEGF 0.5 ml/500 ml, hydrocortisone

0.5 ml/500 ml, gentamicin sulfate amphotericin-B 0.5 ml/500 ml, bovine brain extract 2

ml/500 ml (BioWhittaker, Walkersville, MD) in 24 well plate coated with 0.2% gelatin.

Cells were grown for 2 days >90% confluence, and then treated with SU10944 and

human recombinant VEGF165 (R&D Systems, Minneapolis, MN) at 50 ng/ml or PMA

(phenylmethylsulfonyl fluoride, Sigma, St. Louis, MO) at 10 nM for 4 hours; negative

control cells received media only. Cells were lysed with HNTG lysis buffer (50mM

Hepes, pH 7.4, 150mM NaCl, 1.5mM MgCl2, 10% glycerol, 1% Triton X-100, 1mM

EGTA), plus EDTA (1:100), protease inhibitor (1:100), and PMSF (1:100) in 125 ul per

well for 20 minutes at 4 °C. Cell lysates were were processed using an IMUBIND Tissue

Factor ELISA Kit (American Diagnostica, Greenwich, CT). Briefly, lysates were

transferred to 96 well format precoated micro-test strips and incubated at 4 °C overnight.

The micro-test strips were washed 4 times with wash buffer, 100 uL of biotinylated anti-

human tisuue factor antibody added to each well, and incubated for 1 hour at room

temperature. After washing 4 times, 100 uL of diluted streptavidin-horseradish

peroxidase conjugated antibody was added to each well, and incubated for 1 hour at room

temperature. The wells were washed 4 times with wash buffer and incubated with 100 ul

of TMB (tetramethyl-benzidine, Sigma, St. Louis, MO) substrate solution for 20 minutes


at ASPE



ownloaded from


JPET #52167

12

at room temperature. The reaction was stopped by the additional of 50 ul of H2SO4

solution, and absorbance read on a microplate reader at a wavelength of 450 nm.

Functional Cellular Assays for SCF-R, EGFR, PDGFRβ, and FGFR-1. For EGFR,

PDGFRβ, and FGFR-1, the functional activity of the receptor was measured by a ligand-

induced BrdU incorporation assay in 3T3 cells which endogenously express both FGFR-

1 and PDGFRβ but not EGFR. A single cell line stably transfected with EGFR was used

for all assays. Cells seeded in a 96-well plate were made quiescent by serum-deprivation

for 24 hr and then stimulated with FGF2/bFGF (1.5 nM), EGF (4 nM) or PDGF (3.8 nM)

in the absence or presence of the indicated concentrations of SU10944 for 20 hr. BrdU

was added for a two hr labeling period, and the cells were fixed. The amount of BrdU

incorporation was determined with an anti-BrdU-peroxidase conjugated antibody using

an ELISA kit (Boehringer Mannheim). Cell viability following exposure to compounds in

the assay format was assessed by substituting the addition of BrdU with resazurin (1

mg/ml) at 1:100 after the 20 hour incubation. Following a 3 hr incubation, the absorbance

of the samples are measured at 630 nm in "dual wavelength" mode with a filter reading at

450 nm, as a reference wavelength) on a Dynatech ELISA plate reader. Compound was

added to wells in the dilutions used to determine IC50 values, including a negative

control in which all components were included except for cells and a positive control in

which all components except compound were added. The resazurin assay is based upon

the conversion of the dark blue resazurin dye to a pink dye in proportion to the metabolic

activity of the cells (O’Brien et al. 2000). For each IC50 value, the standard deviation is

reported.


at ASPE



ownloaded from


JPET #52167

13

The functional activity of SCFR was assessed using a SCF-dependent cell proliferation/

survival assay. MO7e cells were grown and expanded in RPMI with 10%FBS in the

presence of IL-3 (10ng/ml) and GM-CSF (10 ng/ml). After counting, cells were pelleted

by centrifugation and washed twice with PBS. Cells were resuspended in medium

containing either IL-3 or SCF (100ng/m) and aliquoted into 96-well plates at 50,000 cells

per well along with varying concentrations of SU10944. After incubation for three days

at 37°C in a humidified incubator with 5% CO2, live cells were quantified by their ability

to metabolize resazurin as described above.

