weekly dosing with the platelet-derived growth factor receptor tyrosine kinase inhibitor su9518...

Weekly Dosing With the Platelet-Derived Growth FactorReceptor Tyrosine Kinase Inhibitor SU9518 Significantly

Inhibits Arterial StenosisYasundo Yamasaki, Kazuhisa Miyoshi, Nobuyuki Oda, Motomu Watanabe, Hidekazu, Miyake,

Julie Chan, Xueyan Wang, Li Sun, Cho Tang, Gerald McMahon, Kenneth E. Lipson

Abstract—The platelet-derived growth factor (PDGF) ligands and their receptors have been implicated as critical regulatorsof 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 selec-tively inhibits the cellular PDGF receptor kinase and PDGF receptor–induced cell proliferation. Inhibition of PDGFreceptor phosphorylation in cell-based assays occurs within 5 minutes after drug exposure and persists for.6 hours afterdrug removal. The pharmacokinetics indicate plasma levels that exceeded the effective concentration required to inhibitthe 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 administrationof SU9518 reduced intimal thickening of the carotid artery (ratio of neointimal to medial area, 1.9460.38 versus1.0360.29 [P,0.01] 2.2160.32 versus 1.3460.45 [P,0.01], respectively). These studies provide the rationale toevaluate PDGF receptor tyrosine kinase inhibitors, including inhibitors related to the indolinone, SU9518, for thetreatment of arterial restenosis.(Circ Res. 2001;88:630-636.)

Key Words: angioplastyn platelet-derived growth factorn restenosisn tyrosine kinasen indolinone

I t is currently estimated that.350 000 coronary angio-plasty procedures will be performed in the United States in

the next few years.1,2 The growth of angioplasty has beensuggested to be the result of improvements in surgicalprocedures, increased success rates, and a reduction in acutecomplications. Percutaneous transluminal coronary angio-plasty (PTCA) for primary treatment of arterial occlusionafter acute myocardial infarction has been demonstrated to besuperior to thrombolytic therapy with regard to the restorationof normal coronary blood flow and may be associated withlower rates of recurrent reinfarction.3,4 However, restenosis atthe site of the primary occlusion can occur within the first 6months after the PTCA procedure leading to additionalprocedures or complications. Of patients who undergo PTCA,35% to 50% will develop angiographic renarrowing, and asmany as 25% to 35% are at risk for clinical recurrence.5–7

The underlying mechanisms of restenosis comprise acombination of effects ranging from vessel recoil, negativevascular remodeling, and thrombus formation to neointimalhyperplasia.8,9 The neointimal hyperplasia process has beensuggested to be associated with the expression of growthfactors and their cognate receptors including tyrosine kinases.The growth factors themselves are released by local thrombi

and the angioplasty-injured arterial segment itself and mayserve to enhance the expression of other growth-regulatoryevents. These processes result in an inflammatory reactionand a myofibroblast proliferative response, which worsenvessel narrowing caused by negative remodeling and result inthe formation of a clinically significant restenotic lesion.10,11

Platelet-derived growth factor (PDGF) is one of severalgrowth factors implicated in the restenosis process. In theanimal balloon-injury model, PDGF acts primarily to inducesmooth muscle migration and secondarily to promote intimalproliferation.12,13 PDGF receptor (PDGFr) autophosphoryla-tion increases within a few days after injury and persists forseveral weeks.14 Furthermore, in human restenotic lesions incomparison with nonlesioned sites, the presence of PDGF-Aand -B as ligands and PDGFrb is detected after PTCA.15,16

The finding of the expression of these factors and receptor invascular damaged and repaired regions strongly suggests thatautocrine or paracrine stimulation of these receptor systemsare important in the biological regulation of this tissue injuryprocess.

Triazolopyrimidine (trapidil), a mild competitive inhibitorof the PDGFr, has been evaluated in 3 clinical trials todate.17–19 In one of these studies, Maresta et al19 found a

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

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 [email protected]© 2001 American Heart Association, Inc.

