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CARDIOVASCULAR

Characterization of the Novel P-Selectin Inhibitor PSI-697 [2-(4-Chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] Quinoline-4-carboxylic acid] in Vitro and in Rodent Models of Vascular Inflammation and Thrombosis

Cardiovascular and Metabolic Disease Research (P.W.B., V.C., N.S., B.T., T.A., C.R., J.C.K., J.K.H., D.L.C., R.G.S., G.D.S., L.L.C.), Chemical and Screening Sciences (N.K., S.D., K.J.), and Drug Safety and Metabolism (Q.W.), Wyeth Research, Cambridge, Massachusetts

Received July 11, 2007; accepted November 14, 2007.

	Abstract

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P-selectin plays a significant and well documented role in vascular disease by mediating leukocyte and platelet rolling and adhesion. This study characterizes the in vitro activity, pharmacokinetic properties, and the anti-inflammatory and antithrombotic efficacy of the orally active P-selectin small-molecule antagonist PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid; molecular mass, 367.83]. Biacore and cell-based assays were used to demonstrate the ability of PSI-697 to dose dependently inhibit the binding of human P-selectin to human P-selectin glycoprotein ligand-1, inhibiting 50% of binding at 50 to 125 µM. The pharmacokinetics of PSI-697 in rats were characterized by low clearance, short half-life, low volume of distribution, and moderate apparent oral bioavailability. A surgical inflammation model, using exteriorized rat cremaster venules, demonstrated that PSI-697 (50 mg/kg p.o.) significantly reduced the number of rolling leukocytes by 39% (P < 0.05) versus vehicle control. In a rat venous thrombosis model, PSI-697 (100 mg/kg p.o.) reduced thrombus weight by 18% (P < 0.05) relative to vehicle, without prolonging bleeding time. Finally, in a rat carotid injury model, PSI-697 (30 or 15 mg/kg p.o.) administered 1 h before arterial injury and once daily thereafter for 13 days resulted in dose-dependent decreases in intima/media ratios of 40.2% (P = 0.025) and 25.7% (P = 0.002) compared with vehicle controls. These data demonstrate the activity of PSI-697 in vitro and after oral administration in animal models of both arterial and venous injury and support the clinical evaluation of this novel antagonist of P-selectin in atherothrombotic and venous thrombotic indications.

PSI-697 is a novel, orally available small molecule developed to antagonize P-selectin/PSGL-1 interactions (Kaila et al., 2007a). The goals of the studies reported here were to characterize the ability of PSI-697 to reduce P-selectin/PSGL-1 interactions and to evaluate PSI-697 in animal models that illustrate key P-selectin-dependent aspects of the atherothrombotic and venous thrombotic processes, namely platelet/leukocyte adhesion, leukocyte rolling and tethering, thrombus growth and stabilization, and the myointimal proliferation associated with atherosclerotic plaque growth.

	Materials and Methods

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Materials. Unless otherwise stated, all chemicals were obtained from Fisher Scientific (Fair Lawn, NJ) and used as received. PSI-697 was synthesized as a free acid at Wyeth Research (Cambridge, MA). The chemical structure for PSI-697 (molecular mass, 367.83) is shown in Fig. 1.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Chemical structure of PSI-697.

Biacore P-Selectin/PSGL-1 Inhibition Assay. Surface plasmon resonance (Biacore) assays were performed on a Biacore 3000 instrument (Biacore Inc., Piscataway, NJ) at 25°C at a flow rate of 30 µl/min and consisted of 60-s equilibration, 60-µl sample injection (K_inject), and 300-s dissociation. Biotinylated SGP3 was immobilized on a Biacore SA sensor chip (Biacore Inc.) using HBS-EP buffer (Biacore Inc.), target 600 to 700 relative units. The coated chip was re-equilibrated with HBS-P buffer (Biacore Inc.). PSI-697 was incubated for 1 h in 1.1x Biacore assay buffer. The solution was centrifuged at 2500g for 7 min through a 96-well, 0.2 mM filter plate (Millipore, Billerica, MA). Glycyrrhizin tri-sodium salt (TCI America, Wellesley Hills, MA) was prepared as a positive control in the same manner as PSI-697. P-selectin, P-LE, was added to each filtered small-molecule solution. Final concentrations of reagents were 500 nM P-LE, 31.25 to 250 µM PSI-697 (or 1 mM glycyrrhizin), 10% dimethyl sulfoxide, and 1x Biacore buffer (100 mM HEPES, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, pH 7.4). Test samples were supplied to the Biacore instrument in a 96-well plate. A 1x Biacore assay buffer blank solution and an "uninhibited" 500 nM P-LE solution without small-molecule inhibitor were used in double reference subtraction and as 100% binding. These were run at the beginning of each run and again after every five sample injections to compensate for any loss in binding over the period of the assay because the chip surface was not regenerated with each injection.

