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(Circulation Research. 2001;88:630.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Weekly Dosing With the Platelet-Derived Growth Factor Receptor Tyrosine Kinase Inhibitor SU9518 Significantly Inhibits Arterial Stenosis

Yasundo Yamasaki, Kazuhisa Miyoshi, Nobuyuki Oda, Motomu Watanabe, Hidekazu Miyake, Julie Chan, Xueyan Wang, Li Sun, Cho Tang, Gerald McMahon, Kenneth E. Lipson

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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Abstract—The platelet-derived growth factor (PDGF) ligands and their receptors have been implicated as critical regulators of the formation of arterial lesions after tissue injury. SU9518 (3[5-{5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl}-2,4-dimethyl-1H-pyrrol-3-yl]propionic acid) is a novel synthetic indolinone that potently and selectively inhibits the cellular PDGF receptor kinase and PDGF receptor–induced cell proliferation. Inhibition of PDGF receptor phosphorylation in cell-based assays occurs within 5 minutes after drug exposure and persists for >6 hours after drug removal. The pharmacokinetics indicate plasma levels that exceeded the effective concentration required to inhibit the PDGF receptor in cells for up to 8 hours or 7 days after a single oral or subcutaneous administration, respectively. In the rat balloon arterial injury–induced stenosis model, once-daily oral or once-weekly subcutaneous administration of SU9518 reduced intimal thickening of the carotid artery (ratio of neointimal to medial area, 1.94±0.38 versus 1.03±0.29 [P<0.01] 2.21±0.32 versus 1.34±0.45 [P<0.01], respectively). These studies provide the rationale to evaluate PDGF receptor tyrosine kinase inhibitors, including inhibitors related to the indolinone, SU9518, for the treatment of arterial restenosis.

Key Words: angioplasty • platelet-derived growth factor • restenosis • tyrosine kinase • indolinone

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is currently estimated that >350 000 coronary angioplasty procedures will be performed in the United States in the next few years.^{1 2} The growth of angioplasty has been suggested to be the result of improvements in surgical procedures, increased success rates, and a reduction in acute complications. Percutaneous transluminal coronary angioplasty (PTCA) for primary treatment of arterial occlusion after acute myocardial infarction has been demonstrated to be superior to thrombolytic therapy with regard to the restoration of normal coronary blood flow and may be associated with lower rates of recurrent reinfarction.^{3 4} However, restenosis at the site of the primary occlusion can occur within the first 6 months after the PTCA procedure leading to additional procedures or complications. Of patients who undergo PTCA, 35% to 50% will develop angiographic renarrowing, and as many as 25% to 35% are at risk for clinical recurrence.^{5 6 7}

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.

View larger version (37K): [in this window] [in a new window]   Figure 1. SU9518 is a potent inhibitor of PDGFr tyrosine kinase. A, Chemical structure of SU9518. B, Quiescent 3T3 cells expressing human PDGFr or PDGFrß were treated with SU9518 at indicated concentrations for 1 hour followed by stimulation of cells with PDGF for 5 minutes. Lysates of treated cells were fractionated by SDS-PAGE and transferred to nitrocellulose. Amount of PDGFr and phosphorylated PDGFr was visualized with anti-PDGFr and anti-phosphotyrosine (Anti-pY) antibodies, respectively. C, Ability of SU9518 to inhibit rat PDGFr kinase in A7r5 aorta cells was tested as described above.

	Materials and Methods

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Cell Lines and Culture
3T3 cell lines overexpressing epidermal growth factor (EGF) receptor (EGFr), PDGFr, 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-PDGFrß, 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 anti–proliferating 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-PDGFrß 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
SU9518 Inhibits PDGFr Kinase Tyrosine Phosphorylation
SU9518 was evaluated for its ability to inhibit PDGFr kinase using mouse fibroblasts expressing human PDGFr 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). Within 5 minutes of exposure to PDGF, significantly increased tyrosine phosphorylation was observed. SU9518 was found to inhibit the autophosphorylation of both receptors in a dose-dependent manner leading to complete inhibition of receptor activity at a concentration of 80 nmol/L.

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.

