Charles Kasiewski

Restenosis Following Angioplasty in the Swine Coronary Artery Is Inhibited By an Orally Active PDGF-Receptor Tyrosine Kinase Inhibitor, RPR101511A

BACKGROUND Platelet-derived growth factor (PDGF), a purported mediator of arterial response to injury, stimulates proliferation, chemotaxis, and matrix production by activation of its membrane receptor tyrosine kinase. Because these activities underlie restenosis, inhibition of the PDGF-receptor tyrosine kinase (PDGFr-TK) is postulated to decrease restenosis. METHODS AND RESULTS RPR101511A is a novel compound which selectively and potently inhibits the cell-free and in situ PDGFr-TK and PDGFr-dependent proliferation and chemotaxis in vascular smooth muscle cells (VSMC). To evaluate the effect of RPR101511A (30 mg. kg-1. d-1 BID for 28 days following PTCA) on coronary restenosis, PTCA was performed in hypercholesterolemic minipigs whose left anterior descending (LAD) coronary artery had been injured by overdilation and denudation, yielding a previously existing lesion. Angiographically determined prePTCA minimal lumen diameters (MLD) were similar in vehicle and RPR101511A-treated pigs (1.98+/-0.09 versus 2.01+/-0.08 mm) and increased to the same extent in the 2 groups following successful PTCA (2.30+/-0.06 versus 2.52+/-0.13). At termination, there was an average 50% loss of gain in the vehicle-treated group but no loss of gain with RPR101511A (2.16+/-0. 05 versus 2.59+/-0.11, P<0.001). Morphometric analysis of the LAD showed that RPR101511A caused a significant decrease in total intimal/medial ratio (0.96+/-0.58 versus 0.67+/-0.09, P<0.05). CONCLUSIONS RPR101511A, which acts by inhibition of the PDGFr-TK, completely prevented angiographic loss of gain following PTCA and significantly reduced histological intimal hyperplasia.

Synthesis, SAR and in vivo activity of novel thienopyridine sulfonamide pyrrolidinones as factor Xa inhibitors

Thienopyridine sulfonamide pyrrolidinones were found to be potent and selective inhibitors of the coagulation cascade enzyme factor Xa. SAR studies led to several compounds that were selected for further in vivo investigation. These novel aryl binding pocket moieties represent a structural modification to a series of fXa inhibitors. Several compounds proved to be efficacious i.v. antithrombotic agents.

Inhibition of Repetitive Thrombus Formation in the Stenosed Canine Coronary Artery by Enoxaparin, But Not by Unfractionated Heparin

Experiments were designed to compare the antithrombotic efficacy of enoxaparin and unfractionated heparin (UH) in a model of platelet-dependent cyclic flow reductions (CFRs) in the stenosed canine circumflex coronary artery. Low-molecular-weight heparins (LMWHs) are safe and effective in the prevention and treatment of venous thromboembolism. The present experiments were designed to evaluate the potential use of LMWHs in arterial thrombotic indications by comparing the antithrombotic effect of an LMWH with that of UH in an animal model of unstable angina. After establishment of consistent CFRs by experimentally induced vascular stenosis and damage, vehicle (saline), enoxaparin, or UH was administered intravenously as a loading dose plus a continuous infusion for 1 hour. The inhibition of CFRs was taken as an indicator of antithrombotic efficacy. Enoxaparin inhibited repetitive platelet thrombus formation in a dose-dependent manner, with significant inhibition of CFRs achieved at 0.5 mg/kg + 5 microg/kg per minute. This dose of enoxaparin resulted in anti-Xa levels of 0.9 to 1.0 IU/mL, anti-IIa levels of 0.2 to 0.3 IU/mL, activated partial thromboplastin time (APTT) of 1.3-fold over baseline, and a 1.4-fold increase (NS) in template bleeding time. Near-complete abolishment of CFRs was achieved with enoxaparin at 1.0 mg/kg + 10 microg/kg per minute. This dose of enoxaparin produced anti-Xa levels of 2 to 2.2 IU/mL, anti-IIa levels of 0.5 to 0.6 IU/mL, an increase in APTT of 1.4- to 1.5-fold over baseline, and a 1.9-fold increase (P<0.05) in template bleeding time. In contrast, UH had no significant effect on CFRs at a dose (100 U/kg + 10 U/kg per minute) that resulted in anti-Xa levels of 1.2 to 1.6 IU/mL, anti-IIa levels of 1.8 to 2.4 IU/mL, an increase in APTT greater than 10-fold over baseline, and a 2.5-fold increase (P<0.05) in template bleeding time. Compared with the vehicle group, circulating platelet count and hematocrit were not changed significantly by any dose of enoxaparin or UH tested. Enoxaparin, unlike UH, prevented repetitive platelet-dependent thrombus formation in the dog, thereby supporting the potential use of enoxaparin as a replacement for heparin in the treatment of arterial thrombotic disorders such as unstable angina.