HL-60/P-Selectin Static Adhesion Assay. sP-selectin-Ig was immobilized on Costar 3590 96-well plates (Corning Life Sciences, Acton, MA; 3 µg/ml, 50 µl/well) at 37°C for 2 h, and the plates were blocked with 1% BSA in HBSS at 37°C for 2 h. HL-60 cells were labeled with Calcein AM (Molecular Probes/Invitrogen, Carlsbad, CA) at 37°C for 30 min, washed, and resuspended in HBSS with 1% BSA and 2 mM CaCl₂. The cells were added to each well (1 x 10⁵ cells per well) and incubated for 20 min at room temperature under rotation (60 rpm). The plates were washed two times with HBSS containing 1% BSA and 2 mM CaCl₂. Bound cells were lysed with 0.1% Igepal CA-630 (Sigma-Aldrich, St. Louis, MO) in water. Fluorescence intensity was measured using a 1430 Multilabel Counter (Perkin Elmer, Wellesley, MA). In some experiments, cells were incubated with 10 µg/ml neutralizing anti-PSGL-1 or anti-P-selectin antibodies for 20 min. For the adhesion assays in the presence of small-molecule P-selectin inhibitor, the binding HBSS buffer contained 1% BSA, 2 mM CaCl₂, and 5% dimethyl sulfoxide.

Animals and Dosing. Male, adult Sprague-Dawley outbred rats, purchased from Taconic Farms (Germantown, NY), were singly housed and provided rat chow and water ad libitum. Study animals for PK were supplied with one jugular vein catheterized. Animal studies were conduced at Wyeth Research (Cambridge, MA, Andover, MA, and Collegeville, PA) under the supervision of the Institutional Animal Care and Use Committee of Wyeth Research. PSI-697 was administered in an aqueous suspension containing 2% (v/v) polysorbate 80 (JT Baker, Phillipsburg, NJ) and 0.5% (v/v) methylcellulose (Fluka Chemical Corp., Milwaukee, WI) at a final volume of 5 ml/kg [4 ml/kg in acute surgical inflammation (ASI)/intravital microscopy (IVM)] by oral gavage. Warfarin (Sigma-Aldrich; A2250) was administered by oral gavage formulated in distilled water. Anti-rat PSGL-1 antibody was administered as a single bolus i.v. injection in a volume of 200 µl of saline (0.9% solution; Baxter, Round Lake, IL).

IVM/ASI. Male Sprague-Dawley rats (4–5 week old rats, weighing 50–100 g) were divided into four treatment groups: vehicle (n = 9), 30 mg/kg PSI-697 (n = 7), or 50 mg/kg PSI-697 (n = 7), and as a positive control, anti-rat-PSGL-1 antibody (n = 3) 4 mg/kg. Twenty minutes after dose administration, rats were anesthetized with an i.p. injection of ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg). The cremaster muscle was surgically exteriorized, placed over a clear viewing window, superfused continuously with bicarbonate buffered-saline, pH 7.4, at 35°C, and observed through an intravital microscope (model, Axioscope FS; Zeiss, Thornwood, NY) with a 40x (numerical aperture, 0.75) water immersion objective lenses and a 10x eyepiece. The images of the postcapillary venules were illuminated using bright-field microscopy and recorded with a video camera and VCR for quantitative analysis of leukocyte rolling.

For analysis, 10 postcapillary venules (20–45-µm diameter) of the cremaster muscle of each rat were selected for observation. Rolling leukocytes were defined as those moving at a velocity less then that of red blood cells in a given vessel and evaluated using frame-by-frame analysis during video playback. The number of rolling leukocytes (flux) was determined by counting all visible cells passing through a plane perpendicular to the vessel axis over 1 min. The average of 10 vessels was reported for each animal. Immediately after measurements (1 h postdosing), blood was collected from the inferior vena cava into tubes containing EDTA from each PSI-697-treated animal for determination of drug concentration (Table 1).