View larger version (69K): [in this window] [in a new window]   Figure 2. Kinetics of SU9518 inhibition of PDGFr tyrosine kinase. A, Rapidity of inhibition of PDGFr kinase was shown by exposing 3T3/PDGFrß cells to SU9518 (2 µmol/L) for indicated times before stimulation with PDGF. B, To test the duration of PDGFr kinase inhibition, quiescent 3T3/PDGFrß cells were exposed to SU9518 (2 µmol/L) for 1 hour. The compound was then removed from cells for indicated times before stimulation with PDGF for 5 minutes. None indicates that the compound was not removed, whereas 0 minutes of wash out indicates that the compound was removed and cells were immediately stimulated with PDGF. For both panels, lysates of treated cells were fractionated by SDS-PAGE and transferred to nitrocellulose. Amount of PDGFr and phosphorylated PDGFr was visualized with anti-PDGFr and anti-phosphotyrosine (Anti-pY) antibodies, respectively.

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.

View larger version (40K): [in this window] [in a new window]   Figure 3. SU9518 is a potent inhibitor of PDGF-induced proliferation in fibroblasts. Quiescent 3T3 cells expressing human PDGFrß were exposed to SU9518 (2 µmol/L) and stimulated with PDGF for 18 hours. Cells in S phase were labeled with BrdU for 1.5 hours and then fixed for immunofluorescence detection.

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.

View larger version (11K): [in this window] [in a new window]   Figure 4. SU9518 exhibits sustained plasma levels in rats after a single oral or subcutaneous administration. Blood samples (1 mL) were obtained at 2, 4, and 8 hours after oral administration (p.o.) of SU9518 at a dose of 50 mg/kg and at 1, 2, 4, and 7 days after subcutaneous injection (s.c.) of SU9518 at a dose of 100 mg/kg. Plasma samples were processed as described and compound concentrations determined using HPLC.

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).

View larger version (60K): [in this window] [in a new window]   Figure 5. Oral delivery of SU9518 inhibits arterial restenosis after balloon injury in the rat. The endothelium of rat carotid arteries was denuded with a balloon catheter. After 14 days of administration of SU9518 by oral gavage (50 mg/kg per day) or vehicle, animals were euthanized and carotid arteries removed and subjected to histological examination. Shown are cross sections of arteries from untreated (panel A) and SU9518-treated (panel B) animals. A indicates adventitia; M, media; and I, intima.

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).

View this table: [in this window] [in a new window]   Table 1. Effect of Subcutaneously Administered SU9518 on Intimal Thickness of Injured Rat Carotid Arteries

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).

View this table: [in this window] [in a new window]   Table 2. Effect of Subcutaneously Administered SU9518 on Migration and Proliferation of Smooth Muscle Cells in Injured Rat Carotid Arteries

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±0.10–fold) and SU9518-treated (1.75±0.18–fold) rats, compared with normal rats. In vehicle-treated rats, PDGFr phosphorylation increased (3.27±0.20–fold) 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).

View larger version (52K): [in this window] [in a new window]   Figure 6. SU9518 is a potent inhibitor of the PDGFr tyrosine kinase in rats. Endothelium of rat carotid arteries was denuded with a balloon catheter. Seven days after injury and subcutaneous administration of SU9518 (100 mg/kg) or vehicle, animals were killed and carotid arteries removed. PDGFr was immunoprecipitated from lysates of arteries, fractionated by SDS-PAGE, and transferred to nitrocellulose. Amount of PDGFr and phosphorylated PDGFr was visualized with anti-PDGFr and anti-phosphotyrosine antibodies, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Synthetic inhibitors of tyrosine kinases have emerged as important new therapies for the potential treatment of human cancers and metastases.²⁸ In addition, there have been several published reports of kinase inhibitors as potential therapeutic agents for the treatment of disease associated with blood vessel injury.^{12 29 30 31 32 33 34 35 36 37 38} In this regard, the PDGFrs have been viewed as attractive targets for intervention in arterial restenosis after angioplasty or allograft-induced vasculopathies using antibodies.^{39 40 41 42} Synthetic tyrosine kinase inhibitors^{12 30 31 33 38} of the quinoxaline,²⁵ phenylaminoquinazoline,³¹ or phenylaminopyridopyrimidine^{33 37 43} classes of compounds have been shown to inhibit this process.

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

 
This work was supported by Taiho Pharmaceutical Co and SUGEN, Inc.

	Footnotes

 
Original received September 7, 2000; resubmission received January 17, 2001; revised resubmission received February 2, 2001; accepted February 2, 2001.

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