Pharmacodynamic activity and antithrombotic efficacy of RPR120844, a novel inhibitor of coagulation factor Xa.

These studies were designed to examine the pharmacodynamic profile and antithrombotic efficacy of RPR120844, a competitive inhibitor of coagulation factor Xa, with a K(i) of 7 nM against human factor Xa. In vitro, RPR120844 doubled activated partial thromboplastin time (APTT) at concentrations of 1.54, 1.48, and 0.74 microM in plasma obtained from humans, dogs, and rats, respectively. Intravenous bolus administration of RPR 120844 at 0.3, 1, and 3 mg/kg to rats resulted in maximal increases in APTT of 1.8-, 2.6-, and 8.4-fold over baseline, respectively. The effect on prothrombin time (PT) was less pronounced, resulting in a 4.4-fold increase at 3 mg/kg. These effects were rapidly reversible; APTT and PT returned to control values by 30 min after dosing. Intragastric administration to rats at 50, 100, and 200 mg/kg resulted in modest increases in APTT and PT of 1.5- and 1.3-fold over baseline at the highest dose. Plasma levels were estimated by anti-Xa activity by using an amidolytic, chromogenic assay. Plasma levels were 0.65, 1.29, and 2.45 microM at 30 min after dosing at 50, 100, and 200 mg/kg, respectively. Intravenous administration to dogs at 0.1 and 0.3 mg/kg produced maximal increases in APTT of 1.7- and 2.4-fold over baseline, respectively. Intragastric administration to dogs at 50 mg/kg resulted in maximal increases in APTT and PT of 1.7- and 1.1-fold over baseline, with peak plasma levels of 3.9 microM observed at 15 min after dosing. In a rat model of FeCl2-induced carotid artery thrombosis, RPR120844 (3 mg/kg, i.v. bolus + 300 microg/kg/min constant infusion; n = 4) significantly increased time-to-occlusion from 18+/-1 min (vehicle, n = 4) to 60 min (maximal observation time) and reduced thrombus mass from 5.5 +/- 0.2 mg (vehicle) to 1.4 +/- 0.2 mg. These results indicate that RPR120844 is a potent, selective inhibitor of Xa that exhibits oral activity and is efficacious in a standard model of arterial thrombosis.

O-benzyl hydroxyproline as a bioisostere for Phe-Pro: Novel dipeptide thrombin inhibitors

Abstract A series of analogs were prepared based on the known thrombin inhibitor PPACK, in which the D-Phe-Pro dipeptide has been replaced by trans-4-O-benzyl hydroxyproline. One of these analogs is a more potent inhibitor of thrombin, and is more selective, than PPACK itself.

Pharmacology of RG W-2938 : a cardiotonic agent with vasodilator activity

Summary The cardiovascular effects of RG W-2938, 6-[6-(3, 4-dihydro-3methyl-2(1H)-2-oxoquinazolinyl)]-4, 5-dihydro-3(2H)-pyridazinone, a new nonglycoside, non-catecholamine cardiotonic/vasodilator agent were examined in vivo in anesthetized and conscious dogs and in vitro in isolated guinea pig hearts; in the latter, RG W-2938 5 nmol-5 μmol increased contractility in a doserelated fashion. RG W-2938 30–300 μg/kg administered intravenously (i.v.) to anesthetized dogs increased contractile force while decreasing arterial pressure and total peripheral resistance (TPR) in a dose-related manner. Heart rate (HR) was only slightly increased, and aortic flow was not appreciably altered. A single oral dose of RG W-2938 0.3 mg/kg administered to conscious chronically instrumented dogs produced a marked and sustained increase in contractility 15–240 min after treatment while only slightly increasing HR. The effects of RG W-2938 30–300 μg/kg, i.v. were studied in a mecamylamine-propranplol-induced model of heart failure. RG W-2938 effectively reversed the drug-induced heart failure by increasing myocardial contractility and decreasing arterial pressure while only slightly affecting HR. These studies show that RG W-2938 is an orally effective positive inotropic/vasodilator agent.