Rat Venous Thrombosis Model. Male Sprague-Dawley rats (weighing 350–490 g) were treated with vehicle (n = 10), PSI-697 100 (n = 7), or 30 (n = 5) mg/kg as a single oral gavage dose the day of the experiment. Warfarin (0.3 mg/kg) (n = 3) was delivered once daily for 4 consecutive days. One hour after dosing, the rats were anesthetized with sodium-pentobarbital (50 mg/kg i.p.), and the left jugular vein was isolated and cannulated with PE-60 tubing for a preinjury drug concentration sampling (3.7% sodium citrate was used as the anticoagulant (1:10 citrate to blood, v/v). The anesthetized rats were placed in dorsal recumbency on a heating pad (37°C), the abdomen was opened by a midline incision, and the inferior vena cava was exposed. A partial stenosis was applied to the vena cava by tying a 4.0 silk suture (Ethicon) around both the vessel and a blunt 20-gauge needle at the renal veins. The needle was removed, and a 3-mm diameter disk of filter paper presoaked with 3.5 µl of a 10% ferric chloride solution was applied to the external surface of the vena cava for 5 min (2 h postdosing). Tail bleeding time was measured on the experimental animals 1 h postdosing by transecting the tail 0.5 cm from the tip using a disposable surgical blade, vertically suspending the tail in 25 ml of saline, and measuring the time until the bleeding had stopped completely. A blood sample was taken from the heart by cardiac puncture 2 h postdosing, and plasma was prepared for prothrombin time and postinjury drug concentration analysis (Table 1). The animals were euthanized by inhalation of 100% CO₂, the vena cava was removed, and the thrombus was extracted from the vessel and weighed immediately.

Prothrombin time was determined using an ST4 Coagulation Instrument (Diagnostica Stago, Asnieres sur Seine, France). Each assay was run according to the reagent kit Neoplastine with addition of plasma to a reconstituted thromboplastin reagent (Diagnostica Stago).

Rat Carotid Balloon Injury Model. Male Sprague-Dawley rats (10–12 weeks old, starting weights 350–380 g) were treated orally with vehicle (n = 40) or PSI-697 at 3 mg/kg (n = 11), 15 mg/kg (n = 31), or 30 mg/kg (n = 12) 1 h before surgery and once daily for 13 days after surgery. After initial dosing, rats were anesthetized via an i.m. injection of xylazine (5 mg/kg) and ketamine (60 mg/kg) and s.c. administration of Buprenex (0.2 mg/kg). Surgeries were systematically performed by the same surgeon. A ventral midline incision in the cervical region was made to expose the left external carotid artery, a 2F Fogarty balloon catheter was introduced through the external carotid artery, and the common left carotid artery was injured by four passages of the inflated balloon catheter. The catheter was withdrawn, the left external carotid artery was ligated, and the subcutis and skin were apposed with subcuticular sutures. After 13 days of recovery and 20 to 24 h after the last drug treatment, blood was collected in serum separator tubes from each PSI-697-treated animal for determination of drug concentrations (Table 1) for comparison with a separate 24-h PK study to estimate drug exposure (see Fig. 6). Animals received and infusion Evan's blue solution (60 mg/kg) via the tail vein and were euthanized 30 min later by inhalation of 100% CO₂. The arterial system was perfused at physiologic pressure with saline solution, followed by 4% paraformaldehyde. The left carotid artery was excised, and the injured portion of the artery was identified as the arterial segment stained in blue.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Pharmacokinetic simulation of plasma concentration-time profile of PSI-697 after 14 days of daily oral administration of 30 mg/kg in the rat carotid balloon injury model. Actual measured values are superimposed on the plot.

Pharmacokinetics and Estimated Plasma Concentrations of PSI-697. Male adult Sprague-Dawley rats with jugular vein catheters (n = 3), weighing 225 to 300 g, were administered 30 mg/kg PSI-697 formulated in 2% polysorbate 80 and 0.5% methylcellulose by gavage. Blood (0.25 ml) samples were collected from each rat at 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 h postdose administration into K₂EDTA-coated sampling tubes.

Plasma or serum was treated with acetylnitrile (1:2 /v), containing an internal standard of similar chemical structure to precipitate plasma protein. After vortexing and centrifugation, supernatants were directly injected into a liquid chromatography/tandem mass spectrometry instrument. High-performance liquid chromatography separation was performed on a Perkin Elmer Series 200 (Perkin Elmer, Norwalk, CT) using an XTerra MS C18 column (2.1 x 20 mm, 2.5 µm; Waters, Milford, MA). Detection of test articles was performed on a PESCIEX API-3000 triple quadrupole mass spectrometer (Applied Biosystems, Concord, ON, Canada; L4K4V8) using a TurboIon Spray source. The concentrations of test articles were estimated by comparison with a standard curve generated by plotting peak area ratio of PSI-697 and an internal standard against nominal concentrations. The curve remained linear from 1 to 5000 ng/ml. Sample concentrations exceeding the linear range of the standard curves were diluted with rat plasma before analysis. The PK parameters were determined using WinNonlin (version 4.1; Pharsight, Mountain View, CA). Calculations were performed using noncompartmental analysis approach. Estimation of area under the plasma concentration versus time curve was based upon log trapezoidal rule. Simulation of the steady-state plasma level was based upon nonparametric superposition of single-dose PK study. No statistical analysis other than descriptive statistics was conducted.

