Molecular Medicine |
From Taiho Pharmaceutical Co, Ltd (Y.Y., K.M., N.O., M.W., H.M.), Hanno Research Center, Saitama, Japan, and SUGEN, Inc (J.C., X.W., L.S., C.T., G.M., K.E.L.), South San Francisco, Calif.
Correspondence to Kenneth E. Lipson, SUGEN, Inc, 230 East Grand Ave, South San Francisco, CA 94080. E-mail ken-lipson{at}sugen.com
| Abstract |
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Key Words: angioplasty platelet-derived growth factor restenosis tyrosine kinase indolinone
| Introduction |
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The underlying mechanisms of restenosis comprise a combination of effects ranging from vessel recoil, negative vascular remodeling, and thrombus formation to neointimal hyperplasia.8 9 The neointimal hyperplasia process has been suggested to be associated with the expression of growth factors and their cognate receptors including tyrosine kinases. The growth factors themselves are released by local thrombi and the angioplasty-injured arterial segment itself and may serve to enhance the expression of other growth-regulatory events. These processes result in an inflammatory reaction and a myofibroblast proliferative response, which worsen vessel narrowing caused by negative remodeling and result in the formation of a clinically significant restenotic lesion.10 11
Platelet-derived growth factor (PDGF) is one of several growth factors implicated in the restenosis process. In the animal balloon-injury model, PDGF acts primarily to induce smooth muscle migration and secondarily to promote intimal proliferation.12 13 PDGF receptor (PDGFr) autophosphorylation increases within a few days after injury and persists for several weeks.14 Furthermore, in human restenotic lesions in comparison with nonlesioned sites, the presence of PDGF-A and -B as ligands and PDGFrß is detected after PTCA.15 16 The finding of the expression of these factors and receptor in vascular damaged and repaired regions strongly suggests that autocrine or paracrine stimulation of these receptor systems are important in the biological regulation of this tissue injury process.
Triazolopyrimidine (trapidil), a mild competitive inhibitor
of the PDGFr, has been evaluated in 3 clinical trials to
date.17 18 19
In one of these studies, Maresta et
al19 found a reduction
(
50%) in restenosis for trapidil when compared with aspirin.
Several PDGFr kinase inhibitors have been evaluated in preclinical
models as a prelude to possible clinical
development.20 21
In this study, we have identified a potent indolinone
inhibitor of the PDGFr kinase, SU9518
(3-[5-{5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl}-2,4-dimethyl-1H-pyrrol-3-yl]propionic
acid)
(Figure 1
). Data in this report suggest that SU9518 rapidly
penetrates the cell membrane, leading to a durable inactivation of the
receptor due to competitive inhibition of the catalytic activity of the
kinase after receptor activation. SU9518 was evaluated for
pharmacological properties, including the potential to inhibit arterial
thickening after tissue injury.
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| Materials and Methods |
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, or PDGFrß were provided by Dr
Axel Ullrich (Max-Planck Institute, Martinsried, Germany). A7r5 rat
aorta smooth muscle cells were purchased from American Type Culture
Collection. DMEM, FBS, and calf serum were purchased from
GIBCO-BRL. 3T3 mouse fibroblasts overexpressing the ß or
form of
the human PDGFr (3T3/PDGFrß or 3T3/PDGFr
cells, respectively) were
grown in DMEM containing 10% calf serum and 2 mmol/L
L-glutamine at 37°C under
an atmosphere of 5% CO2. A7r5 cells were grown
in DMEM containing 10% FBS.
Cellular Kinase Assay
Confluent cells were deprived of serum overnight,
treated with the test compound for 1 hour, and then stimulated with 3.3
nmol/L (100 ng/mL) PDGF-AA or -BB for 5 minutes at 37°C. Cells were
then lysed with SDS sample buffer and fractionated by SDS-PAGE.
Phosphorylated PDGFrs were detected by Western blot analysis using
anti-phosphotyrosine antibodies (4G10) with enhanced chemiluminescence
(ECL) detection. The amount of PDGFr in each lane was determined by
using anti-PDGFr
ß, anti-PDGFr
, or anti-PDGFrß antibodies and
ECL detection.
Ligand-Induced Bromodeoxyuridine (BrdU)
Incorporation Assay
3T3/EGFr cells (3T3 cells overexpressing EGFr) in a
96-well plate were made quiescent by serum deprivation for 24 hours.
