Neuroprotective Profile of Novel Src Kinase Inhibitors in Rodent Models of Cerebral Ischemia

Neuroprotective Profile of Novel Src Kinase Inhibitors inRodent Models of Cerebral Ischemia

Shi Liang, Kevin Pong, Cathleen Gonzales, Yi Chen, Huai-Ping Ling, Robert J. Mark,Frank Boschelli, Diane H. Boschelli, Fei Ye, Ana Carolina Barrios Sosa, Tarek S. Mansour,Philip Frost, Andrew Wood, Menelas N. Pangalos, and Margaret M. ZaleskaDiscovery Neuroscience, Wyeth Research, Princeton, New Jersey (S.L., K.P., C.G., Y.C., H.-P.L., R.J.M., A.W., M.N.P.,M.M.Z.); and Oncology (F.B., P.F.) and Chemical Sciences (D.H.B., F.Y., A.C.B.S., T.S.M.), Wyeth Research, Pearl River, NewYork

Received June 1, 2009; accepted September 8, 2009

ABSTRACTSrc kinase signaling has been implicated in multiple mechanismsof ischemic injury, including vascular endothelial growth factor(VEGF)-mediated vascular permeability that leads to vasogenicedema, a major clinical complication in stroke and brain trauma.Here we report the effects of two novel Src kinase inhibitors,4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propoxy]-3-quinolinecarbonitrile (SKI-606)and 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[4-(4-methypiperazin-1-yl)but-1-ynyl]-3-quinolinecarbonitrile (SKS-927), on ischemia-induced brain infarction and short- and long-term neurological deficits. Two well established transient [transientmiddle cerebral artery occlusion (tMCAO)] and permanent [per-manent middle cerebral artery occlusion (pMCAO)] focal ischemiamodels in the rat were used with drug treatments initiated up to 6 hafter onset of stroke to mimic the clinical scenario. Brain penetra-tion of Src inhibitors, their effect on blood-brain barrier integrity

and VEGF signaling in human endothelial cells were also evalu-ated. Our results demonstrate that both agents potently blockVEGF-mediated signaling in human endothelial cells, penetrate ratbrain upon systemic administration, and inhibit postischemic Srcactivation and vascular leakage. Treatment with SKI-606 or SKS-927 (at the doses of 3–30 mg/kg i.v.) resulted in a dose-dependentreduction in infarct volume and robust protection from neurolog-ical impairments even when the therapy was initiated up to 4- to6-h after tMCAO. Src blockade after pMCAO resulted in acceler-ated improvement in recovery from motor, sensory, and reflexdeficits during a long-term (3 weeks) testing period poststroke.These data demonstrate that the novel Src kinase inhibitors pro-vide effective treatment against ischemic conditions within a clin-ically relevant therapeutic window and may constitute a viabletherapy for acute stroke.

Ischemic stroke is a life-threatening disorder, with no ef-fective therapy except for intravascular thrombolysis withrecombinant tissue plasminogen activator or, in some in-stances, surgical removal of the obstructing blood clot withinterventional devices (Donnan et al., 2008; Zaleska et al.,2009). However, the risk of hemorrhage and a restrictive 3- to4.5-h treatment window (Hacke et al., 2008) reduce the poolof patients eligible for treatment with recombinant tissue

plasminogen activator to only 5 to 8%. Despite continuedimprovement in our understanding of mechanisms underly-ing ischemic damage and the identification of numerous neu-roprotective agents effective in animal models, no agent hasyet demonstrated significant benefit in the clinic (O’Collins etal., 2006; Savitz and Fisher, 2007; Zaleska et al., 2009).Hence, the development of stroke therapeutic agents remainsa daunting challenge as does the urgent need for new agents.

The Src family of nonreceptor protein tyrosine kinases(PTKs) coordinate a broad spectrum of physiological re-sponses including extracellular signals derived from growthfactor and cytokine receptors, G-protein-coupled receptors,ion channels, and cell-cell and cell-matrix adhesion signaling

This work was supported by Wyeth Research.Article, publication date, and citation information can be found at

http://jpet.aspetjournals.org.doi:10.1124/jpet.109.156562.

ABBREVIATIONS: PTKs, protein tyrosine kinases; CNS, central nervous system; NMDA, N-methyl-D-aspartate; VEGF, vascular endothelialgrowth factor; VP, vascular permeability; BBB, blood-brain barrier; PP1, 1-tert-butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine; PP2,4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine; Abl, Abelson; SKI-606, bosutinib, 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propoxy]-3-quinolinecarbonitrile); SKS-927, 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[4-(4-methypiperazin-1-yl)but-1-ynyl]-3-quinolinecarbonitrile; MCA, middle cerebral artery; tMCAO, transient middle cerebral artery occlusion; pMCAO,permanent middle cerebral artery occlusion; TTC, 2,3,5-triphenyl tetrazolium chloride; PBS, phosphate-buffered saline; HUVEC, human umbilicalvein endothelial cell; PP3, 1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine.

0022-3565/09/3313-827–835$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 3Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 156562/3533255JPET 331:827–835, 2009 Printed in U.S.A.

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molecules (Thomas and Brugge, 1997). Five members of theSrc family PTKs (Src, Fyn, Yes, Lck, and Lyn) are highlyexpressed in platelets and in the mammalian CNS. Src fam-ily kinases have been shown to be activated after globalischemia and to potentiate the activity of NMDA receptors(Salter and Kalia, 2004; Guo et al., 2006) and voltage-gatedcalcium and potassium channels (Cataldi et al., 1996; Fadoolet al., 1997), all known contributors to ischemic cell death.