In Vitro PSI-697 Plasma Protein Binding Studies. The binding of PSI-697 to plasma proteins in mouse, rat, rabbit, dog, and human was determined by the ultracentrifugation method at plasma PSI-697 concentrations of 1, 10, and 100 µg/ml based on the PSI-697 maximum concentrations observed in preclinical species after pharmacological dosages. Plasma from male CD 1 mice, Sprague-Dawley rats, New Zealand White rabbits, beagle dogs, and human volunteers was obtained from Bioreclamation, Inc. (Hicksville, NY).

Statistical Analysis. In vitro results are expressed as mean ± S.D. In vivo results are expressed as mean ± S.E.M. Analysis of variance with Tukey post hoc testing was employed for comparison between groups, using GraphPad Prism software version 3.02 (GraphPad Software Inc., San Diego, CA). Differences were considered significant if P < 0.05. Data from the three warfarin-treated rats were assumed to be normally distributed based on agreement with previous data from warfarin-treated animals with demonstrated normality.

	Results

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PSI-697 Inhibits P-Selectin/PSGL-1 Interactions In Vitro. High-throughput screening identified the quinoline carboxylic acid class of compounds as antagonists of P-selectin/PSGL-1 interactions. Subsequent chemistry efforts used a Biacore assay to optimize the activity of this class of compounds based on the ability to prevent P-selectin/PSGL-1 interactions (Kaila et al., 2007a,b). This resulted in the identification of PSI-697 (Fig. 1). PSI-697 inhibited the binding of a soluble human P-selectin to its immobilized ligand, PSGL-1, in a reproducible concentration-dependent manner inhibiting 50% of binding at a concentration of 125 ± 25 µM (Fig. 2A). Glycyrrhizin, a natural product inhibitor of P-selectin, was used as a positive control and had an IC₅₀ of 1 mM, in agreement with values reported in the literature (Kaila et al., 2005).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Effect of PSI-697 on P-selectin/PSGL-1 binding in Biacore and static cell assays. A, PSI-697 inhibited soluble human P-selectin binding to immobilized human PSGL-1 by 50% at 125 µM in a Biacore inhibition assay. The positive control, glycyrrhizin, inhibited the interaction by 50% at 1 mM (left). Sample sensograms illustrate the magnitude of the reference subtracted binding signal (right). B, PSI-697 inhibited HL-60 cells binding to 3 µg/ml coating concentration of sP-selectin-Ig by 68% at 100 µM and 66% at 50 µM relative to vehicle. Neutralizing antibodies to P-selectin and PSGL-1 inhibited binding by 96 and 76% relative to IgG control. Values presented as mean ± S.D.

PSI-697 Inhibits Leukocyte Rolling in the IVM/ASI Model. The surgical trauma caused by the exteriorization of the cremaster muscle evokes an inflammatory response resulting in P-selectin transfer to the endothelial cell surface that promotes increased leukocyte rolling on the activated venous endothelium (Robinson et al., 1999). PSI-697 at an oral dose of 50 mg/kg reduced leukocyte rolling flux by 39% relative to vehicle (P < 0.05, plasma concentration of 28,500 ± 5480 ng/ml). There was a 26% reduction of rolling flux at 30 mg/kg, which was not statistically significant (P > 0.05, plasma concentration of 5900 ± 983 ng/ml). A neutralizing antibody to rat PSGL-1, given i.v. at 4 mg/kg as a positive control, reduced rolling leukocytes by 84% (Fig. 3). A total of three animals treated with antibody were sufficient to obtain a robust and reproducible response, which was consistent with previous results.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Effect of PSI-697 on leukocyte rolling flux in an acute surgical inflammation model. Sixty minutes after oral administration of PSI-697 to Sprague-Dawley outbred rats, the average number of leukocytes per vessel rolling on inflamed endothelium of cremasteric postcapillary venules is reduced 26 ± 1.3% (n = 7, P > 0.05) and 39 ± 1.7% (n = 7, P < 0.05) in animals treated with 30 or 50 mg/kg PSI-697, respectively. Values presented as mean ± S.E.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effects of PSI-697 on thrombus weight and bleeding time in an FeCl3-induced venous stasis model in Sprague-Dawley Outbred rats. A, thrombus weight was reduced 19% (n = 7, P < 0.05) relative to vehicle in animals treated with 100 mg/kg PSI-697. There was no difference between animals treated with 30 mg/kg PSI-697 and vehicle. B, PSI-697 did not have any significant effect on rat tail bleeding time at either 30 or 100 mg/kg. Warfarin significantly increased bleeding time as expected.

