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CARDIOVASCULAR
Cardiovascular and Metabolic Disease Research (J.K.H., G.S.F., G.A.M., R.E.S., T.M.A., A.H., D.L.C.), Chemical and Screening Sciences (H.E.), and Bioanalytical R&D (M.L., A.J.), Wyeth Research, Collegeville, Pennsylvania
Received January 25, 2005; accepted April 26, 2005.
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Abstract |
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,9-epoxymethanoprostaglandin F2 (U46619
[GenBank]
)-, and collagen-induced platelet aggregation, as well as template bleeding and prothrombin time, remained unaffected by PAI-039. Ex vivo clot lysis analysis revealed normal clot formation but accelerated clot lysis in PAI-039-treated groups. The pharmacokinetic profile of PAI-039 indicated an oral bioavailability of 43 ± 15.3% and a plasma half-life of 6.2 ± 1.3 h. In conclusion, PAI-039 is an orally active prothrombolytic drug that inhibits PAI-1 and accelerates fibrinolysis while maintaining normal coagulation in a model of coronary occlusion.
), and its role in the development of acute disorders such as deep vein thrombosis and myocardial infarction has recently been extended to tissue remodeling, atherosclerosis, and cancer (Stefansson et al., 2003). PAI-1 is a member of the serpin family of proteins (Potempa et al., 1994) and exists in multiple conformations of which a minor component, the "active" form, exhibits inhibitory effects against tPA and uPA (Lawrence, 1997). Inhibitory monoclonal antibodies to PAI-1 have been used in preclinical models of acute thrombosis and shown to enhance endogenous fibrinolysis (Biemond et al., 1995; van Giezen et al., 1997). Due in part to its structural complexity, modulation of PAI-1 by orally active drugs has not been fully realized, despite considerable effort in this area over the past decade (Wu and Zhao, 2002; Gils and Declerck, 2004).
We recently identified a small molecule inhibitor directed specifically against the active, inhibitory conformation of the serpin (Elokdah et al., 2004). This molecule, tiplaxtinin (chemical designation PAI-039), performed as an oral profibrinolytic agent in preliminary studies in a rodent model of acute arterial thrombosis. The goal of the present study was to further characterize PAI-039 in a canine model of coronary thrombosis, specifically focusing on endpoints of recanalization and hemostasis. We show that this orally active inhibitor of PAI-1 both stimulates endogenous fibrinolysis and restores coronary blood flow without increasing template bleeding time.
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Materials and Methods |
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). Recombinant human tPA (Activase) was manufactured by Genentech (South San Francisco, CA). All remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO). In Vivo Dog Coronary Thrombosis Model. Dogs were anesthetized with sodium pentobarbital (30 mg/kg i.v.) and ventilated with room air with the use of a cuffed endotracheal tube and a Harvard respirator (Harvard Apparatus Inc., South Natick, MA) adjusted to deliver a tidal volume of 30 ml/kg (12 respirations/min). The right femoral artery and vein were isolated and cannulated for blood pressure monitoring (Millar Mikro-tip catheter; Millar Instruments Inc., Houston, TX) and blood sampling, respectively. Figure 1 illustrates the experimental protocol in detail. Ninety minutes after intragastric administration of vehicle (2% Tween 80/0.5% methylcellulose suspension) or PAI-039, male beagle dogs were subjected to left circumflex (LCX) coronary artery injury via an intravascular electrode. Based on the pharmacokinetics of PAI-039 and a Cmax at 1.8 h, we chose to initiate injury 90 min after dosing.
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The anodal current was initiated (150 µA) and maintained constant for a period of 30 min. All variables (LCX blood flow, heart rate, and arterial pressure) were monitored continuously for 240 min after the initiation of LCX electrode stimulation. Occlusive thrombosis was assessed as a constant reading of zero flow on the flowmeter for 1 min. At the conclusion of the observation period, the LCX coronary artery was excised and examined for electrode penetration and evidence of endothelial injury. The thrombus was extracted from the injured region of the vessel and weighed. Blood draws were performed to investigate ex vivo platelet reactivity, drug plasma concentration, PAI-1 activity, and general hemostatic profiling (PT, APTT, and template bleeding time) according to Fig. 1.