A major role for Src kinase in VEGF-induced vascularpermeability (VP) has also been established (Eliceiri et al.,1999). Focal cerebral ischemia in rodents (Plate et al., 1999;Zhang and Chopp, 2002) and ischemic stroke in humans (Issaet al., 1999) induce expression of VEGF, a key promoter ofVP, leading to breakdown of the BBB and clinical deteriora-tion (Croll and Wiegand, 2001). Antagonism of VEGF reducesstroke-induced injury and edema (van Bruggen et al., 1999)whereas administration of recombinant VEGF shortly afterstroke increases BBB leakage and exacerbates ischemic in-jury (Zhang et al., 2000; Zhang and Chopp, 2002). Paul et al.(2001) first demonstrated that mice deficient in Src or Yesbut not Fyn did not exhibit VP in response to VEGF admin-istration and were resistant to ischemic injury, whereas ad-ministration of Src inhibitors PP1 or PP2 to wild-type ani-mals reduced ischemic injury when assessed within the first24 h (Paul et al., 2001; Lennmyr et al., 2004; Weis et al.,2004a). These data established proof of concept for achievingneuroprotection by targeting Src kinase. In addition, both theSrc inhibitor PP1 and the dual Src/Abl inhibitor SKI-606(bosutinib) (Boschelli and Boschelli, 2007), undergoing clini-cal trials for the treatment of cancer, were shown to blockVEGF-induced endothelial barrier breakdown and reduceinjury in a mouse model of myocardial ischemia (Weis et al.,2004b) and to suppress VEGF-mediated tumor cell extrava-sation and metastasis (Weis et al., 2004a).

In the present study, we report a systematic evaluation ofthe neuroprotective profiles of SKI-606 and the novel Srcinhibitor SKS-927, both with potency superior to that of PP1.Our results demonstrate that both agents block VEGF-me-diated signaling in endothelial cells, penetrate into the CNSupon intravenous administration, inhibit postischemic Srckinase activation and BBB leakage, and provide robust andlong-lasting neuroprotection in two focal cerebral ischemiamodels, with a clinically relevant therapeutic window. Ouranimal efficacy studies were designed to mimic clinical cir-cumstances, and the results obtained strongly support theuse of SKI-606 and SKS-927 in clinical trials for stroke.

Materials and MethodsAnimals. Animal procedures were approved by the institutional

animal care and use committee and conducted in accordance with theguidelines of the National Institutes of Health. Adult male Wistarrats (290–310 g; Charles River Laboratories, Inc., Wilmington, MA)were randomly allocated to treatment groups. Researchers blinded totreatment allocation performed stroke-inducing operations and con-ducted assessments of neurological deficits, infarct volume, andhistology.

Drug Profiles and Administration. SKI-606 and SKS-927 (seeFig. 1A for structures) were synthesized as described previously(Boschelli et al., 2001, 2007). The potency of SKI-606 was 30-foldhigher (IC50 of 1.2 nM) than that of PP1 (35 nM) in an enzyme-linkedimmunosorbent assay-based Src enzymatic assay, whereas in aLANCE-based enzymatic assay, IC50 values for SKI-606 and SKS-

927 were 3.9 and 3.8 nM, respectively. In a cell proliferation assayusing Src-transformed rat fibroblasts, IC50 values for SKI-606, SKS-927, and PP1 were 100, 73 (Boschelli et al., 2001, 2007), and 710 nM(J. Golas and F. Boschelli, unpublished observations), respectively.In cell-based assays, SKI-606 and SKS-927 also inhibited anothermember of the Src kinase family, Lck, with IC50 values of 20 and 52nM, respectively. When tested under identical conditions for activityagainst a number of other kinases, the two compounds shared asimilar profile. Both SKI-606 and SKS-927 displayed IC50 valuesgreater then 5 �M against cyclin-dependent kinase 4, 3-phosphoino-sitide-dependent kinase 1, kinase insert domain receptor (alsoknown as vascular endothelial growth factor receptor 2), and tumorprogression loci 2. Both compounds displayed submicromolar IC50

values against Raf/mitogen-activated protein kinase kinase, 500 and360 nM for SKI-606 and SKS-927, respectively, and against epider-mal growth factor receptor, 350 and 720 nM for SKI-606 and SKS-927, respectively. Both compounds inhibited Abl with IC50 values of1 and 2 nM, respectively, but had no significant activity against 65receptors, ion channels, and transporters when tested at 10 �M byNovaScreen. More extensive enzymatic assay screens at Wyeth andstudies by others indicate that SKI-606 is a multikinase inhibitor,with activity against some mitogen-activated protein, some calcium-dependent, Sterile 20, ephrin, Tec, Trk, and Axl kinase families(Puttini et al., 2006; Bantscheff et al., 2007; Remsing Rix et al.,2009). Only a few of these activities have been verified at the level ofprotein phosphorylation in cells. It is noteworthy that SKI-606 doesnot inhibit platelet-derived growth factor receptor, which is believedto be a source of toxicity for other clinically relevant Abl kinaseinhibitors. SKS-927 has not been profiled as extensively. Althoughthe structures of SKI-606 and SKS-927 share a 4-[(2,4-dichloro-5-methoxyphenyl)amino]-3-quinolinecarbonitrile moiety, which con-fers very similar profiles for the in vitro kinase inhibitory activity,the group at C-7 of the quinoline core plays a key role in the physi-cochemical properties and potential in vivo activities. Both com-pounds were evaluated in our studies because these properties canaffect pharmacokinetics, protein binding, tissue distribution, andtolerability. Inhibitors and vehicle (20 mM citrate-0.85% NaClbuffer) were administered as a slow intravenous bolus into the tailvein in a volume of 2 ml/kg or as an intravenous infusion; dosingregimens are shown in the figure legends.