The serum-deprived cells were then stimulated with (in nmol/L)
fibroblast growth factor (FGF) 2/basic FGF 1.5, EGF 4, or PDGF 3.8 in
the absence or presence of the indicated concentrations of SU9518 for
20 hours. BrdU was added for a 2-hour labeling period, and the cells
were fixed. The amount of BrdU incorporation was determined with an
ELISA kit (Roche Molecular) using anti-BrdU/POD (peroxidase).
Cytotoxicity was assessed under identical conditions by using a Tox-8
kit (Sigma).
Pharmacokinetic Analysis
All experimental protocols were approved by the
Institutional Animal Care and Use Committee of Taiho Pharmaceutical Co.
Male Wistar rats (7 weeks old, Charles River Japan, Inc
[Tsukuba]) were housed in constant temperature facilities and
given standard laboratory chow and water ad libitum. Blood samples (1
mL) were obtained at 2, 4, and 8 hours after oral administration of
SU9518 or 1, 2, 4, and 7 days after subcutaneous administration. Blood
was sampled from a cervical vein, and the plasma was stored at
-20°C until analysis was performed. SU9518 was extracted from the
plasma with acetonitrile, acidified, and extracted with ethyl acetate.
HPLC analysis was performed on a Mightysil RP-18 GR column, 5-µm
particle size, 4.6x150 mm using a Waters HPLC with a gradient
of 90% 5 mmol/L phosphate buffer (pH 2.8) and 10% acetonitrile to
30% 5 mmol/L phosphate buffer (pH 2.8) and 70%
acetonitrile.
Rat Arterial Injury Model
Male Sprague-Dawley rats (12 to 13 weeks old, Clea
Japan Inc, Tokyo, Japan) were housed in constant-temperature facilities
and given standard laboratory chow and water ad libitum. Balloon
catheter denudation of the carotid artery endothelium was performed
according to the method described by Clowes et
al.22 Briefly, the rats were
anesthetized with a gas mixture of
N2O/O2 (30:70) containing
2% halothane. After a median incision of the abdominal skin had been
made, the right iliac artery was dissected and cannulated with a 2F
balloon catheter (embolectomy catheter arterial balloon, Medical
Technology Transfer). The catheter was inflated with saline and passed
through the left common carotid artery 4 times to produce a distending,
de-endothelializing injury.
Histopathologic Evaluation
On the 14th day after balloon injury, the rats were
anesthetized with ether and perfused transcardially with saline,
followed by 10% buffered formalin. The left carotid artery (from the
aortic arch to bifurcation) was removed, postfixed, and embedded in
paraffin. Sections 3 µm thick (5 per artery) were cut and stained
with hematoxylin and eosin. The cross-sectional areas of the intima and
the media on photographs were measured with a digital analyzer
(Digitaizer, Wacom).
PDGFr Protein Expression and
Phosphorylation
Detection of PDGFr phosphorylation in injured
arteries was performed according to the method of Panek et
al.23 The injured artery was
excised and cut into strips 7 days after balloon catheter injury. The
strips were immediately frozen in liquid nitrogen, and protein was
extracted, subjected to PDGFrß immunoprecipitation, and separated by
SDS-PAGE. Phosphorylated PDGFr was visualized by anti-phosphotyrosine
immunoblotting (4G10) with ECL detection. The amount of PDGFr in each
lane was determined using anti-PDGFrß antibody. The signal intensity
of PDGFr and phosphorylated PDGFr in vehicle- or SU9518-treated rats
(subcutaneous injection of 100 mg/kg) was compared with that of
uninjured rat artery PDGFr (n=3 in each group) by
densitometry.
In Vivo Migration Assay
Quantification of smooth muscle cell migration into
the intima was performed by the method of Bendeck et
al.24 Four days after
balloon injury, the artery was fixed with 4% buffered
paraformaldehyde. The middle of the artery was cut lengthwise and
pinned intimal side up onto a Teflon plate. The arteries were incubated
with monoclonal antibody against human nuclei and chromosomes (MAB
1276, 1:100) overnight at 4°C, and immunoreactive cells were
visualized with a commercially available detection system (Vector
Laboratories). The number of cell nuclei per
mm2 was counted by light
microscopy.