Platelet Aggregation and Bleeding Time. To assess ex vivo platelet reactivity, blood (10 ml) was withdrawn from the right femoral vein into a plastic syringe containing 3.7% sodium citrate as the anticoagulant [1:10 citrate to blood (v/v)]. Platelet-rich plasma (PRP) was obtained by collecting the supernatant from whole blood centrifuged at 140g for 10 min. Subsequently, platelet-poor plasma was prepared from the same blood sample by further centrifugation at 2000g for 15 min. Ex vivo platelet aggregation was assessed at 37°C with a four-channel platelet aggregometer (Bio-Data-PAP-4; Bio Data, Hatboro, PA) by recording the increase in light transmission through a stirred suspension of PRP. Aggregation was induced with 20 µM ADP, 4 µM U46619 [GenBank] , and 2.5 µg/ml collagen. A subaggregatory dose of epinephrine (550 nM) was used to prime the platelets before the agonists were added. Bleeding times were determined with the use of a Surgicutt device (International Technidyne Corporation, Edison, NJ), which made a uniform incision 5 mm in length and 1 mm in depth on the upper surface of the tongue. The lesion was blotted with filter paper every 20 s until the transfer of blood to the filter paper ceased.
APTT and PT Determination. Activated partial thromboplastin time and prothrombin time were determined using an ST4 Coagulation Instrument (Diagnostica Stago, Asnières, France) with a four-channel clot detection system. Each APTT assay was run according to the reagent kit PTT Automate with addition of plasma to a reconstituted PTT Automate reagent and 0.025 M CaCl2 (Diagnostica Stago). Each PT assay was run according to the reagent kit Neoplastine with addition of plasma to a reconstituted thromboplastin reagent (Diagnostica Stago). Citrated whole blood was withdrawn from the right femoral vein at the time points specified in Fig. 1. Sodium citrate (3.7%) was used as the anticoagulant [1:10 citrate to blood (v/v)]. Plasma was prepared by centrifugation at 2000g for 15 min. APTT and PT were measured using prepared reagents according to instrument directions.
Plasma PAI-1 Activity Assay. PAI-1 activity was determined using a two-stage indirect back titration method as described by Chandler et al. (1997). In the first stage, a fixed amount of PAI-1 (50 ng/ml) was added to a 75-µl aliquot of citrated plasma that reacts with the tPA present. In the second stage, residual tPA activity was determined by measuring the change in absorbance at 405 nm of 1 mM Spectrozyme tPA (American Diagnostica, Greenwich, CT), with the amount of color developed proportional to the amount of tPA in the sample. The endogenous PAI-1 activity in the plasma is equivalent to the difference in tPA activity with or without the addition of PAI-1 and was compared between plasma samples obtained from control or drug-treated dogs at 3 h postelectrolytic injury.
Ex Vivo Plasma Clot Lysis Assay. Rates of clot lysis were determined in samples collected 2 h after electrolytic injury using the methods described by Robbie et al. (2000). Plasma (100 µl) was added to a 96-well plate, followed by the addition of various concentrations of human recombinant tPA and incubated at room temperature for 5 min. Clot formation was triggered by the addition of bovine thrombin (10 U/ml) dissolved in CaCl2. Lysis of the clot was measured by monitoring the change in absorbance at 405 nm determined over a 60-min period at 37°C, and the time to 50% reduction in absorbance was recorded for each sample with and without tPA.
Plasma Levels of PAI-039. PAI-039 exposure was determined both in anesthetized dogs during electrolytic injury (Fig. 1) and separately in a detailed conscious dog pharmacokinetic study involving dosing at 1 mg/kg i.v. and 3 mg/kg orally. Dogs were administered the oral dose during the first week and the i.v. dose in a crossover design the second week. PAI-039 was delivered orally by gavage with a vehicle consisting of 2.0% polysorbate 80, national formulary, and 0.5% methylcellulose (4000 cps) in purified water. The vehicle for the intravenous formulation was 100% polyethylene glycol200. Blood samples were collected through the jugular vein from four animals per time point using a serial bleeding design at 0.5, 1, 2, 4, 8, 12, and 24 h after oral dosing, and at 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after i.v. dosing. Quantitation of PAI-039 in plasma was determined using a validated liquid chromatography-tandem mass spectrometry method using negative mode of electrospray ionization. Pharmacokinetic parameters were calculated by noncompartmental model analysis using WinNonlin Professional Software, version 2.1 (Pharsight, Cary, NC).