Corneal Angiogenesis Model. An intrastromal pocket was surgically created in one or

both corneas of each anesthetized Sprague Dawley female rat. A slow release

hydron/sucralfate pellet containing 150 ng human recombinant VEGF165 (PeproTech,

Rocky Hill, NJ) was inserted into the pocket as previously described (Kenyon 1996), the

pocket closed to self-seal, and the rats given analgesia and topical antibiotic ointment

applied once to the eye. The rats recovered from anesthesia on a warming pad, and were

returned to their cages. SU10944 was administered daily by gavage in a 1.0 ml

suspension of 0.5% methylcellulose (Sigma Chemical, St. Louis), 0.025% Tween-20

(Sigma) to four to six rats per dose group beginning the day before implant, and

continuing the length of the study. Four days after surgery, the corneas of the re-

anesthetized rats were examined under a slit lamp microscope and the neovascular

response was quantified by measuring the average new vessel length (VL), the corneal

radius (r = 2.6 mm), and the contiguous circumferential zone (CH = clock hours where 1

CH = 30 degrees), and applied to the formula: Area (mm2)= (CH/12) x 3.14(r2- (r-VL)2 ).

The rats were then immediately euthanized. Eyes from rats that developed infection as a


at ASPE



ownloaded from


JPET #52167

14

result of the surgery were not included in the study. Six to eight eyes were included per

dosing group. The neovascular areas of the vehicle and 250mg/kg dosed rat corneas were

dissected from the eye, flat mounted, and photographed at 4x with a digital camera

mounted on a microscope. Rat corneas implanted with pellets containing no growth

factor (placebo pellets) generated no new blood vessel growth (Leahy et al). All animal

treatment protocols were reviewed by and were in compliance with Pharmacia’s

Institutional Animal Care and Use Committee.

Miles assay for vascular permeability. The Miles assay for vascular permeability

(Miles and Miles 1952) was adapted to athymic mice as follows. Mice were given a

single oral dose of SU10944 or vehicle alone. Simultaneously or at designated later

timepoints, 100ul of 0.5% Evan’s blue dye (Sigma-Aldrich, St. Louis, MO) in PBS was

administered intravenously via the tail vein. One hour later, mice were injected

intradermally (in duplicate sites on their backs) with 400 ng of VEGF (human

recombinant VEGF165, R&D Systems, Minneapolis, MN) dissolved in 20 ul of PBS or (in

adjacent duplicate sites) with PBS alone. After an additional 30 minutes, VEGF-

dependent dye leakage from the vasculature into skin was assessed visually and scored

semi-quantitatively (100, 50, or 0% inhibition for each spot). Two spots per animal

allowed each animal to be scored as representing 100%, 75%, 50%, 25%, or 0%

inhibition. Low-level background effects from time-matched vehicle-treated groups were

subtracted out.

Determination of SU10944 Plasma Levels. Plasma samples (100 µl), SU10944 standard

or quality control samples in mouse blank plasma were mixed with acetonitrile (300 µl)

containing DL-propranolol hydrochloride (internal standard) in a 96-well polypropylene


at ASPE



ownloaded from


JPET #52167

15

plate (Orochem Technology, Westmont, IL). The plate was mixed by vortex for 1 minute

and the samples were centrifuged for 10 minutes at 4000 rpm. Ten µL of the supernatant

was injected onto the LC/MS/MS system where separation occurred on a BDS

HYPERSIL C-18 (5 µm, 100 x 4.6 mm) reverse-phase HPLC column (Keystone

Scientific, Foster City, CA). The amount of SU10944 and the internal standard in each

mouse plasma sample was quantified based on standard curves generated using known

amounts of compound ranged from 5 to 10,000 ng/mL. Standard curve samples and

quality control samples of SU010944 were prepared by spiking 10 µL of stock standard

solutions with 90µL of blank plasma.


at ASPE



ownloaded from


JPET #52167

16

RESULTS

We identified SU10944 (Fig. 1) as a potent inhibitor of VEGF-R as part of our efforts to

synthesize and characterize indolin-2-ones as inhibitors of class III receptor tyrosine

kinases (Yarden and Ullrich, 1988). Previous structure-activity relationship studies

revealed that modifications on the indolin-2-one core could generate compounds with

different kinase selectivity profiles (Sun et al. 1998, 1999, 2000). Modifications of the

core have also been found to have a dramatic impact on the pharmaceutical properties of

the compounds in terms of solubility, metabolic stability, permeability, plasma protein

binding, and pharmacokinetic properties.

Following the identification of SU10944 as an inhibitor of VEGFR-2 in biochemical

assays (IC50 96 ± 20 nM), we went on to further assess the biochemical activity of the

compound against other kinases, as well as its potential to act as a competitor for ATP.

SU10944 exhibited competitive inhibition with respect to ATP for VEGFR-2. This is

indicated by the fact that the lines from the double reciprocal plot (Fig. 2) converge on

the Y-axis. The Ki was determined to be 21 + 5 nM. In a panel of kinase assays,

SU10944 potently inhibited VEGFR-2 with an IC50 of 96 ± 20 nM and exhibited even

greater activity against VEGFR-1 IC50 6±1 nM (Fig. 3). It showed some activity against

other closely related family members, for example PDGFRβ and FGFR-1 but exhibited

significantly less activity against other receptors (Table 1). The compound is not a pan-


at ASPE



ownloaded from


JPET #52167

17

kinase inhibitor as no discernible inhibition of more distantly related tyrosine kinases was

evident e.g. EGFR and Src.