In Vivo Proliferation Assay
Quantification of medial and intimal smooth muscle
cell proliferation was performed by the method of Muranaka et
al.25 Four or 7 days after
injury, the artery was fixed with 4% buffered paraformaldehyde and
embedded in paraffin for preparation of 4 sections 3 µm thick. The
sections were incubated with biotinylated antiproliferating cell
nuclear antigen (PCNA) monoclonal antibody (PCNA15, 1:250) overnight at
4°C and counterstained with hematoxylin to visualize all
nuclei.
Drug Administration and Dosage
SU9518 (50 mg/kg in 0.5% hydroxypropyl
methylcellulose solution [10 mL/kg]) was administered orally by
gavage once daily from 1 hour before denudation to the day before
removal of the artery for evaluation. Alternatively, SU9518 (100 mg/kg
in carboxymethylcellulose sodium USP [0.5%], sodium chloride
[0.9%], polysorbate 80 [0.4%], benzyl alcohol [0.9%], and
deionized water [4 mL/kg]) was administered subcutaneously 1 day
before denudation and again 6 days after the
operation.
Reagents
SU9518
(Figure 1A
) was synthesized by SUGEN using methods previously
described.26 ECL reagents
were purchased from Amersham. PDGF-AA, PDGF-BB, and
anti-BrdU/POD conjugate were purchased from Roche Molecular.
Anti-phosphotyrosine monoclonal (4G10) and anti-PDGFr
ß polyclonal
antibodies were from Upstate Biotechnology Inc, whereas anti-PDGFr
and anti-PDGFrß antibodies were purchased from Santa Cruz
Biotechnology. Biotinylated anti-PCNA monoclonal antibody was purchased
from Caltag Laboratories. Anti-nuclei and chromosomes were
purchased from Chemicon International. All other reagents were obtained
from Sigma.
Statistical Analysis
All data are expressed as mean±SD. Statistically
significant differences between 2 groups were calculated by (2-tailed)
Aspin-Welch t test. A
P value <0.05 was considered
to indicate statistical
significance.
| Results |
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or PDGFrß.
Cells were made quiescent by serum deprivation and contact inhibition
and were then stimulated with PDGF and various concentrations of SU9518
(Figure 1B
To demonstrate that SU9518 would also inhibit rat PDGFr
kinase, a similar experiment was performed with A7r5 cells
(Figure 1C
), which only express PDGFrß (not shown).
PDGFrß in A7r5 cells were inhibited with a dose response similar to
that observed in mouse fibroblasts overexpressing human
PDGFrs.
Kinetics of SU9518 Inhibition of PDGFr
Kinase in Fibroblasts
The temporal inactivation of the PDGFr kinase by SU9518
was studied to determine the pharmacological requirements to maintain
the inhibitory effect. In the first experiment, receptor
autophosphorylation was measured using 3T3/PDGFrß fibroblast cells
after exposure of cells to SU9518 for different periods of time
(Figure 2A
). Complete inhibition of PDGF-induced receptor
autophosphorylation was observed after incubation of the cells with
SU9518 at all time points tested, indicating that <5 minutes of
exposure was required to block the activation of PDGFr kinase. The
rapid penetration and inactivation of the receptor may be due in part
to the high relative lipophilicity of the compound and its ability to
penetrate cell membranes.
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Cells were incubated with SU9518 for 1 hour, followed by the
removal of the media, to determine the persistence of the inhibition.
They were then incubated for various periods of time before stimulation
with PDGF to induce receptor autophosphorylation.
Figure 2B
illustrates that PDGFr kinase was completely
inhibited for up to 2 hours after compound removal. This durable
inhibitory effect was further substantiated for up to 6 hours after
SU9518 had been removed from the medium (not
shown).