Anti-thrombotic activity of RG13965, a novel platelet fibrinogen receptor antagonist

RG13965, a pseudotetrapeptide analogue of Arg-Gly-Asp (RGD), inhibited collagen-induced dog, monkey, human, hamster, mouse, and pig platelet aggregation in vitro with IC50 values of 3.7, 4.6, 6.3, 126, 136 and 1600 microM, respectively. RG13965 (3, 10, and 30 mg/kg, i.v.) decreased the incidence of collagen/epinephrine-induced thrombosis in mice from 90% in untreated animals to 63, 37, and 0%, respectively. In hamsters, RG13965 (10 and 30 mg/kg, i.v.) prolonged the time required for formation of a hemostatic plug in severed mesenteric arteries by 1.6- and 3.6-fold, respectively. In a canine model of repetitive platelet thrombus formation in the coronary artery, RG13965 (0.1, 0.3, and 1 mg/kg, i.v.) reversibly inhibited cyclic flow reductions (CFRs) and inhibited ADP-induced ex vivo platelet aggregation by 29, 57, and 77%, respectively. RG13965 (1 mg/kg) completely inhibited CFRs for at least 40 min. Platelet count was not altered at any dose and template bleeding time was prolonged modestly (1.8-fold) at only the highest dose. RG13965 dose-dependently and reversibly inhibited thrombus formation at doses which did not completely inhibit ex vivo platelet aggregation and only modestly prolonged template bleeding time.

Role of short-term inhibition of factor Xa by FXV673 in arterial passivation: a study in a chronic model of thrombosis in conscious dogs.

Summary: Factor Xa (fXa) plays a pivotal role in the activation of the coagulation system during thrombosis, but, unlike GPIIb/IIIa receptor antagonists, the role of fXa inhibition in arterial passivation is not well defined. We compared the long‐term antithrombotic efficacy of a direct fXa inhibitor, FXV673, and heparin after short‐term infusion in conscious dogs. Dogs were instrumented surgically to induce carotid artery thrombosis by electrolytic injury. On day 1, dogs received a 3‐h infusion of placebo (n = 10), FXV673 (100 &mgr;g/kg + 10 &mgr;g/kg/min, n = 7), or heparin (60 U/kg + 0.7 U/kg/min, n = 7). Injury (100 &mgr;A) was initiated concomitantly for 1 h. The procedure was repeated on day 2 with injury of 200 &mgr;A for 3 h. Carotid artery blood flow (CBF) and coagulation parameters were monitored continuously for 3 h on days 1 and 2 and for 30 min on days 3, 4, and 5. On day 1 at 3 h, CBF in the placebo‐treated group was 26% of baseline with 70% incidence of occlusion. None of the vessels occluded in the heparin and FXV673 groups; however, the CBF was significantly higher in the FXV673 group (92 ± 8 ml/min versus 39 ± 12 ml/min). Before injury on day 2, CBF recovered in all groups to 71‐89% of baseline. After the second injury, all vessels in the placebo‐treated group progressed to complete occlusion by 3 h. CBF was significantly higher in FXV673 group compared with heparin throughout the 3‐h period. On days 3, 4, and 5 the placebo‐treated vessels remained occluded, but the CBF in the heparin group was 33 ± 20 ml/min, 55 ± 11 ml/min and 68 ± 12 ml/min, respectively, compared with 84 ± 10 ml/min, 98 ± 7 ml/min, and 99 ± 10 ml/min in the FXV673 group. The arterial thrombus mass was significantly lower in FXV673 group (13 ± 4 mg) compared with placebo (103 ± 10 mg) and heparin (44 ± 11 mg). In summary, these data demonstrate that short‐term infusion of FXV673 was associated with long‐term efficacy that was superior to standard heparin and underscore the role of direct fXa inhibition in arterial passivation.

Electrode cuff-induced changes in DNA and PDGF gene expression in the rat carotid artery

Low current (0.25, 3 mA) stimulation through a miniature electrode cuff encased around the carotid artery of the rat was used to induce intimal hyperplasia, an important feature of the atherosclerotic plaque and a phenomenon limiting the long term success of angioplasty. Compared to contralateral unstimulated arteries, 11-14 days of daily transmural stimulation of cuffed arteries (20 min period) significantly increased the amount of extracted DNA (diphenylamine colorimetric assay). Low current (0.25 mA) was as effective as 3 mA in producing an increase in extractable DNA. The cuff alone without applied current also stimulated an increase in DNA content but to a smaller degree than in arteries receiving current. Infusion of a calcium channel antagonist, diltiazem, at a dose which achieved therapeutic drug levels, significantly reduced the amount of electrode cuff-induced DNA content but had no effect on the increase in DNA induced by the presence of the cuff without applied current. Gene expression of PDGF-A chain, PDGF-B chain and PDGF-beta receptor (beta r) (Northern analysis of extracted carotid RNA) increased within 4 h after electrical stimulation with 3 mA. Lower current (0.25 mA) and the presence of the cuff also enhanced PDGF gene expression but with a delayed onset of several days. The pattern of gene expression for PDGF ligands and beta r during the 11-14 days of stimulation differed, but each remained above contralateral control levels. It is concluded that the continued coexpression of PDGF and one of its receptors may contribute to induced hyperplastic changes.