We then used a panel of three cell-based assays to determine whether the compound

could cross the cellular membrane and inhibit VEGFR-2 within cells. In 293T cells

transiently transfected with mouse VEGFR-2, SU10944 exhibited an IC50 value of 227 ±

80 nM for receptor autophosphorylation as measured by ELISA (Figure 4A). Similarly,

in an assay to assess the functional activity of endogenous VEGFR-2 in HUVECs, tissue

factor production stimulated by VEGF was inhibited with an IC50 of 102 ± 27 nM (Figure

4B). However SU10944 did not inhibit TNF-stimulated release of tissue factor (Figure

4B). Final confirmation that the compound inhibits VEGFR-2 receptor phosphorylation

was obtained by western blot analysis of 3T3 cells engineered to express mouse VEGFR-

2 (Figure 4C). After cells were pre-treated with compound and stimulated with VEGF,

VEGFR-2 was immunoprecipitated and then detected on the blot with an anti-

phosphotyrosine antibody. SU10944 inhibited receptor autophosphorylation confirming

the results of the 293 assay in a different cell type and assay format.

For kinases against which SU10944 was active in biochemical assays, cellular assays

were performed to determine whether the activity was maintained in a more physiological

setting (Table 2). Similar to the observations in the panel of biochemical assays,

significantly higher concentrations of compound were required to inhibit closely related

subgroup of family members such as FGFR-1, SCF-R, and PDGFRβ than was required

for the inhibition of VEGFR-2 in functional assays. The functional activities of the


at ASPE



ownloaded from


JPET #52167

18

FGFR-1, PDGFRβ, and EGFR were measured by ligand-induced cell proliferation of

3T3 cells as measured by BrdU incorporation. For SCFR, the activity was measured by

the SCF-dependent survival of MO7e cells. SU10944 did not exhibit detectable inhibition

of EGFR or FGFR-1 (IC50 values >50 uM). Furthermore SU10944 was not cytotoxic: the

LD50 was >50 µM for 3T3 cells.

We then assessed the ability of SU10944 to inhibit angiogenesis and vascular

permeability in vivo when administered by the oral route. In the rat corneal micropocket

model of angiogenesis, a VEGF pellet is implanted in the cornea to stimulate

neovascularization. In this model, the compound significantly decreased the

angiogenesis, both the number of vascular sprouts as well as the length of the sprouts

(Fig. 5). Moreover, SU10944 gave a clear dose response with an ED50 of ~ 50 mg/kg for

inhibition of neovascularization (Fig. 6). In order to increase potential exposure levels

upon oral administration by decreasing dissolution rate limitations of the compound, we

prepared an in situ sodium salt of SU10944 and repeated the experiment. The sodium salt

decreased the apparent ED50 slightly, to approximately 30 mg/kg. In addition a

maximum inhibition of 95% was achieved at the highest dose of 300 mg/kg. Increased

exposure upon administration of the sodium salt was confirmed in pharmacokinetic

studies (data not shown).

In the Miles assay for vascular permeability (Miles and Miles, 1952), a visualization dye

is administered intravenously, followed by a bolus administration of VEGF by the

intradermal route. Leakage of the dye in the skin indicates a local increase in vascular


at ASPE



ownloaded from


JPET #52167

19

permeability. In this assay, SU10944 inhibited VEGF-induced vascular permeability in a

time and dose dependent manner (Fig 7a). Maximum inhibition was observed at the

earliest timepoint (1 hr), with decreasing levels of inhibition over the course of 24 hr. At

100 mg/kg the maximum inhibition of vascular permeability was sustained to 2 hr, and a

50% reduction in response was still apparent 24 hr post-dose. In contrast, at the 30 mg/kg

dose maximum inhibition was seen at 1 hr and decreased dramatically over 24 hr to zero.

Plasma concentrations of SU01944 were also determined and correlated with inhibition

(Fig 7b). Based on the total data set that reflects a range of doses and times post-

administration of compound, we conclude that SU10944 plasma exposures of 250 ng/ml

(844 nM) result in 50% inhibition of VEGF-mediated vascular permeability in vivo.


at ASPE



ownloaded from


JPET #52167

20

DISCUSSION

We have identified and characterized a new small molecule inhibitor of VEGF signaling

which inhibits both angiogenesis and vascular permeability when administered orally.