SU9518 Inhibits PDGF-Induced Cell
Proliferation
The observation that SU9518 potently inhibited
PDGF-induced receptor autophosphorylation suggested that the compound
should also inhibit cellular responses mediated by PDGFrs. Mouse
fibroblasts containing expressed human PDGFrß were stimulated with
PDGF in the absence or presence of SU9518 (2 µmol/L) for 18 hours,
after which they were exposed to BrdU for an additional 1.5 hours to
label cells in the S phase of the cell cycle. The cells were
subsequently fixed and stained for total DNA or BrdU incorporation
(Figure 3
). Very few of the serum-deprived cells were in S
phase at the time of BrdU labeling. In contrast, the majority of
PDGF-simulated cells incorporated BrdU at 18 hours, indicating that
most had entered the cell cycle and progressed to the S phase. SU9518
was a very effective inhibitor of this process, which resulted in the
blockade of PDGF-induced cell-cycle progression. The relative
IC50 values for SU9518 inhibition of PDGF-, FGF-
and EGF-induced BrdU incorporation using mouse fibroblasts were
0.053±0.04 (n=5), 4.40±1.13 (n=13), and 9.63±2.98 (n=7),
respectively. SU9518 demonstrated excellent potency for inhibition of
PDGF-induced proliferation and good selectivity relative to that for
FGF- and EGF-induced proliferation. This latter finding was expected,
because SU9518 exhibited no inhibition of isolated EGFr kinase using a
biochemical assay (not shown). SU9518 demonstrated no cytotoxicity at
concentrations of up to 50 µmol/L (the highest concentration tested;
n=3). Together, these data demonstrate that SU9518 is a potent and
selective inhibitor of PDGFr kinase activity in
cells.
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Plasma Pharmacokinetic Analysis of SU9518
in Rats
Pharmacokinetic studies
(Figure 4
, left panel) conducted with SU9518 at a single oral
dose of 50 mg/kg in rats indicated plasma levels of 0.96±0.23,
1.76±0.64, and 1.06±0.09 µg/mL at 2, 4, and 8 hours, respectively.
This analysis indicated that plasma levels above 1 µmol/L were
maintained for >8 hours after a single oral administration of this
compound. These data along with the durable inactivation of the
receptor provided a rationale to administer the compound on a daily
basis by the oral route at a dose of 50 mg/kg. A similar analysis for
subcutaneous delivery
(Figure 4
, right panel) showed plasma levels of SU9518 after
a single administration at 100 mg/kg to be 0.87±0.10, 0.63±0.14,
0.55±0.09, and 0.46±0.09 µg/mL at 1, 2, 4, and 7 days,
respectively. In this latter case, plasma levels exceeded 1 µmol/L
for >7 days after the single subcutaneous injection.
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SU9518 Inhibits Intimal Thickening of Rat
Carotid Artery
Because PDGFr has been strongly implicated as a
contributor to the thickening of arterial walls after balloon injury in
the rat, SU9518 was evaluated in a neointimal thickening model. For
these experiments, rat carotid arteries were denuded of endothelium
with a balloon catheter, and then SU9518 was administered orally at 50
mg/kg per day after the procedure. After 14 days, the arteries were
removed and examined for the degree of neointimal thickening.
Figure 5
shows a cross section of arteries from
vehicle-treated control (panel A) or SU9518-treated (panel B) rats.
Without SU9518 treatment, substantial thickening was observed by 14
days after vascular injury in vehicle-treated control rats. In this
case, the ratio of neointimal area to medial area (I/M ratio) was
1.94±0.38. Oral administration of SU9518 at a dose of 50 mg/kg
significantly reduced the I/M ratio by 46.7% to 1.03±0.29
(P<0.01). Moreover, this
reduction of neointimal thickening was accomplished with no apparent
changes in clinical symptoms, body weight, or organ morphology or color
(not shown).
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For evaluation of efficacy after subcutaneous administration
of SU9518 at 100 mg/kg, the first dose was administered 1 day before
balloon catheter denudation of the carotid artery endothelium, and the
second dose was administered 6 days after arterial injury. Control
untreated animals experienced substantial intimal thickening 14 days
after vascular injury (I/M ratio, 2.21±0.32)
(Table 1
). Weekly administration of SU9518 led to a
significant reduction (39.4%) in arterial thickening, which was mainly
due to inhibition of intimal thickening without statistically
significant changes of the medial and luminal areas. As in the case of
the oral administration, no obvious changes in symptoms, body weight,
or organs were observed (not shown).
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SU9518 Inhibits Migration and Proliferation of
Smooth Muscle Cells in Rats
Smooth muscle cell migration and proliferation are
thought to be the main processes leading to arterial thickening in the
rat balloon injury model. To examine the early steps in arterial
thickening, the number of migrating cells was examined 4 days after the
initial trauma. After endothelial denudation, the intima is usually
devoid of cells. However, 4 days after balloon injury, cells were
observed in the intima, as previously
reported.27 Treatment with
SU9518 significantly reduced the number of intimal cells per
mm2 compared with vehicle-treated control
rats
(Table 2
).