Circulation Researchis available at http://www.circresaha.org

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reduction ('50%) in restenosis for trapidil when comparedwith aspirin. Several PDGFr kinase inhibitors have beenevaluated in preclinical models as a prelude to possibleclinical development.20,21

In this study, we have identified a potent indolinoneinhibitor 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 reportsuggest that SU9518 rapidly penetrates the cell membrane,leading to a durable inactivation of the receptor due tocompetitive inhibition of the catalytic activity of the kinaseafter receptor activation. SU9518 was evaluated for pharma-cological properties, including the potential to inhibit arterialthickening after tissue injury.

Materials and Methods

Cell Lines and Culture3T3 cell lines overexpressing epidermal growth factor (EGF) recep-tor (EGFr), PDGFra, or PDGFrb were provided by Dr Axel Ullrich(Max-Planck Institute, Martinsried, Germany). A7r5 rat aortasmooth muscle cells were purchased from American Type CultureCollection. DMEM, FBS, and calf serum were purchased fromGIBCO-BRL. 3T3 mouse fibroblasts overexpressing theb or a formof the human PDGFr (3T3/PDGFrb or 3T3/PDGFra cells, respec-tively) were grown in DMEM containing 10% calf serum and 2mmol/L L-glutamine at 37°C under an atmosphere of 5% CO2. A7r5cells were grown in DMEM containing 10% FBS.

Cellular Kinase AssayConfluent cells were deprived of serum overnight, treated with thetest compound for 1 hour, and then stimulated with 3.3 nmol/L (100ng/mL) PDGF-AA or -BB for 5 minutes at 37°C. Cells were thenlysed with SDS sample buffer and fractionated by SDS-PAGE.Phosphorylated PDGFrs were detected by Western blot analysisusing anti-phosphotyrosine antibodies (4G10) with enhanced chemi-luminescence (ECL) detection. The amount of PDGFr in each lanewas determined by using anti-PDGFrab, anti-PDGFra, or anti-PDGFrb antibodies and ECL detection.

Ligand-Induced Bromodeoxyuridine (BrdU)Incorporation Assay3T3/EGFr cells (3T3 cells overexpressing EGFr) in a 96-well platewere made quiescent by serum deprivation for 24 hours. Theserum-deprived cells were then stimulated with (in nmol/L) fibro-blast growth factor (FGF) 2/basic FGF 1.5, EGF 4, or PDGF 3.8 inthe absence or presence of the indicated concentrations of SU9518for 20 hours. BrdU was added for a 2-hour labeling period, and thecells were fixed. The amount of BrdU incorporation was determinedwith an ELISA kit (Roche Molecular) using anti-BrdU/POD (per-oxidase). Cytotoxicity was assessed under identical conditions byusing a Tox-8 kit (Sigma).

Pharmacokinetic AnalysisAll experimental protocols were approved by the InstitutionalAnimal Care and Use Committee of Taiho Pharmaceutical Co. MaleWistar rats (7 weeks old, Charles River Japan, Inc [Tsukuba]) werehoused in constant temperature facilities and given standard labora-tory chow and water ad libitum. Blood samples (1 mL) wereobtained at 2, 4, and 8 hours after oral administration of SU9518 or1, 2, 4, and 7 days after subcutaneous administration. Blood wassampled from a cervical vein, and the plasma was stored at220°Cuntil analysis was performed. SU9518 was extracted from the plasmawith acetonitrile, acidified, and extracted with ethyl acetate. HPLCanalysis was performed on a Mightysil RP-18 GR column, 5-mmparticle size, 4.63150 mm using a Waters HPLC with a gradient of

Figure 2. Kinetics of SU9518 inhibition of PDGFr tyrosinekinase. A, Rapidity of inhibition of PDGFr kinase was shown byexposing 3T3/PDGFrb cells to SU9518 (2 mmol/L) for indicatedtimes before stimulation with PDGF. B, To test the duration ofPDGFr kinase inhibition, quiescent 3T3/PDGFrb cells wereexposed to SU9518 (2 mmol/L) for 1 hour. The compound wasthen removed from cells for indicated times before stimulationwith PDGF for 5 minutes. None indicates that the compoundwas not removed, whereas 0 minutes of wash out indicates thatthe compound was removed and cells were immediately stimu-lated with PDGF. For both panels, lysates of treated cells werefractionated by SDS-PAGE and transferred to nitrocellulose.Amount of PDGFr and phosphorylated PDGFr was visualizedwith anti-PDGFr and anti-phosphotyrosine (Anti-pY) antibodies,respectively.