Pharmacokinetic Analysis. At various time intervals after ad-ministration of SKI-606 or SKS-927 at doses of 5 and 10 mg/kg,animals (n � 3/group) were anesthetized with isoflurane (IsoFlo;Abbott Laboratories, Chicago, IL), and plasma and brain sampleswere harvested. A quantitative analysis of drug concentration insample extracts was accomplished by liquid chromatography com-bined with tandem mass spectrometry using an Agilent 1100 liquidchromatography system (Agilent Technologies, Palo Alto, CA) inter-faced with a Micromass Ultima mass spectrometer (Waters, Beverly,MA).

Measurement of Total and Phosphorylated Src (pY418 Src)Kinase Levels in Blood and Brain. At various time intervals aftersystemic treatment with SKS-927, control and ischemic (tMCAO)animals were anesthetized with isoflurane, and 1 ml of blood wasdrawn from the heart and centrifuged to obtain a platelet-enrichedfraction. The ipsilateral cerebral cortex and striatum were removedfrom a 6-mm mid-forebrain section and homogenized in 1:10 (w/v)ice-cold radioimmunoprecipitation assay buffer (Sigma-Aldrich, St.Louis, MO) containing protease (Complete Mini; Roche Diagnostics,Indianapolis, IN) and phosphatase inhibitors (cocktail set 2; Calbio-chem, La Jolla, CA). Src levels were measured using total Src andpY418 Src (autophosphorylated) enzyme-linked immunosorbent as-say kits (BioSource International, Camarillo, CA) following the man-ufacturer’s recommended protocols. Src concentrations were calcu-lated according to appropriate standard curves run within eachassay. The pY418 Src concentration (units) was normalized to thetotal Src level (nanograms) in each sample, and results are presentedas mean ratios of pY418 Src/total Src.

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tMCAO. Rats were anesthetized with 3% isoflurane in a mixtureof 70% nitrous oxide and 30% oxygen through a nose cone. Core bodytemperature was maintained at 37°C throughout the surgery usinga heating lamp. tMCAO was induced for 90 min using intraluminalsuture according to the established procedure (Longa et al., 1989). Inbrief, an 18-mm length of 4-0 nylon monofilament with a flame-rounded tip was inserted into the external carotid artery and ad-vanced into the internal carotid artery to occlude the origin of theMCA. Ninety minutes later rats were reanesthetized, the suture waswithdrawn, reperfusion was confirmed visually under the operatingstereomicroscope, and the incision was closed. Successful inductionof focal ischemia was confirmed by contralateral hemiparesis. Ani-mals were tested for motor deficits at 90 min and at 24 and 48 hpoststroke using an established scoring system (Bederson et al.,1986), and a maximum score of 5 at 90 min after induction of tMCAOwas a criterion for inclusion in the study. SKI-606, SKS-927, orvehicle was administered at various time intervals (0.5–6.0 h) afterthe induction of stroke at the doses indicated in the figure legends(n � 16/group). Core body temperature was measured within 30 minof drug dosing to monitor for a potential drug effect on temperature.Sham-operated controls were subjected to identical surgical proce-dures but without advancement of the monofilament into the MCAbranching point. Rats were sacrificed at 48 h after tMCAO. Animals

that died before 48 h or displayed hemorrhage upon harvesting of thebrain were excluded.

pMCAO. A craniotomy was performed under isoflurane anesthe-sia followed by electrocauterization of the distal portion of the MCAunder a stereomicroscope according to the procedure established byChen et al. (1986). Core body temperature was maintained at 37°Cthroughout the surgery using a heating lamp. This procedure resultsin a consistent infarction in the area of the sensorimotor cortexgoverning forelimb and hindlimb function and excellent survival ofanimals allows for long-term assessment of neurological outcomes.Sham surgery included craniotomy without cauterization of theMCA. SKI-606, SKS-927 (10 mg/kg i.v.), or vehicle was administeredat the time intervals indicated in figure legends. In one study,animals (n � 12/group) were sacrificed at 48 h after induction ofpMCAO for measurement of infarct volume. In a second study,animals (n � 10/group) were subjected to testing of sensorimotor andreflex deficits at 3-day intervals for 3 weeks according to De Ryck etal. (1989). Each trial included a postural test, two visual and twotactile forelimb placement tests, and a hindlimb placement test.Performance on each test was scored from 0 (no deficit) to 2 (severedeficit) for a maximum combined score of 12. Naive and sham-operated animals displayed no deficits (score � 0)

Fig. 1. A, chemical structures of Src ki-nase inhibitors SKI-606 and SKS-927. B,effect of SKI-606 (f) or SKS-927 (o) onpSrc levels in cortical lysates of naive ratsat 1 h after single intravenous bolus dos-ing. C, dose- and time-dependent reduc-tion of pSrc levels in platelets after a 1-hintravenous infusion of SKS-927 com-pared with pretreatment levels. D, tem-poral profile of Src kinase activation inthe ischemic striatum and cortex and theinhibitory effect of in vivo administrationof SKS-927 (10 mg/kg i.v.) at 90 min afterinduction of tMCAO. Results are pre-sented as the percentage of pSrc levels inbrain lysates derived from control (non-ischemic) animals. �, p � 0.05; ��, p �0.01 statistical significance of the effect ofdrug treatment. #, statistical significance(p � 0.05) of the effect of ischemia on Srckinase activation. Data are presented asmeans � S.E.M.; n � 3 to 5 animals/group.