PSI-697 Decreases Myointimal Proliferation in a Rat Carotid Balloon Injury. Myointimal proliferation develops in this model after de-endothelialization and circumferential distension of the carotid artery produced by the intraluminal passage of a balloon catheter. Ten- to 12-week-old male Sprague-Dawley rats were treated orally with PSI-697 at 3, 15, or 30 mg/kg or dose vehicle 1 h before surgery and once per day for 13 days after surgery. Histomorphometric analysis (Fig. 5) of the injured carotid arteries of rats that underwent angioplasty of the left carotid artery revealed a dose-dependent inhibition of intimal hyperplasia when dosed orally with PSI-697, compared with vehicle-treated animals. The intima/media ratios were decreased by 40.2% (P = 0.025) and 25.7% (P = 0.002) in animals treated with PSI-697 at 30 or 15 mg/kg, respectively, compared with animals treated with vehicle. No significant effect was observed in animals treated with PSI 697 at 3 mg/kg. These values, expressed as percentage of vehicle control, are shown in Fig. 5C.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Effect of PSI-697 on myointimal proliferation in the rat carotid balloon injury model as demonstrated by histomorphology. A, left, Evans blue dye shows the area of injury and areas of cross-sectioning (1, distal; 2, medial; 3, proximal). Right, carotid cross-sections of vehicle-treated rats corresponding to: 1, distal; 2, medial; and 3, proximal portions of the injured area showing significant neointimal thickening. Carotid cross-sections of 30 mg/kg PSI-697-treated rats showing significantly less neointimal thickening than vehicle-treated animals. B, quantitative evaluation of the effect PSI-697 on myointimal proliferation in the rat carotid balloon injury model. Intimal and medial areas were measured, and the result was expressed as the ratio of the two values. C, dose-dependent inhibition of myointimal proliferation shown as intima/media ratio percentage of vehicle.

In a separate single rat PK study at oral dose of 30 mg/kg (n = 3), the average plasma exposure (the area under the concentration-time curve) was 8 µg · h/ml. On the last day of rat carotid injury study, plasma samples were collected at 20 to 24 h after last dose administration. The plasma levels collected were in agreement with the projection from the single-dose PK study (Fig. 6). Although this comparison was not made for all dose groups, it can be concluded that daily dosing of 30 mg/kg, resulting in 40% reduction of the intima/media ratios, was associated with a daily plasma exposure of 8 µg · h/ml.

Single-Dose Pharmacokinetic Study of PSI-697 in Rat. The pharmacokinetic profile of PSI-697 (Table 2) when administered 30 mg/kg p.o. or 1 mg/kg i.v. shows that the drug has moderate apparent oral bioavailability (18%), low volume of distribution (0.32 l/kg) consistent with high plasma protein binding (described below), low clearance (5 ml/min/kg), moderate p.o. exposure (8 µg · h/ml), and short half-life (3 h).

PSI-697 Is Highly Bound to Plasma Protein in Five Species. PSI-697 was highly bound to protein in mouse, rat, rabbit, dog, and human plasma at nominal concentrations of 1, 10, and 100 µg/ml, as measured by ultracentrifugation method. The mean (±S.D.) percentages of PSI-697 bound to proteins were 98.7, 98.8, and 98.8%, respectively, in mouse plasma; 99.5, 99.5, and 99.3%, respectively, in rat plasma; 99.3, 99.2, and 99.3%, respectively, in rabbit plasma; 99.6, 99.6, and 99.7%, respectively, in dog plasma; and 99.1, 99.0, and 99.0%, respectively, in human plasma. PSI-697 protein binding was slightly lower in mouse plasma than in rat, rabbit, dog, or human plasma. A minor degradation of PSI-697 in mouse plasma was demonstrated in stability studies (data not shown) that may have resulted in an underestimation of PSI-697 binding in mouse plasma. There was no change in the percentage of PSI-697 protein binding at plasma PSI-697 concentrations between 1 and 100 µg/ml among the species tested.