The compound, SU10944, is a potent ATP-competitive inhibitor of the kinase activity of

purified VEGFR-2 with a Ki of 21± 5 nM and an IC50 of 96± 20 nM. SU10944 also

exhibits potent activity against VEGFR-1, with an IC50 of 6± 1nM. The compound

maintains activity in cellular assays against both the mouse(flk-1) and human (KDR)

forms of VEGFR-2. In the cellular assay for inhibition of VEGFR-2 autophosphorylation

the compound exhibited an IC50 of 227± 80 nM, a value in good agreement with the IC50

obtained in our HUVEC assay for receptor function (IC50 102± 27 nM). In the functional

assay, we demonstrated that the inhibition of tissue factor production resulted from

inhibition of VEGFR by showing that TNF-stimulated release of tissue factor was not

affected by the compound. The HUVEC data suggest that the compound will not only be

functionally active against human VEGFR-2 but should be active in endothelial cells

which are the target for inhibition of angiogenesis and vascular permeability. We were

further able to confirm the nature of the cellular activity of SU10944 by visualizing

inhibition of receptor phosphorylation by Western analysis.

From a panel of biochemical and cellular assays we conclude that SU10944 is a relatively

selective inhibitor with a strong preference for VEGFRs. The kinase activity of both

VEGFR-1 and VEGFR-2 are potently inhibited by the compound, with low to mid

nanomolar IC50 values. Some activity was observed in the biochemical assays,

particularly against other members of the class III receptor tyrosine kinases, for example,


at ASPE



ownloaded from


JPET #52167

21

PDGFRβ 1000±� 83 nM and SCFR 1580± 270 nM. SU10944 exhibited little or no

activity against the other kinases surveyed, which represent a range of tyrosine kinases as

well as some serine/ threonine kinases. We limited our cellular assays to those kinases

which had shown activity in biochemical assays, plus one more distantly related kinase.

Consistent with the biochemical observations, the compound displayed very limited cross

reactivity in the cellular functional assays. The most notable cross reactivity occurred

against SCFR (IC50 1600± 300 nM; however, compared to VEGFR functional readout

(IC50 102± 27 nM), there was an 18-fold selectivity. Selectivity at the cellular level was

good compared to PDGFRβ (IC5030.6±�13.3 uM), with a ratio of 340x.

While SU10944 inhibited the kinase activity of purified VEGFR-2 with an IC50 of 96± 20

nM, when the compound was tested in cellular assays the apparent IC50 value shifted to

227± 80 nM in the autophosphorylation assay but was in very close agreement with the

functional assay IC50 of 102 ±27 nM. A more notable discrepancy was observed between

the IC50 values for the kinase activity of purified PDGFRβ (1000±�83 nM) compared to

the cellular assay (IC5030.6±�13.3 uM). Modest discrepancies between the biochemical

and cellular values are not uncommon findings with small molecule enzyme inhibitors

since the physical properties of the compounds as well as the assay format become

important when translating from biochemical to cellular activity. To inhibit activity of

the kinase within a cellular context, the compound must cross the cell membrane and

retain activity in the presence of cellular proteins. In addition, differences in assay

formats can influence the observed IC50 values.


at ASPE



ownloaded from


JPET #52167

22

In the context of potential treatments for exudative AMD and diabetic retinopathy, a

therapeutic molecule should inhibit both neovascularization as well as increased vascular

permeability in order to be maximally effective. In pre-clinical models, SU10944

potently inhibited both VEGF-induced angiogenesis and vascular permeability following

administration by the oral route. In the corneal micropocket model of angiogenesis, we

observed a clear dose response with a maximal inhibition of nearly 100%. Compound

levels obtained by oral administration clearly achieved sufficient exposure levels to

inhibit the functional activity of the receptor. Similarly, in the Miles assay of vascular

permeability, a time and dose dependent response was observed. The maximum response

was nearly 100%, confirming that in a second species and different model,

pharmacologically relevant levels of drug were achieved by oral administration.

We note that there is a discrepancy between the in vitro potency of SU10944 (227± 80

nM in the autophosphorylation assay, 102±27 nM in the functional assay) and its in vivo

activity as measured in the vascular permeability assay (EC50 cutoff estimated to be 833

nM). While there can be many reasons for such observed differences in the translation

from in vitro to in vivo results, the main source in this case is likely to be high plasma

protein binding of the compound. Other compounds in the series have been shown to

have high protein binding; our data suggest protein binding of >98% (data not shown).