Specificity Assays. PAI-039 was assessed in vitro for potential inhibitory activity against structurally related serpins and serine proteases. PAI-039 was dissolved in dimethyl sulfoxide at a final concentration of 10 mM and then diluted 200x in buffer containing 50 mM Tris base, 150 mM NaCl, 10 µg/ml bovine serum albumin, and 0.01% Tween 80, pH 7.5. The effect of PAI-039 on the inhibition of 1-antitrypsin (Lawrence and Loskutoff, 1986), antithrombin III (Bjork et al., 1992), 2-antiplasmin (Wang et al., 2003), and uPA and tPA (Crandall et al., 2004) was assessed at concentrations of the compound ranging from 2.5 to 25 µM. In addition, the effect of PAI-039 on other coagulation proteases was tested using purified human enzymes (Enzyme Research Laboratories Inc., South Bend, IN) and synthetic peptide substrates in kinetic assays. Buffer (for 100% activity control) or PAI-039 was incubated with enzyme for 10 min at ambient temperature, followed by the addition of substrate. The initial rate of substrate cleavage was measured on a Spectromax Plus 384 or Gemini EM plate reader (Molecular Devices, Sunnyvale, CA). Thrombin and Factor Xa assays were performed with a final concentration of 1 nM enzyme and 300 µM S-2366 or 150 µM S-2765, respectively (DiaPharma Group, West Chester, OH), in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 0.005% Triton X-100. The Factor VIIa assay was performed with a final concentration of 3 nM FVIIa/10 nM tissue factor (rTF 1-219, from S. Krishnaswamy, Children's Hospital of Philadelphia, PA) and 100 µM Pefafluor tPA (Centerchem, Norwalk, CT) in 50 mM HEPES, pH 7.4, 5 mM CaCl2, 100 mM NaCl, and 0.005% Triton X-100 buffer.
Statistical Analysis. The data are expressed as mean ± S.E.M. Time to thrombosis and thrombus weight between control and PAI-039-treated dogs were compared using a one-way ANOVA followed by Dunnett's post hoc test. Total volume of blood flow after initial thrombotic occlusion was calculated from the area under the curve of the coronary blood flow over time. Area was determined using the measurements function in the Ponemah physiograph software. Area was converted to total blood flow by calculating the area of a 1-min tracing at 10 ml/min using the calibration feature of the flowmeter. Total blood flow during spontaneous reperfusion was compared with control using a one-way ANOVA followed by Dunnett's post hoc test. Any change in blood flow >0.5 ml/min after sustained zero blood flow that lasted longer than 1 min was recorded as an episode of spontaneous reperfusion. Mean bleeding time, percentage of platelet aggregation, ex vivo clot lysis time, PAI-1 inhibited-tPA activity, and APTT and PT in control and PAI-039-treated dogs were compared with each group's respective baseline using a one-way ANOVA followed by Dunnett's post hoc test. Statistical significance was reported when p < 0.05. Heart rate and mean arterial blood pressure in the dog were compared using a repeated measures one-way ANOVA.
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Results |
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Effect of PAI-039 on Platelet Aggregation, Bleeding Time, PT, and APTT. Ex vivo platelet responses were measured throughout the experiment. PRP prepared from blood of control dogs showed typical ex vivo aggregation responses to 20 µM ADP, 4 µM U46619 [GenBank] , and 2.5 µg/ml collagen over the entire course of the experiment (Table 2). PAI-039 treatment at 10 mg/kg had no effect on platelet responses to any of these agonists, indicating that reperfusion in drug-treated groups was not related to inhibition of these common platelet activation pathways. Similar responses were observed at the 1- and 3-mg/kg dose (data not shown). The additional hemostatic measurements of APTT, PT, and template bleeding time were also determined throughout the experiment. At the highest dosage of PAI-039, there was no observed effect on template bleeding time, APTT, or PT (Table 3). Similar results were obtained at the lower doses (data not shown).