Figure 1. SU9518 is a potent inhibitor of PDGFr tyrosine kinase.A, Chemical structure of SU9518. B, Quiescent 3T3 cellsexpressing human PDGFra or PDGFrb were treated withSU9518 at indicated concentrations for 1 hour followed by stim-ulation of cells with PDGF for 5 minutes. Lysates of treated cellswere fractionated by SDS-PAGE and transferred to nitrocellu-lose. Amount of PDGFr and phosphorylated PDGFr was visual-ized with anti-PDGFr and anti-phosphotyrosine (Anti-pY) anti-bodies, respectively. C, Ability of SU9518 to inhibit rat PDGFrkinase in A7r5 aorta cells was tested as described above.

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90% 5 mmol/L phosphate buffer (pH 2.8) and 10% acetonitrile to30% 5 mmol/L phosphate buffer (pH 2.8) and 70% acetonitrile.

Rat Arterial Injury ModelMale Sprague-Dawley rats (12 to 13 weeks old, Clea Japan Inc,Tokyo, Japan) were housed in constant-temperature facilities andgiven standard laboratory chow and water ad libitum. Ballooncatheter denudation of the carotid artery endothelium was performedaccording to the method described by Clowes et al.22 Briefly, the ratswere anesthetized with a gas mixture of N2O/O2 (30:70) containing2% halothane. After a median incision of the abdominal skin hadbeen made, the right iliac artery was dissected and cannulated witha 2F balloon catheter (embolectomy catheter arterial balloon, Med-ical Technology Transfer). The catheter was inflated with saline andpassed through the left common carotid artery 4 times to produce adistending, de-endothelializing injury.

Histopathologic EvaluationOn the 14th day after balloon injury, the rats were anesthetized withether and perfused transcardially with saline, followed by 10%buffered formalin. The left carotid artery (from the aortic arch tobifurcation) was removed, postfixed, and embedded in paraffin.Sections 3mm thick (5 per artery) were cut and stained withhematoxylin and eosin. The cross-sectional areas of the intima andthe media on photographs were measured with a digital analyzer(Digitaizer, Wacom).

PDGFr Protein Expression and PhosphorylationDetection of PDGFr phosphorylation in injured arteries was per-formed 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 proteinwas extracted, subjected to PDGFrb immunoprecipitation, andseparated by SDS-PAGE. Phosphorylated PDGFr was visualized byanti-phosphotyrosine immunoblotting (4G10) with ECL detection.The amount of PDGFr in each lane was determined using anti-PDGFrb antibody. The signal intensity of PDGFr and phosphory-lated PDGFr in vehicle- or SU9518-treated rats (subcutaneousinjection of 100 mg/kg) was compared with that of uninjured ratartery PDGFr (n53 in each group) by densitometry.

In Vivo Migration AssayQuantification of smooth muscle cell migration into the intima wasperformed by the method of Bendeck et al.24 Four days after ballooninjury, the artery was fixed with 4% buffered paraformaldehyde. Themiddle of the artery was cut lengthwise and pinned intimal side uponto a Teflon plate. The arteries were incubated with monoclonalantibody against human nuclei and chromosomes (MAB 1276,1:100) overnight at 4°C, and immunoreactive cells were visualizedwith a commercially available detection system (Vector Laborato-ries). The number of cell nuclei per mm2 was counted by lightmicroscopy.

In Vivo Proliferation AssayQuantification of medial and intimal smooth muscle cell prolifera-tion was performed by the method of Muranaka et al.25 Four or 7days after injury, the artery was fixed with 4% buffered paraformal-dehyde and embedded in paraffin for preparation of 4 sections 3mmthick. The sections were incubated with biotinylated anti–proliferat-ing cell nuclear antigen (PCNA) monoclonal antibody (PCNA15,1:250) overnight at 4°C and counterstained with hematoxylin tovisualize all nuclei.