	Discussion

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Inflammation plays a significant role in both arterial and venous vascular disease (Schaub et al., 1984; Ross, 1999) Key cells involved in the proinflammatory pathophysiology of atherothrombosis and deep vein thrombosis express P-selectin and its ligand PSGL-1 (Ross, 1999; Wagner and Burger, 2003; Myers and Wakefield, 2005). In addition, elevated soluble P-selectin levels have been associated with increased cardiovascular events (Ridker et al., 2001), suggesting that inhibition of the P-selectin/PSGL-1 interaction could be an important target for the treatment of atherothrombotic vascular disease.

Studies using gene-deficient mice or antagonists of P-selectin/PSGL-1 interactions support a role for this pathway. Atherosclerotic lesion development and arterial injury are reduced in apolipoprotein-E-deficient mice that are also deficient in P-selectin (Dong et al., 2000; Manka et al., 2001). Neutralizing antibodies to P-selectin or PSGL-1 blocked neointima formation after arterial injury in apolipoprotein E-deficient mice (Phillips et al., 2003). P-selectin-deficient mice have decreased thrombosis in models of inferior vena cava ligation (Sullivan et al., 2003) and decreased neointimal hyperplasia after carotid artery ligation or femoral artery injury (Kumar et al., 1997; Smyth et al., 2001). An antibody to P-selectin reduced neointimal formation in rat (Hayashi et al., 2000) and selectin antagonism with a soluble, recombinant form of PSGL-1 (rPSGL-Ig) decreased restenosis in rat and pig models of arterial balloon injury (Wang et al., 2001; Zhou et al., 2002). rPSGL-Ig also decreased thrombosis and increased spontaneous recanalization in a baboon DVT model and reduced postthrombotic vein wall fibrosis in a rat stasis-induced deep vein thrombosis (Thanaporn et al., 2003).

Although antibodies or other protein antagonists demonstrate therapeutic benefit, their utility in treatment of chronic human disease like atherothrombosis can be limited by the need for parenteral administration and cost. Several nonprotein compounds have demonstrated activity in animal models, OC229-648, KF38789, and Efomycine M, and a few have made it into human trials, Cylexin, bimosiamose, and rPSGL-Ig. However, before our recent work with quinoline salicylates (Myers et al., 2006; Kaila et al., 2007a,b), efforts to discover an orally available, noncarbohydrate, small-molecule inhibitor of the P-selectin/PSGL-1 interaction have been unsuccessful. The studies presented here describe the in vitro and in vivo characterization of PSI-697, the first orally active small-molecule antagonist of P-selectin/PSGL-1 with demonstrated activity in in vivo models of both arterial and venous injury.

Assays using purified proteins under flow and cells under static conditions were used to evaluate the inhibitory activity of this compound in vitro. Biacore analysis demonstrated that PSI-697 effectively inhibited binding of P-selectin to PSGL-1 with 50% inhibition of binding at 125 µM. A 2-fold lower inhibitory concentration of PSI-697 was observed when PSGL-1-expressing cells were subjected to a static adhesion assay. This high concentration in vitro and difference between the monomeric protein/protein interactions of the Biacore assay and multimeric cell/protein interaction of the static cell assay probably reflect the difficulty in reproducing in vivo flow rates, selectin densities, and other contributing cellular interactions in vitro. It suggests that in vivo receptor occupancy requirements may be lower than those in vitro, and as expected for a flow-dependent interaction, the EC₅₀ of PSI-697 decreased further from the Biacore and static adhesion assay to the in vivo models described below. These data are consistent with previous selectin studies comparing in vitro protein/protein interaction assays with cell-based assays, static and under flow, using sLex, rPSGL-Ig, or small molecules. sLex, a natural ligand of P-selectin, has an IC₅₀ of 10 mM in the P-selectin Biacore assay. Poor pharmacokinetic properties prohibit the use of sLex in vivo. However, an sLex mimetic bimosiamose (TBC-1269) has an IC₅₀ of 70 µMinan HL-60 cell binding assay to P-selectin, is active preclinically at 10 mg/kg, and is currently in phase II for psoriasis. The monomeric SGP3 construct of PSGL-1 has an IC₅₀ of 900 nM in the Biacore assay and 25 nM in a cell-based flow assay. Again, poor pharmacokinetics prevent use of this monomeric construct in disease models, although a recombinant mutated IgG chimera shows activity in a variety of cardiovascular models. Perhaps the most thorough, and relevant to the current studies, demonstration of the impact of monomeric versus multimeric presentation and difficulty in presenting the appropriate receptor and ligand densities in vitro comes from data reported in Kaila et al. (2007a). PSI-697 analogs were evaluated in Biacore (monomeric), cell-based flow assay (multimeric under physiologic flow), and adjuvant-induced arthritis in rat (in vivo). The absolute value of IC₅₀ was reduced approximately 10-fold from one assay to the next from monomeric to in vivo. However, the rank order of potency remained the same, demonstrating the predictivity of the in vitro assays.