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Physiological Monitoring. The effect of drug administration onphysiological parameters of rats subjected to tMCAO was investi-gated in a separate group of animals. After cannulation of the fem-oral artery and induction of tMCAO, arterial blood pressure andheart rate were measured before drug administration and at 5 and30 min after drug treatment with the use of a Biopac System accord-ing to the manufacturer’s specification. Animals remained undercontinuous isoflurane anesthesia during the entire procedure. SKI-606 and SKS-927 at the doses of 10 and 30 mg/kg (n � 3/group) wereadministered as a single intravenous bolus at 30 min after inductionof tMCAO.

Infarct Volume Analysis. After sacrifice, brains were extracted,and 2-mm coronal sections were cut starting at 4 mm from thefrontal pole using a rat brain matrix (Stoelting, Wood Dale, IL).Sections were stained by immersion in a 2% solution of 2,3,5-triph-enyl tetrazolium chloride (TTC) at 37°C for 10 min and then in 10%neutral formalin for preservation. Digitized images of sections werecollected using the MCID image analysis system (Interfocus Imag-ing, Ltd., Cambridge, UK), and the area of infarct was determined foreach slice. Total infarct volume was calculated by multiplying a sumof areas by the distance between them in millimeters.

Evans Blue Extravasation. The integrity of the BBB was as-sessed by measuring extravasation of Evans blue dye into the brainparenchyma. A 2% solution of Evans blue in saline was administeredintravenously at a volume of 4 ml/kg at 4 or 22 h after induction oftMCAO. Two hours later, rats were anesthetized with isoflurane andperfused with 40 ml of saline through the left ventricle of the heart.Brains were removed; either 2-mm coronal sections were sliced forphotography or the striatum and cortex were dissected and weighed,and Evans blue content was measured by fluorometry (Belayev et al.,1996).

Immunohistochemistry. Three days after induction of tMCAO,vehicle- and SKS-927 (10 mg/kg i.v.)-treated rats (n � 4/group) wereanesthetized with isoflurane and perfused transcardially with PBS,followed by 100 ml of 4% paraformaldehyde. Brains were postfixedovernight at 4°C in the same fixative, soaked overnight in 30%buffered sucrose, frozen in CO2 snow, and cut on a cryostat in 40-�msections throughout the area of the infarct. After preincubation with2.5% normal horse serum (Vector Laboratories, Burlingame, CA),sections were incubated with mouse (monoclonal) anti-rat leukocytecommon antigen CD-45 (OX-1; BioSource) 1:100 with 2.5% horseserum in 1% Triton X-100 in PBS overnight at room temperature.The sections were then processed with biotinylated secondary anti-body followed by biotin-avidin solution (ABC-Elite Kit; Vector Lab-oratories) and exposure to diaminobenzidine tetrahydrochloride (0.5mg/ml; Polysciences, Warrington, PA). For quantification, multipleregions (0.5 mm2 area per region) within the ischemic striatum andcortex were sampled from matching planes of sections in each animaland photomicrographs were taken. The sum of immunopositive cellswas generated from four regions of the striatum (dorsolateral, me-dial, central, and ventral areas) and three regions of the cortex

(dorsal, lateral, and ventral areas) to determine the total cell countper striatum and cortex. Two sections were used to derive an averagecell count within each brain structure per animal. Labeled cells werecounted in a blinded manner. The anti-CD-45 (OX-1) antibody rec-ognizes leukocytes and activated microglia, both markers of inflam-matory response in brain parenchyma.

VEGF-Mediated Effects in Human Umbilical Vein Endothe-lial Cells, Annexin V Staining, and Flow Cytometry. Inhibitionof VEGF-mediated Src-dependent rescue of serum-starved HUVECs(Abu-Ghazaleh et al., 2001; Eliceiri et al., 2002) was used as an assayto test the potency and efficacy of Src inhibitors against VEGFsignaling in human endothelial cells. The importance of using thisassay is 2-fold: 1) it involves the same signaling pathways that areactivated in brain vasculature during VEGF-mediated VP, and 2) itprovides an opportunity to test the activity of Src inhibitors againsthuman target tissue. HUVECs (Cambrex, East Rutherford, NJ) wereplated at 5.0 � 104 cells/well in gelatin-coated six-well plates andmaintained for 24 h in complete media containing serum as de-scribed (Abu-Ghazaleh et al., 2001). To induce apoptosis, serum-containing medium was replaced with serum-free medium supple-mented with 25 ng/ml VEGF in the presence or absence of 30 or 300nM SKI-606 or SKS-927 or PP1 or PP3, the inactive analog of PP1(Calbiochem). Twenty-four hours later, cells were trypsinized andcentrifuged at 200g, and pellets were resuspended in annexin Vstaining solution (Roche Molecular Biochemicals, Indianapolis, IN)and incubated for 15 min at room temperature. After a wash in PBS,annexin V-positive cells were quantified by flow cytometry (FACS-Calibur; BD Biosciences, San Jose, CA).

Statistical Analysis. All measurements in the studies reportedhere were performed blindly. Results are expressed as means �S.E.M. The statistical significance of differences between means wascalculated by one-way analysis of variance with a post hoc Fisher’stest or Dunnett’s comparison to control or with Student’s t testanalysis, as appropriate. A linear mix model was used to assessdifferences between treatment groups during 3-week neurologicalfunction recovery testing after pMCAO. Statistical significance wasassumed at p � 0.05.