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Plasma PAI-1 Activity. The PAI-1 inhibitory effect of PAI-039 was determined in citrated plasma at 3 h postelectrolytic injury. As shown in Fig. 3, each dosage of PAI-039 reduced plasma PAI-1 activity, with statistical significance observed in the 10-mg/kg group (p < 0.05).
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). Three hours postinjury, the plasma concentration in the 3-mg/kg group was 0.630 ± 0.168 µg/ml and in the 10-mg/kg group was 1.021 ± 0.378 µg/ml.
In addition to measuring plasma concentrations at 3 h after dosing, a full pharmacokinetic profile of PAI-039 was assessed under good laboratory practice conditions in the conscious dog. The mean plasma concentrations of PAI-039 after a single 3-mg/kg oral dose or 1 mg/kg i.v. dose are shown in Fig. 5. The observed mean (± S.D.) peak plasma concentration (Cmax) for PAI-039 after oral administration was 2125 ± 937 ng/ml and was observed at 1.8 h after dosing, which was higher than the value observed in the anesthetized dogs at the same dose. The C5min value after iv. dosing was 6863 ± 1234 ng/ml. The mean (± S.D.) AUC0-
values for PAI-039 were 17,818 ± 9047 ng · h/ml after oral dosing and 13,274 ± 3799 ng · h/ml after i.v. dosing. The mean (± S.D.) clearance (CLT) and the steady-state volume of distribution (Vdss) after i.v. dosing were 0.0800 ± 0.0216 l/h/kg and 0.340 ± 0.130 l/kg, respectively. The mean (± S.D.) bioavailability of PAI-039 was 43.9 ± 15.3%. The mean (± S.D.) t1/2 values of PAI-039 were 6.2 ± 1.3 h after i.v. dosing and 6.2 ± 0.8 h after oral dosing.
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Discussion |
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). PAI-039 is the second in a series of orally active PAI-1 inhibitors that we have identified by high throughput screening and structure-based medicinal chemical synthesis (Crandall et al., 2004; Elokdah et al., 2004). The screening format was designed to specifically account for the multiple conformations of PAI-1 (Lawrence, 1997) and to identify molecules with higher binding affinity for the active rather than the latent form.
In this article, we report for the first time the effect of PAI-039 on prevention of canine coronary arterial thrombosis after electrolytic injury, a model that has been used extensively in cardiovascular pharmacology laboratories for profiling first-in-class drugs, including antiplatelet agents and thrombin inhibitors (Mickelson et al., 1989; Cook et al., 1999). PAI-039 administration was associated with plasma PAI-1 inhibition, delayed time to occlusive thrombosis, and recanalization of the coronary artery. The quality of flow after recanalization was reduced compared with baseline. However, the highest dose of PAI-039 generated 99 ml of blood flow during recanalization compared with zero recanalization in vehicle-treated controls and the improved coronary flow occurred without associated effects on platelet aggregation or template bleeding. Additionally, the rate of ex vivo clot lysis was accelerated in PAI-039-treated groups, further supporting a drug-mediated inhibition of endogenous PAI-1. Although this is the first report of the effect of pharmacological inhibition of PAI-1 in a large animal model of arterial thrombosis, these results parallel previous observations in PAI-1 knockout mice, which include accelerated clot lysis without associated bleeding (Carmeliet et al., 1993; Farrehi et al., 1998), prolonged time to arterial occlusion (Matsuno et al., 2002), and an improved potency of tPA in arterial recanalization (Zhu et al., 1999). Clot lysis has also recently been used to predict risk for venous thrombosis in a large population-based case-control study, suggesting that PAI-039 could potentially prove beneficial in this disease (Lisman et al., 2005).