Figure 3. SU9518 is a potent inhibitor ofPDGF-induced proliferation in fibroblasts.Quiescent 3T3 cells expressing humanPDGFrb were exposed to SU9518 (2mmol/L) and stimulated with PDGF for 18hours. Cells in S phase were labeled withBrdU for 1.5 hours and then fixed forimmunofluorescence detection.

Figure 4. SU9518 exhibits sustained plasma levels in rats aftera single oral or subcutaneous administration. Blood samples (1mL) 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 7days after subcutaneous injection (s.c.) of SU9518 at a dose of100 mg/kg. Plasma samples were processed as described andcompound concentrations determined using HPLC.

Figure 5. Oral delivery of SU9518 inhibits arterial restenosisafter balloon injury in the rat. The endothelium of rat carotidarteries was denuded with a balloon catheter. After 14 days ofadministration of SU9518 by oral gavage (50 mg/kg per day) orvehicle, animals were euthanized and carotid arteries removedand subjected to histological examination. Shown are crosssections of arteries from untreated (panel A) and SU9518-treated (panel B) animals. A indicates adventitia; M, media; andI, intima.

632 Circulation Research March 30, 2001



Drug Administration and DosageSU9518 (50 mg/kg in 0.5% hydroxypropyl methylcellulose solution[10 mL/kg]) was administered orally by gavage once daily from 1hour before denudation to the day before removal of the artery forevaluation. Alternatively, SU9518 (100 mg/kg in carboxymethylcel-lulose sodium USP [0.5%], sodium chloride [0.9%], polysorbate 80[0.4%], benzyl alcohol [0.9%], and deionized water [4 mL/kg]) wasadministered subcutaneously 1 day before denudation and again 6days after the operation.

ReagentsSU9518 (Figure 1A) was synthesized by SUGEN using methodspreviously described.26 ECL reagents were purchased from Amer-sham. PDGF-AA, PDGF-BB, and anti-BrdU/POD conjugate werepurchased from Roche Molecular. Anti-phosphotyrosine monoclonal(4G10) and anti-PDGFrab polyclonal antibodies were from UpstateBiotechnology Inc, whereas anti-PDGFra and anti-PDGFrb antibod-ies were purchased from Santa Cruz Biotechnology. Biotinylatedanti-PCNA monoclonal antibody was purchased from Caltag Labo-ratories. Anti-nuclei and chromosomes were purchased from Chemi-con International. All other reagents were obtained from Sigma.

Statistical AnalysisAll data are expressed as mean6SD. Statistically significant differ-ences between 2 groups were calculated by (2-tailed) Aspin-Welchttest. A P value ,0.05 was considered to indicate statisticalsignificance.

ResultsSU9518 Inhibits PDGFr KinaseTyrosine PhosphorylationSU9518 was evaluated for its ability to inhibit PDGFr kinaseusing mouse fibroblasts expressing human PDGFra orPDGFrb. Cells were made quiescent by serum deprivationand contact inhibition and were then stimulated with PDGFand various concentrations of SU9518 (Figure 1B). Within 5minutes of exposure to PDGF, significantly increased ty-

rosine phosphorylation was observed. SU9518 was found toinhibit the autophosphorylation of both receptors in a dose-dependent manner leading to complete inhibition of receptoractivity at a concentration of 80 nmol/L.

To demonstrate that SU9518 would also inhibit rat PDGFrkinase, a similar experiment was performed with A7r5 cells(Figure 1C), which only express PDGFrb (not shown).PDGFrb in A7r5 cells were inhibited with a dose responsesimilar to that observed in mouse fibroblasts overexpressinghuman PDGFrs.