ResultsSystemic Administration of SKI-606 or SKS-927 Re-

sults in Dose-Dependent Brain Exposure and Reduc-tion of pY418 Src Levels in Platelets and Brain. After asingle intravenous bolus administration at the doses of 5 or10 mg/kg, both compounds were found to penetrate the BBBin a dose-proportional manner with SKS-927 transientlyreaching a higher brain concentration than SKI-606 at com-parable doses (Table 1). At 1 and 4 h after administration ofthe 10 mg/kg dose, the brain/plasma concentration ratios forSKI-606 were 1.1 and 2.6, whereas for SKS-927, the ratios

TABLE 1Plasma and brain tissue drug exposure levels after intravenous administration of SKI-606 and SKS-927 in ratSKI-606 or SKS-927 was administered as a single intravenous bolus at doses of 5 and 10 mg/kg. At the time intervals indicated, animals were anesthetized, and blood sampleswere obtained through cardiac puncture. After perfusion with PBS to remove blood from the central nervous system, compound levels were determined by liquidchromatography and mass spectrometry with the use of appropriate standards. Results are presented as the means � S.E.M.; n � 3/group.

Dose and TimeSKI-606 SKS-927

Plasma Brain Plasma Brain

ng/ml ng/g ng/ml ng/g

5.0 mg/kg i.v.0.5 h 365 � 36 200 � 27 239 � 17 757 � 1931.0 h 330 � 57 139 � 24 145 � 11 588 � 264.0 h 85 � 6 94 � 11 54 � 20 80 � 24

10.0 mg/kg i.v.0.5 h 726 � 53 865 � 65 531 � 29 2086 � 2011.0 h 578 � 47 654 � 214 364 � 42 1703 � 1904.0 h 208 � 8 554 � 212 119 � 32 250 � 32

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were 4.6 and 2.1, respectively. However, 4 h after dosing, theconcentration of SKS-927 in the brain was 50% lower thanthat of SKI-606. Comparison of concentrations of compoundsin brain tissue with their IC50 values for Src enzyme inhibi-tion (3.9 and 3.8 nM for SKI-606 and SKS-927, respectively)and activity against Src in cell-based assays (IC50 values of100 and 73 nM for inhibition of Src-dependent proliferationfor SKI-606 and SKS-927, respectively) indicated that thebrain exposures achieved were in considerable excess of lev-els needed to block Src activity. Brain penetration of PP1 wasnot assessed in our studies; however, its reported efficacy inrodent stroke models upon intraperitoneal administration(Paul et al., 2001) suggests that this compound is crossing theBBB.

Because the phosphorylation of Src at Y418 within theactivation loop of the kinase domain is required for full acti-vation (Sun et al., 1998), the level of pY418 Src in relation tototal Src is therefore a good measure of its activation state.To evaluate the in vivo inhibitory effects of SKI-606 andSKS-927 on Src activation state in the brain, either agentwas administered to naive rats as a single intravenous bolusat 10 or 30 mg/kg, and cortical lysates were assayed 1 h aftertreatment. Reductions of 31 and 61% and 64 and 84% in pSrclevels were found for SKS-927 and SKI-606, respectively(Fig. 1B).

As shown in Fig. 1C, intravenous infusion of SKS-927resulted in a dose-proportional and long-lasting reduction ofpY418 Src levels in the platelet-enriched blood fraction. Atdoses of 1, 10, and 30 mg/kg SKS-927, Src autophosphoryla-tion was inhibited by approximately 50, 80, and 100%, re-spectively, 2 h after initiation of infusion, and remained atsimilar levels 4 h after infusion. At 6 to 8 h after infusion,pSrc levels returned to baseline values in animals receiving 1and 10 mg/kg dose, whereas at a 30 mg/kg dose, pSrc levelswere still reduced by 50% at 8 h after infusion. These resultsindicate long-lasting inhibition of Src kinase in blood afterintravenous administration of an Src inhibitor.

SKS-927 Abolishes Ischemia-Induced Activation ofSrc Kinase in the Brain. To investigate whether focalischemia induces activation of Src kinase in the brain andwhether that activation could be inhibited by systemic ad-ministration of SKS-927, we evaluated the temporal profile ofSrc activation during the initial 6-h period after induction ofstroke. Four groups of animals were used in this study: 1)naive (control), 2) sham-operated, 3) subjected to 90 min oftMCAO with vehicle administration as an intravenous bolusat reperfusion time, and 4) subjected to 90 min of tMCAOfollowed by administration of a single intravenous bolus of 10mg/kg SKS-927. Sham surgery did not affect pSrc levels inthe cortex or striatum. A 2- to 4-fold increase in the level ofactivated Src kinase, sustained for 6 h after induction oftMCAO was observed in ischemic striatum (Fig. 1D). AfterSKS-927 treatment (10 mg/kg i.v.) this increase in pSrc levelswas dramatically reduced by 75 and 60% at 2 and 6 h,respectively. In cortical lysates derived from ischemic ani-mals, elevation of activated Src did not reach statistical sig-nificance. However, after SKS-927 treatment, a significantreduction in the cortical pY418 Src level was found 6 h afterinduction of tMCAO, with a trend toward lowered pY418 Srclevels observed at 2 h (Fig. 1D). Taken together, these resultsshow that a single intravenous bolus dose of SKS-927 confers

a clear inhibitory action on postischemic activation of brainSrc kinase over at least a 6-h period after MCA occlusion.