The unbound concentration of SU10944 in vivo would therefore be roughly 16 nM, a

value in better agreement with our in vitro observations.


at ASPE



ownloaded from


JPET #52167

23

To develop the compound for potential use in the treatment of human ocular disease, the

efficacy of the compound in more relevant disease models, as well as the potential safety

of the compound with systemic administration will require careful investigation. The

activity of the compound in human retinal endothelial cells and in more physiologically

relevant animal models such as retinal vascular permeability in streptozoticin-induced

rats, hypoxia-driven retinal angiogenesis in mouse neonates, and laser injury-induced

choroidal neovascular model of AMD should be determined. In addition, the therapeutic

index of such compounds will be essential to establish because of potential mechanism-

based toxicities with systemically administered VEGFR inhibitors, particularly in the

context of diabetic co-morbidities. One area of particular concern is whether VEGFR

inhibitors will further impair coronary collateral formation in diabetic patients, in

response to myocardial ischemia. Collateral vessel development occurs by the process of

arteriogenesis, the expansion of existing arterioles, a process which is differentially

regulated from angiogenesis. Monocyte migration into the area of ischemia is a key

process in the formation of the collateral vessels. Current evidence suggests that

impaired collateral vessel formation in diabetic individuals results from a signaling defect

downstream of VEGFR-1 in monocytes (Waltenberger, 2001), which normally respond

to VEGF-A by increased migration. Since the signaling via VEGFR-1 is already impaired

in diabetic patients, it remains to be determined whether a VEGFR-1 inhibitor would be

additive or neutral for collateral formation, or whether a selective VEGFR-2 inhibitor

would be preferred for treatment of diabetic retinopathy.


at ASPE



ownloaded from


JPET #52167

24

Unlike VEGFR-2, VEGFR-1 has not been implicated directly in mediating VEGF-

induced angiogenesis or vascular permeability. However, inhibition of VEGFR-1 may be

beneficial in AMD where macrophage infiltration has been suggested to play a role in the

etiology of the disease. VEGF produced by hypoxic retinal pigmented epithelial cells has

been postulated to act as a chemotactic factor for macrophages, which can then secrete

additional VEGF as well as other pro-angiogenic factors.

We have identified and characterized a novel small molecule inhibitor of VEGFR-2,

SU10944. This compound is a potent, ATP-competitive inhibitor of VEGFR-2

biochemical activity and is active in the nanomolar range in cellular assays. SU10944 can

be administered in vivo by the oral route and achieves sufficient exposure to inhibit

nearly all VEGF-stimulated neovascularization and vascular permeability. A selective,

well-tolerated VEGFR inhibitor, administered orally or by local delivery, should be of

therapeutic benefit in both diabetic retinopathy and exudative age-related macular

degeneration. In addition we believe compounds of this nature will be valuable in

delineating the role of VEGF in various forms of pathological angiogenesis where

multiple kinases may play contributing roles. Clinical trials will be necessary to show the

potential prophylactic or therapeutic utility of these novel and selective VEGFR-

inhibitors in human eye diseases. The discovery of a multiple VEGFR inhibitors

representing a variety of pharmacophores offers the opportunity to generate new

molecules with increased potency or improved pharmaceutical properties by rational

design based on co-crystals with VEGFR-2.


at ASPE



ownloaded from


JPET #52167

25

ACKNOWLEDGMENTS

We thank Steve Settle for excellent technical work on the corneal angiogenesis model,

the Discovery Technology Group at SUGEN for biochemical cross screening data, Ken

Lipson for assistance with data analysis, and Anne-Marie O’Farrell for suggestions

regarding the manuscript.


at ASPE



ownloaded from


JPET #52167

26

References

Adamis AP, Miller JW, Bernal MT, Damico DJ, Folkman J, Yeo TK, and Yeo KT (1994)

Increased vascular endothelial growth factor levels in the vitreous of eyes with

proliferative diabetic retinopathy. Am J Ophthal 118:445-450.

Aiello LP, Avery RL, Arrigg PG, Keyt BA. Jampel HD, Shah ST, Pasquale LR, Thieme

H, Iwamoto MA, Park JE, Nguyen HV, Aiello LM, Ferrara N, King GL et al. (1994)

Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy

and other retinal disorders. New Eng J Med 331:1480-1487.

Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L, Ferrara N, King GL, and

Smith LEH (1995) Suppression of retinal neovascularization in vivo by inhibition of

vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric

proteins. Proc Nat Acad Sci 92: 10457-10461.

Alikacem N, Yoshizawa T, Nelson KD, and Wilson CA (2000). Quantitative MR

imaging study of intravitreal sustained release of VEGF in rabbits. Invest Ophthalmol Vis

Sci 41:1561-1569.

Amersham Pharmaceutical Assays Development Group (1995) Measurement of cdk2

activity using a [33P]ATP Scintillation Proximity Assay. Proximity News 18:1-4.


at ASPE



ownloaded from


JPET #52167

27

Blake RA, Broome MA, Liu X, Wu J, Gishizky M, Sun L, and Courtneidge SA (2000)

SU6656, a Selective Src Family Kinase Inhibitor, Used To Probe Growth Factor

Signaling. Mol Cell Biol 20:9018-9027.

Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (Ki) and the

concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic

reaction. Biochem Pharmacol 22:3099-108.

Frank RN, Amin RH, Eliott D, Puklin JE, and Abrams GW (1996) Basic fibroblast

growth factor and vascular endothelial growth factor are present in epiretinal and

choroidal neovascular membranes. Am J Ophthal 122: 393-403.

Funatsu H, Yamashita H, Ikeda T, Nakanishi Y, Kitano S, and Hori S (2002) Increased

levels of vascular endothelial growth factor and interleukin-6 in the aqueous humor of

diabetics with macular edema. Am J Ophthal 133:70-77.

Gilbert RE, Vranes D, Berka JL, Kelly DJ, Cox A, Wu LL, Stacker SA, and Cooper ME

(1998) Vascular endothelial growth factor and its receptors in control and diabetic rat

eyes. Lab Invest 78:1017-1027.

Hammes H-P, Lin J, Renner O, Shani M, Lundqvist A, Betsholtz C, Brownlee M, and

Deutsch U (2002) Pericytes and the pathogenesis of diabetic retinopathy. Diabetes

51:3107-3112.


at ASPE



ownloaded from


JPET #52167

28

Kenyon BM, Voest EE, Chen CC, Flynn E, Folkman J, and D’Amato RJ (1996) A model

of angiogenesis in the mouse cornea. Invest Ophthalmol Vis Sci. 37:1625-32.

King LA and Possee RD (1992) The Baculovirus Expression System: A Laboratory

Guide. Chapman and Hall, Ltd., London.

Kodama T, Oku H, Kawamura H, Sakagami K, and Puro DG (2001) Platelet-derived

growth factor-BB: a survival factor for the retinal microvasculature during periods of

metabolic compromise. Current Eye Res 23: 93-97.

Kolb AJ, Kaplita PV, Hayes DJ, Park Y-W, Pernell C, Major JS, and Mathis G (1998)

Tyrosine kinase assays adapted to homogeneous time-resolved fluorescence. Drug Disc

Today 3:333-342.

Kwak N, Okamoto N, Wood JM, and Campochiaro PA (2000) VEGF is a major

stimulator in model of choroidal neovascularization. Invest Ophthalmol Vis Sci 41:3158-

3164.

Laird AD, Vajkoczy P, Shawver LK, Thurnher A, Liang C, Mohammadi M, Schlessinger

J, Ullrich A, Hubbard SR, Blake RA, Fong TAT, Strawn LM, Sun L, Tang C, Hawtin R,

Tang F, Shenoy N, Hirth HP, McMahon G, and Cherrington J (2000) SU6668, a broad


at ASPE



ownloaded from


JPET #52167

29

spectrum angiogenesis inhibitor, exhibits potent anti-tumor activity in xenograft models,

including regression of established tumors. Cancer Res 60:4152-4160.

Leahy KM, Ornberg RL, Wang Y, Zweifel BS, Koki AT, Masferrer JL. (2002)

Cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in

angiogenic endothelial cells in vivo. Cancer Res 62(3):625-31.

Miles AA and Miles EM (1952) Vascular reactions to histamine, histamine-liberator and

leukotaxine in the skin of guinea pigs. J Physiol 118: 228-257.

Moshinsky DJ, Ruslim L, Blake RA, and Tang F (2003) A widely applicable, high-

throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGF

receptor 2 as a prototype. J Biomol Screening In Press.

O’Brien J, Wilson I, Orton T, and Pognan F (2000) Investigation of the Alamar Blue

(resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J

Biochem 267: 5421-5426.

Ozaki H et al (1997) Intravitreal sustained release of VEGF causes retinal

neovascularization in rabbits and breakdown of the blood-retinal barrier in rabbits and

primates. Exp Eye Res 64:505-517.


at ASPE



ownloaded from


JPET #52167

30

Ozaki H, Okamoto N, Ortega S, Chang M, Ozaki K, Sadda S, Vinores MA, Derevjanik

N, Zack D, Basilico C, Campochiaro PA (1998) Basic fibroblast growth factor is neither

necessary nor sufficient for the development of retinal neovascularization. Am J Path

153: 757-765.

Ozaki H, Seo MS, Ozaki K, Yamada H, Yamada E, Okamoto N, Hofmann F, Wood JM,

and Campochiaro PA (2000) Blockade of vascular endothelial growth factor receptor

signaling is sufficient to completely prevent retinal neovascularization. Am J Path

156:697-707.

Robinson GS, Pierce EA, Rook SL, Foley E, Webb R, and Smith LEH (1996)

Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of

proliferative retinopathy. Proc Nat Acad Sci 93:4851-4586.