PAI-039 prolonged time to occlusive thrombosis and produced spontaneous clot lysis after complete coronary occlusion. Although PAI-1 is synthesized by a number of different tissues, the platelet pool is considered to be the predominant source of PAI-1 during acute thrombotic events (Potter van Loon et al., 1992; Stringer et al., 1994). Despite a delay in time to thrombotic occlusion after treatment with the two highest doses of PAI-039, occlusive thombus formation occurred in all groups. Interestingly, reperfusion was enhanced in the PAI-039-treated groups. This is supported by the coronary blood flow data, because the time to complete occlusion is dose dependently prolonged by PAI-039, but not prevented. The other indices of PAI-1 inhibition, plasma PAI-1 and clot lysis time, were also positively affected by PAI-039 treatment, but with less separation between doses. Because PAI-1 has a short plasma half-life due to the metastable conformation of the active form, PAI-1 inactivation would occur by two additive mechanisms, namely, spontaneous conversion to the latent conformation and pharmacological inhibition by PAI-039. The inactivation of concentrated platelet PAI-1 at the site of the coronary lesion may therefore not be equally reflected by PAI-1 values in the peripheral blood. This hypothesis is supported by recanalization studies in mice reported by Zhu et al. (1999), where an infusion of tPA was more effective in restoring blood flow in the PAI-1 null than wild-type mice, despite producing plasma levels many times that of circulating PAI-1. Additionally, in a recent in vivo study in humans, the regulation of local fibrinolysis was not accurately reflected by peripheral plasma levels of PAI-1 and tPA (Hrafnkelsdottir et al., 2004). Ultimately, these data suggest that a functional endpoint, such as coronary blood flow, is required in addition to biochemical measurements in peripheral blood to truly establish efficacy of PAI-1 inhibitors.
Endogenous PAI-1 inhibition allows active availability of unbound circulating tPA and acceleration of fibrinolysis. Accordingly, the initial clinical indication for a PAI-1 inhibitor would be similar to that of thrombolytic agents, including treatment of acute myocardial infarction and peripheral arterial occlusion (Verstraete, 1998; Armstrong and Collen, 2001). Since the elevation in circulating PAI-1 has been shown to contribute to failure of tPA to stimulate peripheral arterial thrombolysis (Nicholls et al., 2003), the use of a PAI-1 inhibitor together with a lower dose of tPA could prove both effective and reduce the risk of intracerebral hemorrhage and bleeding observed with standard tPA therapy alone. In addition to indications in acute settings, it is important to note that PAI-1 is elevated in chronic diseases such as type 2 diabetes and atherosclerosis (Festa et al., 2002; Sobel et al., 2003), and preclinical studies suggest that PAI-1 inhibition may be beneficial in their treatment (Eitzman et al., 2000, Ma et al., 2004). Finally, PAI-1 is regulated by proinflammatory cytokines, which may further contribute to insulin resistance and vascular risk (Juhan-Vague et al., 2003). Inhibition of PAI-1 may therefore have potential therapeutic benefit in both acute and chronic diseases. The current study provides evidence of preclinical utility in acute coronary thrombosis. Future experiments will focus on the potential beneficial effect of PAI-039 in models of chronic cardiovascular and metabolic disease.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: PAI-1, plasminogen activator inhibitor-1; tPA, tissue plasminogen activator; uPA, urokinase plasminogen activator; PAI-039, {1-benzyl-5-[4-(trifluoromethoxy)phenyl]-1H-indol-3-yl}(oxo)acetic acid; LCX, left circumflex; PT, prothrombin time; APTT, activation partial thromboplastin time; ANOVA, analysis of variance; PRP, platelet-rich plasma; U46619
[GenBank]
, 9,11-dideoxy-11,9-epoxymethanoprostaglandin F2; S-2366, L-pyro-glutamyl-L-prolyl-L-arginine-p-nitroaniline hydrochloride; S-2765, N--benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroaniline-dihydrochloride.
1 Current address: Safety Pharmacology, Schering-Plough Research Institute, Lafayette, NJ. 
Address correspondence to: Dr. James K. Hennan, Wyeth Research, N2252A, P.O. Box 42528, Philadelphia, PA 19101. E-mail: hennanj{at}wyeth.com
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