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To assess the extent of cell proliferation induced by
balloon injury, the medial and intimal layers were examined for
PCNA-positive cells after 4 and 7 days, respectively
(Table 2
). The labeling index in the medial layer 4 days
after injury was 24.1±5.3%, which is comparable with previous
reports.22 25 At
7 days after injury,
60% of intimal SMCs were in S phase, which is
also comparable with a previous
report.22 SU9518 treatment
significantly reduced the labeling index in both the media and the
intima
(Table 2
).
SU9518 Inhibits Arterial PDGFr
Phosphorylation
PDGFr autophosphorylation has been reported to increase
within a few days after balloon injury and to persist for several
weeks.14 To determine the
effect of administration of SU9518 on PDGFr expression and
phosphorylation, immunoblots were examined 7 days after carotid artery
injury
(Figure 6
). Balloon injury increased PDGFrß expression
2-fold in vehicle-treated (1.81±0.10fold) and SU9518-treated
(1.75±0.18fold) rats, compared with normal rats. In vehicle-treated
rats, PDGFr phosphorylation increased (3.27±0.20fold) in parallel
with the increase of PDGFrß expression. Administration of SU9518
decreased the level of PDGFr phosphorylation without significantly
affecting the receptor expression (1.11±0.26, 95.2% inhibition,
P<0.01).
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| Discussion |
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We have synthesized a novel series of PDGFr tyrosine kinase inhibitors derived from the 3-substituted indolinone chemical class of compounds.26 From this series, SU6668 has recently entered clinical development for the treatment of human cancers.44 SU9518 is an analog of SU6668 containing a bromo substitution at the C-5 position on the oxindole core. As in the case of SU6668, SU9518 has been shown to exhibit potent inhibitory activity toward isolated PDGFr kinases (IC50=0.06 µmol/L) and has shown ATP-competitive properties with the enzyme. Computational models based on cocrystallography of a related indolinone in the catalytic core of the FGF receptor45 suggest that the propionate substituent at the C-4' position of the pyrrole ring may contribute to the increased inhibitory potency observed for the PDGFr. This appears to result from interaction of the propionate with a basic arginine residue, which in other protein kinases, such as FGFr1 and vascular endothelial growth factor receptor 2, is a lysine residue.
The results of the present study demonstrate the first use of a proven, indolinone-based PDGFr kinase inhibitor to show antirestenotic activity. An indolinone compound has previously demonstrated efficacy in a stenosis model25 ; however, it has not yet been determined whether it inhibits any kinases. In contrast, we have shown that SU9518 potently inhibits the kinase activity of, and cellular responses mediated by, PDGFrs. Pharmacokinetic analysis has indicated that SU9518 can attain plasma levels that should be sufficient to block the cellular activity associated with the PDGFr when given by the oral or subcutaneous route. In addition, it appears that the subcutaneous route allows for less frequent administration of compound to achieve the antirestenotic effect.
The rat carotid injury model is known to better model the contribution of smooth muscle migration and proliferation than some other aspects of the clinical manifestations observed in humans such as the contribution of inflammatory processes. Nonetheless, a comparison of SU9518 with other synthetic kinase inhibitors, or other restenotic agents, in the same model suggests pharmacological features that are comparable or superior to other treatment modalities. In this regard, SU9518 represents a novel indolinone prototype that can be used to test the contribution of PDGFr to the tissue injury process and may provide a means to develop a novel therapeutic agent for the treatment of restenosis with distinct pharmacological features that may provide a more favorable clinical utility than that of other therapeutic approaches reported to date.
| Acknowledgments |
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| Footnotes |
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| References |
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and -ß blockade
on flow-induced neointimal formation in endothelialized baboon vascular
grafts. Circ Res. 2000;86:779786.This article has been cited by other articles:
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Y. Chang, D. Zhuang, C. Zhang, and A. Hassid Increase of PTP levels in vascular injury and in cultured aortic smooth muscle cells treated with specific growth factors Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2201 - H2208. [Abstract] [Full Text] [PDF] |
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