In general, leukocytes are swept rapidly through vessels in freely flowing blood. However, at sites of injury or inflammation these cells are tethered initially via the P-selectin/PSGL-1 interaction and begin a characteristic rolling along the vessel wall. In this study, IVM was used to assess the effects of PSI-697 on leukocyte rolling on inflamed vascular endothelium because it models the early events in the inflammatory process that lead from rolling to firm adhesion and extravasation into the subendothelial matrix (Ross, 1999). IVM is particularly useful because it allows real-time assessment of the anti-inflammatory efficacy of P-selectin inhibitors by looking at changes in rolling flux of leukocytes in postcapillary venules in vivo. In this system using endogenous cells and flow, PSI-697 administered orally by gavage at a dose of 50 mg/kg (35774 ± 5480 ng/ml) significantly decreased the rolling flux of leukocytes in the cremasteric blood vessels compared with vehicle control. These data suggest that PSI-697 is effective in blocking the initial phase of leukocyte recruitment during an inflammatory process and confirm its ability to disrupt a P-selectin-dependent interaction (Robinson et al., 1999). Moreover, this acute model offered real-time assessment of the affect of drug levels on leukocyte rolling and adhesion, the basic biological mechanism mediated by P-selectin/PSGL-1 interaction. Although not visualized to the same extent here, it should be noted that others have shown that this characteristic rolling of leukocytes relies on formation of multiple cooperative tethers (Schmidtke and Diamond, 2000). It is hypothesized that this necessary cooperativity of tethers offers a potential explanation for in vivo efficacy of a compound with high protein binding and micromolar in vitro IC₅₀ values. Inhibition of a small fraction of tethers may prevent other tethers from holding under physiologic flow.

The role of inflammation in deep vein thrombosis, specifically leukocyte adhesion and extravazation into subendothelial tissues, has long been recognized (Schaub et al., 1984). More recently P-selectin's role in both arterial and venous thrombosis has been clearly demonstrated in vitro and in vivo. P-selectin has been shown to promote fibrin deposition via tissue factor-bearing microparticles contributing to thrombus growth (Furie and Furie, 2004), and inhibition of P-selectin with rPSGL-Ig was effective in reducing induced venous thrombosis in baboon (Myers et al., 2002). Here, PSI-697 was evaluated in a partial stasis and injury model of venous thrombosis. This model was selected based on the clinical relevance of stasis induction of the thrombus (Peternel et al., 2005) and on the fibrin deposition dependence on the model (Lorrain et al., 2003), which is consistent with the P-selectin mechanism of action. PSI-697 produced a modest reduction in thrombus weight 18% (P < 0.05) relative to vehicle. The complexity of the ferric chloride model has been previously described in a recent study (Furie and Furie, 2007), suggesting that the endothelial denudation produced in this model mediates both cellular and biochemical activation of clot formation. The extent of this injury involves pathways that include but do not exclusively involve P-selectin. The modest reduction in clot size observed with PSI-697 treatment is consistent with the role of P-selectin in mediating clot growth (Celi et al., 2004).

Finally, PSI-697 was tested in a rat carotid balloon injury model to evaluate its effects on a severe and chronic arterial injury. The rat carotid injury model is a widely used animal model of intimal hyperplasia or hyperproliferation of the smooth muscle cells lining the injured artery after angioplasty (Clowes et al., 1983) and is also representative of the myointimal process involved with formation of the atherosclerotic lesion. In both situations, this proliferation results from an abundance of growth factors secreted by activated platelets and monocytes adhering to damaged endothelium (Shah, 2003) P-selectin dependence in this model has been previously demonstrated. A neutralizing antibody to rat P-selectin reduced neointimal proliferation 43% (Hayashi et al., 2000), and a recombinant soluble form of the natural ligand, rPSGL-Ig, resulted in 63% reduction (Zhou et al., 2002) in similar models. Inhibiting P-selectin reduces the binding of leukocytes to platelets adhered to injured endothelium and inhibits the binding of platelets to bound and free leukocytes, thereby preventing exposure of the damaged endothelium to platelet and monocyte growth factors, ultimately preventing or reducing restenosis (Zhou et al., 2002). Treatment with PSI-697 produced significant reductions in intimal hyperplasia when dosed orally once/day at 15 (25.7%) and 30 (40.2%) mg/kg. These data demonstrate that PSI-697 can reduce myointimal proliferation, a process associated with atherosclerotic plaque growth, in a model with established P-selectin dependence.