Kinetics of SU9518 Inhibition of PDGFr Kinasein FibroblastsThe temporal inactivation of the PDGFr kinase by SU9518was studied to determine the pharmacological requirementsto maintain the inhibitory effect. In the first experiment,receptor autophosphorylation was measured using 3T3/PDGFrb fibroblast cells after exposure of cells to SU9518 fordifferent periods of time (Figure 2A). Complete inhibition ofPDGF-induced receptor autophosphorylation was observedafter incubation of the cells with SU9518 at all time pointstested, indicating that,5 minutes of exposure was requiredto block the activation of PDGFr kinase. The rapid penetra-tion and inactivation of the receptor may be due in part to thehigh relative lipophilicity of the compound and its ability topenetrate cell membranes.

Cells were incubated with SU9518 for 1 hour, followed bythe removal of the media, to determine the persistence of theinhibition. They were then incubated for various periods oftime before stimulation with PDGF to induce receptor auto-phosphorylation. Figure 2B illustrates that PDGFr kinase wascompletely inhibited for up to 2 hours after compoundremoval. This durable inhibitory effect was further substan-

TABLE 1. Effect of Subcutaneously Administered SU9518 on Intimal Thickness of Injured RatCarotid Arteries

Treatment Group n Intima/Media Medial Area, mm2Intimal

Area, mm2 Lumen Area, mm2

Vehicle-treated 6 2.2160.32 0.11060.026 0.22960.029 0.21060.017

SU9518-treated (100 mg/kg) 6 1.3460.45* 0.12160.020 0.15460.052* 0.24360.034

Percentage inhibition 39.4% z z z 32.8% z z z

I/M ratio was measured as described in Materials and Methods. I/M, medial area, intimal area, and lumen areavalues are given as mean6SD. SU9518 was administered to rats by subcutaneous injection once weekly for 14 days(2 injections).

*P,0.01 (vs vehicle, Aspin-Welch’s t test).

TABLE 2. Effect of Subcutaneously Administered SU9518 on Migration andProliferation of Smooth Muscle Cells in Injured Rat Carotid Arteries

Treatment Group n

Migration (Cell No.) Labeling Index (%)

Intima (4 days) Media (4 days) Intima (7 days)

Vehicle-treated 5 7269 24.165.3 59.6614.7

SU9518-treated, 100 mg/kg 5 3367* 16.964.2† 36.8614.2†

Percentage inhibition 54.2% 29.9% 38.3%

Number of migrating smooth muscle cells and labeling index (%) in medial and intimal layers weremeasured as described in Materials and Methods and are presented as mean6SD. SU9518 wasadministered to rats by subcutaneous injection once weekly for 14 days (2 injections).

*P,0.01, †P,0.05 (vs vehicle, Aspin-Welch t test).




tiated for up to 6 hours after SU9518 had been removed fromthe medium (not shown).

SU9518 Inhibits PDGF-Induced Cell ProliferationThe observation that SU9518 potently inhibited PDGF-induced receptor autophosphorylation suggested that thecompound should also inhibit cellular responses mediated byPDGFrs. Mouse fibroblasts containing expressed humanPDGFrb were stimulated with PDGF in the absence orpresence of SU9518 (2mmol/L) for 18 hours, after whichthey were exposed to BrdU for an additional 1.5 hours tolabel cells in the S phase of the cell cycle. The cells weresubsequently fixed and stained for total DNA or BrdUincorporation (Figure 3). Very few of the serum-deprivedcells were in S phase at the time of BrdU labeling. In contrast,the majority of PDGF-simulated cells incorporated BrdU at18 hours, indicating that most had entered the cell cycle andprogressed to the S phase. SU9518 was a very effectiveinhibitor of this process, which resulted in the blockade ofPDGF-induced cell-cycle progression. The relative IC50 val-ues for SU9518 inhibition of PDGF-, FGF- and EGF-inducedBrdU incorporation using mouse fibroblasts were0.05360.04 (n55), 4.4061.13 (n513), and 9.6362.98(n57), respectively. SU9518 demonstrated excellent potencyfor inhibition of PDGF-induced proliferation and good selec-tivity relative to that for FGF- and EGF-induced proliferation.This latter finding was expected, because SU9518 exhibitedno inhibition of isolated EGFr kinase using a biochemicalassay (not shown). SU9518 demonstrated no cytotoxicity atconcentrations of up to 50mmol/L (the highest concentrationtested; n53). Together, these data demonstrate that SU9518is a potent and selective inhibitor of PDGFr kinase activity incells.