Rofstad EK and Halsor EF (2000) Vascular endothelial growth factor, interleukin 8,

platelet-derived endothelial growth cell factor, and basic fibroblast growth factor promote

angiogenesis and metastasis in human melanoma xenografts. Cancer Res 60: 4932-4938.

Shaheen RM, Tseng WW, Davis DW, Liu W, Reinmuth N, Vellagas R, Wieczorek AA,

Ogura Y, McConkey DJ, Drazan KE, Bucana CD, McMahon G, and Ellis LM (2001)

Tyrosine kinase inhibition of multiple angiogenic growth factor receptors improves

survival in mice bearing colon cancer liver metastases by inhibition of endothelial cell

survival mechanisms. Cancer Res 61:1464-1468.


at ASPE



ownloaded from


JPET #52167

31

Smith DB and Johnson KS (1988) Single-step purification of polypeptides expressed in

Escherichia coli as fusions with glutathione S-transferase. Gene 67:31-40.

Sone H, Kawakami Y, Okuda Y, Sekine Y, Honmura S, Matsuo K, Segawa T, Suzuki H,

and Yamashita K (1997) Ocular vascular endothelial growth factor levels in diabetic rats

are elevated before observable retinal proliferative changes. Diabetologia 40:726-730.

Sun L, Tran N, Tang F, App H, Hirth P, McMahon G, and Tang C (1998) Synthesis and

Biological Evaluations of 3-Substituted Indolin-2-ones: A Novel Class of Tyrosine

Kinase Inhibitors That Exhibit Selectivity toward Particular Receptor Tyrosine Kinases. J

Med Chem 41:2588-2603.

Sun L, Tran N, Liang C, Tang F, Rice A, Schreck R, Waltz K, Shawver LK, McMahon

G, Tang C. (1999) Design, synthesis, and evaluations of substituted 3-[(3- or 4-

carboxyethylpyrrol-2-yl)methylidenyl]indolin-2-ones as inhibitors of VEGF, FGF, and

PDGF receptor tyrosine kinases. J Med Chem 42:5120-30.

Sun L, Tran N, Liang C, Hubbard S, Tang F, Lipson K, Schreck R, Zhou Y, McMahon

G, and Tang C (2000) Identification of Substituted 3-[(4,5,6,7-Tetrahydro-1H-indol-2-

yl)methylene]-1,3-dihydroindol-2-ones as Growth Factor Receptor Inhibitors for VEGF-

R2 (Flk-1/KDR), FGF-R1, and PDGF-R Tyrosine Kinases. J Med Chem 43:2655-2663.

Vinores SA, Kuchle M, Mahlow J, Chiu C, Green WR, and Campochiaro PA (1995)

Blood-ocular barrier breakdown in eyes with ocular melanoma. A potential role for


at ASPE



ownloaded from


JPET #52167

32

vascular endothelial growth factor/ vascular permeability factor. Am J Path 147: 1289-

1297.

Waltenberger J (2001) Impaired collateral vessel development in diabetes: potential

cellular mechanisms and therapeutic implications. Cardiovasc Res 49:554-560.

Wong CG, Rich KA, Liaw L-HL, Hsu HT, and Berns MW (2001) Intravitreal VEGF and

bFGF produce florid retinal neovascularization and hemorrhage in the rabbit. Current

Eye Res 22:140-147.

Yarden Y and Ullrich A (1988) Growth factor receptor tyrosine kinases. Annual Review

of Biochemistry 57: 443-478.


at ASPE



ownloaded from


JPET #52167

33

Legends for figures

Fig. 1. Chemical structure of SU10944.

Fig. 2. Double reciprocal plot of SU10944 inhibition of VEGFR-2 at 160nM (∇), 80nM

( ��Q0�� DQG�QR�� FRPSRXQG��7KH�LQVHW�GLVSOD\V�D�JUDSK�RI�WKH�VORSHV�IURP�WKH�

double reciprocal plot vs. inhibitor concentration. The –(Ki) value is indicated by an

arrow. Data are representative from 5 independent experiments.

Fig. 3. Dose response curves for SU10944 inhibition of VEGFR-2 (a) and VEGFR-1 (b).

Error bars represent standard deviations from at least 3 replicate measurements.

Fig. 4. SU10944 inhibits VEGFR-2 receptor phosphorylation and functional activity in

cells. (a) 293T cells transiently transfected with mouse VEGFR-2 were exposed to

varying concentrations of SU10944 and receptor phosphorylation detected by ELISA.