Src Kinase Inhibitors Reduce VEGF-Mediated Ef-fects in HUVECs and Ischemia-Induced Vascular Per-meability in the Brain. It has been shown that VEGFtreatment induces rapid Src-mediated phosphorylation ofY861 within the C terminus of the focal adhesion kinase bothin cultured HUVECs and in brain vasculature during VEGF-mediated VP or chemotactic signaling (Eliceiri et al., 1999,2000; Abu-Ghazaleh et al., 2001). Because the same mecha-nism is involved in VEGF-induced Src-dependent rescue ofHUVECs in a serum deprivation paradigm, we have usedthis assay to examine the ability of novel Src inhibitors toblock VEGF effects on vasculature. HUVECs grown in thepresence of 2% fetal bovine serum displayed limited celldeath, whereas removal of fetal bovine serum from the me-dium induced apoptotic cell death, as determined by in-creased annexin V staining. Addition of 25 ng/ml VEGF sig-nificantly attenuated this cell death. In the presence of 30 or300 nM PP1, VEGF-mediated rescue was inhibited by 17%,whereas PP3, an inactive analog of PP1, had no effect. SKI-606, at concentrations of 30 or 300 nM blocked VEGF effectsin HUVECs by 93 and 100%, respectively (Fig. 2A). In thepresence of the same concentrations of SKS-927, the VEGF-mediated antiapoptotic effects were reduced by 37 and 70%,respectively. These results demonstrate that both agents in-terfere with Src-dependent VEGF-mediated signaling in hu-man endothelial cells with efficacy and potency superior tothat of PP1.

The effect of Src inhibition on cerebral ischemia-inducedBBB permeability is shown in Fig. 2B. In vehicle-treatedanimals, a pronounced extravasation of Evans blue was ob-served 6 h after stroke in the region of the ipsilateral basalganglia (ischemic core). In contrast, administration of a sin-gle intravenous dose of SKS-927 (10 mg/kg) almost entirelyeliminated the extravasation of the dye into brain paren-chyma (Fig. 2B, top). When brains derived from a ran-domly chosen subset of animals from this experimentalgroup were examined at 48 h after induction of tMCAO, aremarkable reduction of infarct volume was observed (Fig.2B, bottom). Findings after SKI-606 treatment were simi-lar; quantitative analysis indicated 60% reduction ofEvans blue levels in brain tissue after administration of asingle intravenous dose of SKI-606 (10 mg/kg) (data notshown). Leukocyte infiltration into brain parenchyma wasexamined as an alternative means of assessing the BBBintegrity 3 days poststroke. In vehicle-treated animals, nu-merous clusters of OX-1-positive cells were detected withinmicrovessels and infiltrating into cortical parenchyma (Fig.2C). In addition to leukocytes, anti-OX-1 antibody also rec-ognizes activated microglia, both markers of inflammatoryresponse in the brain. Quantitative assessment of infiltratingleukocytes revealed a 62% reduction within the striatumafter treatment with SKS-927, whereas the decrease in thetotal cortical cell count did not reach statistical significance(Fig. 2D). OX-41 staining was absent in brain sections fromnaive animals.

Protection against tMCAO. Treatment with SKI-606 30min after occlusion, at doses of 3, 10, or 30 mg/kg, signifi-cantly reduced infarct volume by 24, 57, and 44%, respec-tively (Fig. 3A). No significant effects on blood pressure,heart rate, or body temperature were observed at these doses


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(data not shown). Initiation of treatment with SKI-606 at adose of 10 mg/kg at 0.5, 1.5, 3, and 4 h after tMCAO resultedin a robust reduction of infarct volumes by 57, 42, 65, and47%, respectively (Fig. 3B). Although further delay of treat-ment to 5 or 6 h after tMCAO resulted in a trend towardreducing infarct volume, these changes did not reach statis-tical significance. However, a significant reduction of neuro-logical deficits was observed when treatment was delayed upto 6 h, with the highest reduction of 67% in the group ofanimals treated with a 3-h window and strong trends at earlydosing times (Fig. 3C). Ischemia-induced weight loss wassignificantly attenuated by SKI-606, when given up to 5 hafter tMCAO, whereas the 6-h treatment window resulted ina nonsignificant trend (Fig. 3D).

Based on the results obtained with SKI-606, the protectiveefficacy of SKS-927 was evaluated in the tMCAO model witha 4-h postocclusion window. No significant effects of SKS-927on blood pressure, heart rate, or body temperature wereobserved at the doses of 10 and 30 mg/kg i.v. (data not shown)except for a transient decrease of heart rate from 404 � 30 to300 � 31 beats/min during the first 5 min after intravenousadministration of the 30 mg/kg dose. SKS-927 treatmentresulted in a dose-dependent reduction of infarct volume by32 and 40% at the doses of 10 and 30 mg/kg, respectively (Fig.4, A and B). In the same animals, a robust dose-dependentattenuation of neurological deficits was observed at all dosestested (Fig. 4C). After 48 h of recovery, neurological deficitscores were reduced by 35% in the 3 and 10 mg/kg dosetreatment groups, an effect comparable to that observed with10 mg/kg SKI-606 administered at 4 h after occlusion (45%)(Fig. 3C). Administration of SKS-927 at a dose of 30 mg/kgresulted in a 53% decrease in neurological deficit scores rel-ative to those for vehicle-treated animals (Fig. 4C). When

SKS-927 was administered as a 1-h intravenous infusion (10mg/kg), outcome measures comparable with those obtainedafter a single intravenous bolus administration at the samedose were achieved (data not shown). This finding indicatesthat the neuroprotection is driven by total exposure to agentover time (area under the curve) rather than by peak plasmaconcentration, a finding of significance for designing poten-tial clinical dosing paradigms.

Protection and Improvement of Long-Term Neuro-logical Recovery after pMCAO. As shown in Fig. 5A,treatment with SKS-927 resulted in a statistically significantreduction of infarct volume (by 24%), within 48 h after in-duction of pMCAO. SKS-927-treated animals initially dis-played the same level of severity of neurological deficits (amaximum score of 12) as did vehicle-treated animals (Fig.5B). Poststroke evaluation of animals for sensorimotor func-tion and a reflex limb placement battery of tests showed adegree of spontaneous recovery in the vehicle-treated group.However, in the SKS-927-treated group, the neurological re-covery was considerably accelerated, resulting in a highlysignificant increase in the slope of recovery (p � 0.009),starting on the 7th day after pMCAO and continuingthroughout the 3-week assessment period. Similar resultswere found in the analogous experiments conducted withSKI-606 (data not shown).