Plasma Pharmacokinetic Analysis of SU9518in RatsPharmacokinetic studies (Figure 4, left panel) conducted withSU9518 at a single oral dose of 50 mg/kg in rats indicatedplasma levels of 0.9660.23, 1.7660.64, and 1.0660.09mg/mL at 2, 4, and 8 hours, respectively. This analysisindicated that plasma levels above 1mmol/L were maintainedfor .8 hours after a single oral administration of thiscompound. These data along with the durable inactivation of

the receptor provided a rationale to administer the compoundon a daily basis by the oral route at a dose of 50 mg/kg. Asimilar analysis for subcutaneous delivery (Figure 4, rightpanel) showed plasma levels of SU9518 after a singleadministration at 100 mg/kg to be 0.8760.10, 0.6360.14,0.5560.09, and 0.4660.09mg/mL at 1, 2, 4, and 7 days,respectively. In this latter case, plasma levels exceeded 1mmol/L for .7 days after the single subcutaneous injection.

SU9518 Inhibits Intimal Thickening of RatCarotid ArteryBecause PDGFr has been strongly implicated as a contributorto the thickening of arterial walls after balloon injury in therat, SU9518 was evaluated in a neointimal thickening model.For these experiments, rat carotid arteries were denuded ofendothelium with a balloon catheter, and then SU9518 wasadministered orally at 50 mg/kg per day after the procedure.After 14 days, the arteries were removed and examined forthe degree of neointimal thickening. Figure 5 shows a crosssection of arteries from vehicle-treated control (panel A) orSU9518-treated (panel B) rats. Without SU9518 treatment,substantial thickening was observed by 14 days after vascularinjury in vehicle-treated control rats. In this case, the ratio ofneointimal area to medial area (I/M ratio) was 1.9460.38.Oral administration of SU9518 at a dose of 50 mg/kgsignificantly reduced the I/M ratio by 46.7% to 1.0360.29(P,0.01). Moreover, this reduction of neointimal thickeningwas accomplished with no apparent changes in clinicalsymptoms, body weight, or organ morphology or color (notshown).

For evaluation of efficacy after subcutaneous administra-tion of SU9518 at 100 mg/kg, the first dose was administered1 day before balloon catheter denudation of the carotid arteryendothelium, and the second dose was administered 6 daysafter arterial injury. Control untreated animals experiencedsubstantial intimal thickening 14 days after vascular injury(I/M ratio, 2.2160.32) (Table 1). Weekly administration ofSU9518 led to a significant reduction (39.4%) in arterialthickening, which was mainly due to inhibition of intimalthickening without statistically significant changes of themedial and luminal areas. As in the case of the oral admin-istration, no obvious changes in symptoms, body weight, ororgans were observed (not shown).

SU9518 Inhibits Migration and Proliferation ofSmooth Muscle Cells in RatsSmooth muscle cell migration and proliferation are thought tobe the main processes leading to arterial thickening in the ratballoon injury model. To examine the early steps in arterialthickening, the number of migrating cells was examined 4days after the initial trauma. After endothelial denudation, theintima is usually devoid of cells. However, 4 days afterballoon injury, cells were observed in the intima, as previ-ously reported.27 Treatment with SU9518 significantly re-duced the number of intimal cells per mm2 compared withvehicle-treated control rats (Table 2).

To assess the extent of cell proliferation induced byballoon injury, the medial and intimal layers were examinedfor PCNA-positive cells after 4 and 7 days, respectively

Figure 6. SU9518 is a potent inhibitor of the PDGFr tyrosinekinase in rats. Endothelium of rat carotid arteries was denudedwith a balloon catheter. Seven days after injury and subcutane-ous administration of SU9518 (100 mg/kg) or vehicle, animalswere killed and carotid arteries removed. PDGFr was immuno-precipitated from lysates of arteries, fractionated by SDS-PAGE,and transferred to nitrocellulose. Amount of PDGFr and phos-phorylated PDGFr was visualized with anti-PDGFr and anti-phosphotyrosine antibodies, respectively.