It should be noted that although the 30 mg/kg dose reduced intima/media ratio as well as a P-selectin antibody in the rat carotid injury model, this dose was not sufficient to see efficacy in either the ASI/IVM or venous stasis models. This is probably due to the acute versus chronic nature of the models, the early stage at which P-selectin plays a role in disease and the chronic amplifying quality of inflammation. The ASI/IVM model looks solely at leukocyte attachment and rolling, it does not allow measurement of downstream events resulting from this attachment. Likewise, the rat venous stasis model is an early snapshot of thrombus formation and does not address the increasing effects of inflammation over time. In the more chronic setting of the rat carotid injury model, blocking the P-selectin/PSGL-1 interaction shows the downstream effects of inhibiting the adhesion of the entire triad of leukocyte, endothelium, and platelet. PSI-697 seems to have a greater measurable effect at a lower dose in this chronic setting than it does in reducing one or more of the acute early events themselves. In addition, one might speculate that achieving a critical concentration before injury is more relevant than maintaining a concentration above a certain minimum in this setting, given the low trough values in the rat carotid model and known early time course of P-selectin up-regulation in restenosis. Future studies directed at more complex and chronic models are indicated to determine drug exposure requirements under these conditions.

In conclusion, PSI-697 is a novel P-selectin inhibitor with positive activity in vitro and in vivo after oral administration at doses ranging between 15 and 100 mg/kg in these rodent models of both arterial and venous injury. These results are consistent with data presented using other methods to inhibit P-selectin/PSGL-1 interaction and suggest that PSI-697 has therapeutic potential in the treatment of atherothrombotic and venous thrombotic diseases through a unique mechanism of action. PSI-697 is currently in phase 1 clinical trials.

	Acknowledgements

 
We thank Nevena Mollova for analytical work in the pharmacokinetic analysis, Jennifer Tavares, Kim Harding, and Alison Darby for animal surgery skills, and Zeen Tong and Joe McDevitt for plasma protein binding analysis.

	Footnotes

 
This study was funded entirely by Wyeth Research.

P.W.B. and V.C. contributed equally to this work.

The following oral presentations included data in this manuscript for Biacore and the rat carotid model. Data from static cell assay, acute surgical inflammation, and venous thrombosis models have not been presented earlier. Bedard PW, Sushkova N, Eppihimer MJ, Tavares J, Harding K, Darby A, Keith JC Jr, Kaila N, et al. (2006) A novel P-selectin inhibitor, PSI-697, demonstrates positive activity after oral administration in Rodent Models of Vascular Inflammation. 35th Annual New England Pharmacologists Meeting; 2006 Waltham, MA (oral presentation); and Bedard PW, Clerin V, Sushkova N, Darby A, DeBernardo S, Eppihimer MJ, Harding K, Janz K, Kaila N, Keith JC Jr, et al. (2005) A novel P-selectin inhibitor, PSI-697, demonstrates positive activity after oral administration in rodent models of vascular inflammation. International Society of Thrombosis and Haemostasis Meeting; 2005 Sydney, Australia (oral presentation).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.128124.

ABBREVIATIONS: PSGL, P-selectin glycoprotein ligand; PSI-697, 2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid; BSA, bovine serum albumin; HBSS, Hanks' balanced salt solution; PK, pharmacokinetics; ASI, acute surgical inflammation; IVM, intravital microscopy; rPSGL-Ig, recombinant PSGL-1 Ig chimera; KF389789, (E)-3-[7-(2,4-dimethoxyphenyl)-2,3,6,7-tetrahydro-1,4-thiaz epin-5-yl]-4-hydroxy-6-methyl-2H-pyran-2-one; OC229-648, 3-(4-{4-[4-((E)-2-carboxy-vinyl)-phenyl]-5-[4-(2-hexadecylcarbamoyl-vinyl)-phenyl]-1H-imidazol-2-yl}-phenyl)-4,5-dihydro-isoxazole-5-carboxylic acid; TBC-1269 (bimosiamose), 1,6-bis[3-(3-carboxymethylphenyl)-4-(O-alpha-D-mannopyranosyl)-phenyl] hexane.

Address correspondence to: Patricia W. Bedard, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140. E-mail: pbedard{at}wyeth.com

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