DiscussionIn this study, we demonstrate that the Src inhibitors SKI-

606 and SKS-927 inhibit ischemia-induced activation of Srckinase in the brain, ameliorate vascular leakage and inflam-mation, and provide robust and dose-dependent neuroprotec-tion in two well established rodent models of focal cerebral

Fig. 2. A, inhibition of VEGF-mediated rescue ofserum-deprived HUVECs by PP1, SKI-606, andSKS-927 at concentrations of 30 nM (�) and 300nM (f). Results are expressed as percent inhibi-tion of VEGF-induced endothelial cell (EC) sur-vival under serum deprivation conditions. �, p �0.05 versus PP1or PP3 (inactive analog of PP1).Data are presented as the means � S.E.M. fromthree to five independent experiments. B, treat-ment with Src inhibitor blocks ischemia-inducedvascular leak and reduces infarct volume. SKS-927 was administered as a single intravenousbolus at a dose of 10 mg/kg. Evans blue extrav-asation at 6 h (top) and TTC staining at 48 hafter induction of tMCAO (bottom). C, treatmentwith SKS-927 inhibits inflammatory responsesin ischemic brain tissue. OX-1 immunostainingfor infiltrating leukocytes and activated micro-glia in brain cortex of animals treated with ve-hicle or SKS-927. Image magnification: barshown is equivalent to 100 �m. D, quantificationof OX-1-positive cells in cortical and striatal re-gions at 3 days poststroke after vehicle or SKS-927 (10 mg/kg i.v.) treatment, respectively (twosections per animal were averaged; n � 4 ani-mals per treatment group). Results are pre-sented as means � S.E.M. �, p � 0.05.

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ischemia, significantly enhancing neurological recovery post-stroke. It is important to note that intravenous administra-tion of SKI-606 and SKS-927 results in compound concentra-tions in the brain in excess of the respective in vitro IC50

values for both inhibition of Src enzymatic activity and Src-based cellular assays.

Use of an intravenous route of administration initiatedseveral hours after onset of the ischemic insult and followed

by an assessment of functional recovery was critical for de-termining the activity of our compounds in an experimentalsetting more relevant to the human clinical condition. Re-sults from our studies show directly for the first time thatfocal ischemia with reperfusion induces activation of Srckinase in the area of the ischemic core for at least 6 hpoststroke, with some up-regulation of activated Src in sur-rounding cortical areas. These data add to previous reportsdemonstrating an up-regulation of Src activity in a globalischemia model that mimics cardiac arrest rather than isch-emic stroke (Zalewska et al., 2005; Guo et al., 2006). Ourresults show that the focal ischemia-induced up-regulation ofSrc is significantly reduced throughout this 6-h period after asingle intravenous bolus of SKS-927. VEGF-mediated activa-tion of Src is directly involved in several signaling pathwaysin endothelial cells affecting induction of VP, migration, andantiapoptosis (Eliceiri et al., 1999; Abu-Ghazaleh et al.,2001). SKI-606 and SKS-927 were shown to potently inhibitVEGF-mediated human endothelial cell rescue from serumwithdrawal and reduced vascular leak and leukocyte infiltra-tion poststroke, confirming both their ability to target Src inhuman endothelium and their mechanism of action in vivo.The emerging understanding of the mechanisms involvedimplicate a hypoxia-inducible factor-driven and Src-depen-

Fig. 4. Treatment with SKS-927 protects from ischemic injury and improves neurological deficits with at least a 4-h therapeutic window aftertransient focal ischemia. SKS-927 was administered as a single intravenous bolus at 4 h after induction of tMCAO at doses indicated. A,dose-dependent reduction of infarct volume at 48 h after MCAO (�, p � 0.05). B, representative brain slices stained with TTC demonstrating adose-related reduction in the area of injured brain tissue. C, treatment with SKS-927 results in significant reduction of neurological deficits at 48 hpoststroke (�, p � 0.05) Data are presented as means � S.E.M.; n � 13 to 16 animals/group.

Fig. 3. Poststroke administration of SKI-606 reduces the volume ofischemic injury, improves neurological deficits, and protects from post-ischemic weight loss with a clinically relevant therapeutic window. A, asingle intravenous bolus of SKI-606 was administered at 30 min afterinduction of a transient focal ischemia (tMCAO). The mean infarct vol-ume at 48 h after MCAO was reduced in response to treatment withdifferent doses of SKI-606 (�, p � 0.05). B, delayed treatment with asingle dose of 10 mg/kg SKI-606 administered up to 4 h poststroke resultsin significant infarct volume reduction (�, p � 0.05). C, a significantreduction in neurological deficits is still observed when the treatmentwith SKI-606 is delayed by 6 h (�, p � 0.05). D, significant protection fromweight loss (�, p � 0.05) when treatment is delayed up to 5 h. Data arederived from three separate studies, each expressed as a percentage ofthe appropriate vehicle-treated group. Results are presented as means �S.E.M.; n � 12 to 16 animals/group. Veh, vehicle.

Fig. 5. Treatment with SKS-927 reduces infarct volume and improveslong-term neurological recovery after permanent focal ischemia (pM-CAO). SKS-927 was administered at a dose of 10 mg/kg i.v. at 2, 4, 24, and28 h after induction of pMCAO. Vehicle was administered in the samemanner. A, results show a significant reduction of infarct volume inanimals treated with SKS-927 (�, p � 0.05, n � 11–12 animals/group) at48 h after pMCAO. B, improvement of neurological recovery demon-strated as an accelerated slope of decreasing sensorimotor deficit scoresover the course of 21 days poststroke in animals treated with SKS-927 (�,p � 0.009 for slope and day 21 scores; n � 10 animals/group). Results arepresented as means � S.E.M.