(Table 2). The labeling index in the medial layer 4 days afterinjury was 24.165.3%, which is comparable with previousreports.22,25 At 7 days after injury,'60% of intimal SMCswere in S phase, which is also comparable with a previousreport.22 SU9518 treatment significantly reduced the labelingindex in both the media and the intima (Table 2).

SU9518 Inhibits Arterial PDGFr PhosphorylationPDGFr autophosphorylation has been reported to increasewithin a few days after balloon injury and to persist forseveral weeks.14 To determine the effect of administration ofSU9518 on PDGFr expression and phosphorylation, immu-noblots were examined 7 days after carotid artery injury(Figure 6). Balloon injury increased PDGFrb expression'2-fold in vehicle-treated (1.8160.10–fold) and SU9518-treated (1.7560.18–fold) rats, compared with normal rats. Invehicle-treated rats, PDGFr phosphorylation increased(3.2760.20–fold) in parallel with the increase of PDGFrbexpression. Administration of SU9518 decreased the level ofPDGFr phosphorylation without significantly affecting thereceptor expression (1.1160.26, 95.2% inhibition,P,0.01).

DiscussionSynthetic inhibitors of tyrosine kinases have emerged asimportant new therapies for the potential treatment of humancancers and metastases.28 In addition, there have been severalpublished reports of kinase inhibitors as potential therapeuticagents for the treatment of disease associated with bloodvessel injury.12,29–38 In this regard, the PDGFrs have beenviewed as attractive targets for intervention in arterial reste-nosis after angioplasty or allograft-induced vasculopathiesusing antibodies.39–42 Synthetic tyrosine kinase inhibi-tors12,30,31,33,38 of the quinoxaline,25 phenylaminoquinazo-line,31 or phenylaminopyridopyrimidine33,37,43 classes ofcompounds have been shown to inhibit this process.

We have synthesized a novel series of PDGFr tyrosinekinase inhibitors derived from the 3-substituted indolinonechemical class of compounds.26 From this series, SU6668 hasrecently entered clinical development for the treatment ofhuman cancers.44 SU9518 is an analog of SU6668 containinga bromo substitution at the C-5 position on the oxindole core.As in the case of SU6668, SU9518 has been shown to exhibitpotent inhibitory activity toward isolated PDGFr kinases(IC5050.06 mmol/L) and has shown ATP-competitive prop-erties with the enzyme. Computational models based oncocrystallography of a related indolinone in the catalytic coreof the FGF receptor45 suggest that the propionate substituentat the C-49position of the pyrrole ring may contribute to theincreased inhibitory potency observed for the PDGFr. Thisappears to result from interaction of the propionate with abasic arginine residue, which in other protein kinases, such asFGFr1 and vascular endothelial growth factor receptor 2, is alysine residue.

The results of the present study demonstrate the first use ofa proven, indolinone-based PDGFr kinase inhibitor to showantirestenotic activity. An indolinone compound has previ-ously 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, PDG-Frs. Pharmacokinetic analysis has indicated that SU9518 canattain plasma levels that should be sufficient to block thecellular activity associated with the PDGFr when given by theoral or subcutaneous route. In addition, it appears that thesubcutaneous route allows for less frequent administration ofcompound to achieve the antirestenotic effect.

The rat carotid injury model is known to better model thecontribution of smooth muscle migration and proliferationthan some other aspects of the clinical manifestations ob-served in humans such as the contribution of inflammatoryprocesses. Nonetheless, a comparison of SU9518 with othersynthetic kinase inhibitors, or other restenotic agents, in thesame model suggests pharmacological features that are com-parable or superior to other treatment modalities. In thisregard, SU9518 represents a novel indolinone prototype thatcan be used to test the contribution of PDGFr to the tissueinjury process and may provide a means to develop a noveltherapeutic agent for the treatment of restenosis with distinctpharmacological features that may provide a more favorableclinical utility than that of other therapeutic approachesreported to date.

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

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

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

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