Error bars represent standard deviations from n=3. (b) HUVECs were stimulated with

either PMAn or VEGFu. Production of tissue factor was measured by ELISA. Error

bars represent standard deviations from duplicate samples. (c) 3T3 cells stably transfected

with mouse VEGFR-2 were stimulated with VEGF. Anti-mouse VEGFR-2 antibody was

used for immunoprecipitation from cellular lysates. Western analysis of

immunoprecipated proteins was performed using anti-phosphotyrosine antibody or anti-

mouse VEGFR-2 antibody.


at ASPE



ownloaded from


JPET #52167

34

Fig. 5. SU10944 inhibits VEGF-induced neovascular growth in rat corneas. Four days

after VEGF pellet implant, the neovascular area of the rat cornea was measured, dissected

from the eye, flat mounted, and photographed at 4x with a digital camera mounted on a

microscope. Panel A shows representative corneas from rats dosed with vehicle. Panel B

shows corneas from rats dosed with SU10944 free acid at 250 mg per kg.

Fig. 6. Dose response of sodium salt or free acid SU-10944 on VEGF-induced

neovascular growth in rat corneas. Four days after VEGF pellet implant, the neovascular

area of the rat cornea was measured using a calibrated eye piece mounted on a slit lamp

microscope. Vessel length, and the contiguous circumferential angle of

neovascularization were noted and used to calculate the area of each cornea that was

covered with neovessels. This area was expressed as a percent of the average vehicle-

dosed, VEGF-implanted (Control) corneas. Error bars represent SEM for n=6-8 eyes.

*p

JPET #52167

35

three animals), with any low-level background effects from time-matched vehicle-treated

groups subtracted out. Error bars represent SE. Vascular permeability in animals treated

with SU10944 at 100 mg/kg and 30 mg/kg was significantly inhibited (* indicates p

JPET #52167

36

TABLE 1

Biochemical Activity of SU10944

Kinase Mean IC50,

µM

Kinase IC50, µM

VEGFR-2 0.096 ± 0.020

(n=16)

EGFR

(n=4)

>20

VEGFR-1 0.006 ±0.001

(n=5)

Src

(n=8)

>20

FGFR1 1.60±0.87

(n=5)

Lyn

(n=3)

>20

PDGFRβ 1.00±0.08

(n=4)

Fyn

(n=5)

>20

SCFR 1.58±0.27

(n=5)

Cdk2

(n=4)

>20

HGFR

(n=8)

>20

The IC50 values for SU10944 were determined by measuring autophosphorylation or

substrate phosphorylation, as specified in Materials and Methods.


at ASPE



ownloaded from


JPET #52167

37

TABLE 2

Cellular Activity of SU10944

Receptor (cell type) IC50, µM

SCFRa (MO7e) 1.6 ±0.3

(n=2)

PDGFRβ b (3T3) 30.6±13.3

(n=6)

EGFRb (3T3) >50

(n=3)

FGFR-1b (3T3) >50

(n=3)

a Functional activity of SCF-R was measured in a survival assay for MO7e cells.

bFunctional activity was measured by BrdU incorporation in 3T3 cells stably transfected

with the receptor.


at ASPE



ownloaded from


NH

O

NH

OHO

Figure 1

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

Fast Forward. Published on M

ay 23, 2003 as DO

I: 10.1124/jpet.103.052167 at ASPET Journals on June 26, 2021 jpet.aspetjournals.org Downloaded from



at ASPE



ownloaded from


0.001 0.01 0.1 1 10

0

50

100

Concentration (µM)

% in

hibi

tion

Figure 4

a

c

b

VEGF (100ng/ml)

anti-pTyr

anti-Flk-1

25 15 0.2

- +++++

SU10944 [µM]

0.001 0.01 0.1 1 100.0

0.5

1.0

1.5

Concentration (µM)

O.D

. at

450

nm




JPET


ay 23, 2003 as DO



Figure 5

VEGF pelleted corneas—systemic treatment with vehiclea

b VEGF pelleted corneas—systemic treatment with SU10944




JPET


ay 23, 2003 as DO



Figure 6

�

��

��

��

��

��

��

1 �� Dose (mg/kg)

% C

on

tro

lNeo

vasc

ula

rA

rea

�

*

**

*

****

**

**




JPET


ay 23, 2003 as DO



1

10

100

1000

10000

100000

100 75 50 25 0

% InhibitionP

lasm

a S

U10

944

conc

. (ng

/mL)

250 ng/mL

Figure 7

0

10

20

30

40

50

60

70

80

90

100

0 4 8 12 16 20 24

100 mg/kg

30 mg/kg

3 mg/kg

% In

hibi

tion

Time Post Administration (h)

* * **

*




JPET


ay 23, 2003 as DO


A Selective and Oral Small Molecule Inhibitor of VEGFR-2 and … · 2003/5/23 · 6 Intervention...

Documents

Transcript of A Selective and Oral Small Molecule Inhibitor of VEGFR-2 and … · 2003/5/23 · 6 Intervention...