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dent expression of VEGF, predominantly in microglia andastrocytes with a parallel up-regulation of VEGF receptorson the abluminal side of endothelial cells (Brillault et al.,2002; Zhang and Chopp, 2002). VEGF is thought to act in aparacrine fashion on endothelial cells and, upon engaging itsreceptors, recruits Src kinase into a signaling complex medi-ating direct phosphorylation of cell-matrix or cell-cell adhe-sion proteins leading to reduced blood flow, tight junctiondisassembly, vascular leak, and expansion of injury (Weisand Cheresh, 2005). In a mouse myocardial infarction model,both SKI-606 and PP1 were shown to stabilize preformedFlk-cadherin-catenin complexes, maintaining the integrity ofendothelial cell contacts, reducing edema and infarct volume,and improving functional recovery (Weis et al., 2004b). Dis-rupting VEGF-mediated Src activation with SKI-606 wasalso shown to suppress tumor cell extravasation and metas-tasis in vivo (Weis et al., 2004a). Contrary to VEGF-inducedVP in the acute phase, VEGF-mediated angiogenesis hasbeen shown to contribute to recovery and remodeling of neu-rovascular units days after stroke (Croll and Wiegand, 2001;Zhang and Chopp, 2002). However, angiogenesis is not af-fected by a deficiency in individual Src family PTKs, andSrc-deficient mice display a normal angiogenic response toVEGF (Eliceiri et al., 1999).

Of the many reasons attributable to the resounding failureof stroke therapeutic agents in the clinic, one clear shortcom-ing has been the lack of a suitable temporal treatment win-dow poststroke. It is well known that most patients takemany hours to reach hospitals and be treated, so investiga-tional therapeutic agents need to be, at a minimum, effectivehours rather than minutes poststroke in preclinical animalmodels. In this report, we have shown that Src inhibitionsupports a therapeutic treatment window of at least 4 to 6 h,depending on the efficacy measure in a tMCAO rat model ofstroke. This therapeutic window, based on the in vivo out-come measures including impairment of neurological func-tion, is clearly matched by our data showing a temporalprofile of postischemic Src activation in the brain and itsinhibition by a single intravenous bolus of the drug. Like-wise, it is supported by a long-lasting inhibition of Src kinaseobserved in vivo in platelets, another cellular compartmentenriched in Src family PTK that may potentially serve as aclinical biomarker of the drug-target interaction, at least inthe periphery. We have also shown that treatment initiated2 h after pMCAO results in significant reduction of infarctvolume and accelerated improvement in neurological recov-ery throughout the 3-week recovery period. Among the fewstudies reporting protective effects of PP1 or PP2 in ischemicstroke, although within a short 24-h period, only one studyshowed a comparable 6-h therapeutic window in a mousepMCAO model (Paul et al., 2001). Preclinical efficacy profilesof SKI-606 and SKS-927 are also superior to those of otherrecently reported Src inhibitors (Mukaiyama et al., 2007), forwhich treatment was started 15 min poststroke, and no neu-rological recovery was evaluated, both findings with no clin-ical relevance. The predictive value of preclinical stroke mod-els for a successful clinical trial has been poor even for agentsthat have shown efficacy when administered within severalhours poststroke (O’Collins et al., 2006; Savitz and Fisher,2007). We believe that the combined profile of our Src inhib-itors differentiate their therapeutic potential by demonstrat-ing a measurable temporal relationship between neuropro-

tective efficacy and drug-target engagement, a gap identifiedin most published preclinical studies (Feuerstein et al.,2008).

Other cellular mechanisms, independent of VEGF anddownstream of Src, may also contribute to the neuroprotec-tion against ischemic injury. Src kinase has been shown topotentiate NMDA receptor function by direct phosphoryla-tion of the NR2A subunit, to regulate glutamate release fromsynaptic vesicles (Salter and Kalia, 2004), and to activatecalcium channels (Cataldi et al., 1996). Blocking Src activitymay therefore reduce NMDA function and modulate a vari-ety of CNS proteins associated with propagation of excito-toxic injury in ischemic stroke. Supporting this notion is thefact that more robust neuroprotection is achieved by Srcinhibition than by direct anti-VEGF therapies in acute stroke(Kimura et al., 2005). Accordingly, combined effects of Srckinase inhibition within the parenchymal and endothelialcompartments may provide protection of the neurovascularunit poststroke and may therefore be a viable therapeuticapproach. The additional inhibitory activities of SKI-606 sug-gest that other mechanisms important for its neuroprotectiveactivity might as yet be unidentified. Whether these addi-tional activities will be displayed by SKS-927 to the sameextent is another important question for future studies. Theacceptable drug-like properties of these compounds suggesttheir considerable potential as candidates for clinical testingin stroke patients.

Acknowledgments

We thank Dr. Giora Feuerstein, Dr. Peter Reinhart, and ProfessorA. Randall for critical review of this manuscript, Dr. Lin Xu forphysiological monitoring of the animals, Dr. Marc Tesconi for formu-lation support, Dr. Christine Huselton, Dr. Susan Lockhead, andRana Ramdas for pharmacokinetic analyses, and Dr. Youping Huangfor statistical analysis of the data.

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Address correspondence to: Dr. Margaret M. Zaleska, Discovery Neuro-science, Wyeth Research, CN-8000, Princeton, NJ 08543. E-mail:[email protected]


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Neuroprotective Profile of Novel Src Kinase Inhibitors in Rodent Models of Cerebral Ischemia

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