SSR182289A enhances thrombolysis induced by fibrinolytic agents in rabbit models of venous and arterial thrombosis.

BACKGROUND AND OBJECTIVES: The purpose of this work was to investigate whether thrombolysis induced by recombinant tissue plasminogen activator (rt-PA) or streptokinase (SK) was enhanced in different rabbit models of thrombolysis by SSR182289A, a novel synthetic direct thrombin inhibitor which has been shown to possess potent antithrombotic properties in several experimental animal models. METHODS AND RESULTS: Human rt-PA alone (0.125 mg kg(-1) h(-1) for 2 h) induced a significant thrombolysis (18%, P < 0.05) of a venous-type thrombus in the rabbit jugular vein. Under these conditions, SSR182289A (3 mg kg(-1) i.v. bolus) inhibited 125I-fibrin accretion onto a preformed thrombus in the rabbit jugular vein by 72%, but was unable to induce thrombolysis on its own. However, coadministration of SSR182289A and rt-PA strongly enhanced rt-PA-induced thrombolysis (38.4%, P < 0.01). The effect of SSR182289A was further assessed in a model of thrombolysis of an electrical injury-induced, stable (occlusion duration > 2 h) thrombus formed in the rabbit femoral artery. Whereas local intra-arterial infusion of high doses of SSR182289A (3 mg kg(-1) h(-1) for 1 h) alone was able to restore flow, SK (12,000 U kg(-1) h(-1)) and a low dose of SSR182289A (0.3 mg kg(-1) h(-1)) were ineffective. However, intra-arterial coadministration of SSR182289A (0.3 mg kg(-1) h(-1)) and SK (12,000 U kg(-1) h(-1)) induced significant reflow (time to reflow was shortened by 34.7 +/- 7.5 min, P < 0.05). In the same model, systemic i.v. administration of high doses of SSR182289A (10 mg kg(-1) i.v. bolus) and rt-PA (1 mg kg(-1) h(-1)) alone did not induce any thrombolysis. However, the association of both compounds quickly (30 +/- 6 min) restored and maintained flow (duration > 2 h) in all animals. CONCLUSIONS: The present results show that bolus i.v. injection of SSR182289A is able to potentiate thrombolysis induced by two fibrinolytic agents whether the thrombus is of venous or arterial origin, thus suggesting that SSR182289A may be of use as an adjunct to thrombolysis.

Antithrombotic properties of SSR182289A, a new, orally active thrombin inhibitor.

N-[3-[[[(1S)-4-(5-Amino-2-pyridinyl)-1-[[4-difluoromethylene)-1-piperidinyl]carbonyl]butyl]amino]sulfonyl][1,1'-biphenyl]-2-yl]acetamide hydrochloride (SSR182289A) is a novel, potent, and selective thrombin inhibitor. We have examined the antithrombotic properties of SSR182289A administered by i.v. and p.o. routes in several different animal thrombosis models in comparison with reference antithrombotic agents. Oral administration of SSR182289A produced dose-related antithrombotic effects in the following models; rat venous thrombosis (ED(50) 0.9 mg/kg p.o.), rat silk thread arterio-venous (AV) shunt (ED(50) 3.8 mg/kg p.o.), rat thromboplastin-induced AV shunt (ED(50) 3.1 mg/kg p.o.), rat carotid artery thrombosis (ED(200) 5.9 mg/kg p.o.), and rabbit venous thrombosis (ED(50) 7.5 mg/kg p.o.). Administered as an i.v. bolus, SSR182289A showed antithrombotic activity in the above models with ED(50)/ED(200) values in the range of 0.2 to 1.9 mg/kg i.v. SSR182289A increased rat tail transection bleeding time at doses > or =10 mg/kg p.o. In the rat thromboplastin-induced AV shunt model, SSR182289A 10 mg/kg p.o. produced marked antithrombotic effects at 30, 60, 120, and 240 min after administration. Hence, SSR182289A demonstrates potent oral antithrombotic properties in animal venous, AV-shunt, and arterial thrombosis models.

Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of E1-deleted adenovirus vectors.

Replication-defective E1-deleted adenoviruses are attractive vectors for gene therapy or live vaccines. However, manufacturing methods required for their pharmaceutical development are not optimized. For example, the generation of E1-deleted adenovirus vectors relies on the complementation functions present in 293 cells. However, 293 cells are prone to the generation of replication competent particles as a result of recombination events between the viral DNA and the integrated adenovirus sequences present in the cell line. We report here that human lung A549 cells transformed with constitutive or inducible E1-expression vectors support the replication of E1-deficient adenoviruses. E1A transcription was elevated in most of the cell lines, and E1A proteins were expressed at levels similar to those of 293 cells. However, the levels of expression of E1A did not correlate with the efficiencies of complementation of E1-deleted viruses in A549 clones, since some clones complemented replication in the absence of induction of E1A expression. In addition, complementation of E1-deficient adenoviruses did not require expression of the E1B 55-kDa protein. Although these cell lines contain the coding and cis-acting regulatory sequences of the structural protein IX gene, they are not able to complement viruses in which this gene has been deleted. In contrast to 293 cells, such new complementation cell lines do not contain the left end of the adenoviral genome and thus represent a significant improvement over the currently used 293 cells, in which a single recombination event is sufficient to yield replication competent adenovirus.

Low doses of endotoxin potentiate venous thrombosis in the rabbit.

Procoagulant activity of the endothelium and of monocytes was induced by an intravenous administration of a low dose of endotoxin in the rabbit. Venous thrombosis induced by a 45-min stasis of the jugular vein was dramatically increased after the administration of endotoxin. Endotoxin enhanced venous thrombosis in a dose-dependent manner which was the highest after the administration of 1 microgram/kg. This effect increased with time but was optimal when endotoxin was injected 4 h before stasis induction. Leukocytes appear to play a key role in this process since mechlorethamine-induced leukopenia totally inhibited thrombus formation. Hirudin and heparin, administered intravenously 5 min before stasis, were both potent inhibitors of endotoxin-induced venous thrombosis exhibiting ED50 values of 3.8 +/- 1.0 and 2.3 +/- 1.1 microgram/kg, respectively.

Potentiating effect of clopidogrel and SR 46349, a novel 5-HT2 antagonist, on streptokinase-induced thrombolysis in the rabbit.

Current thrombolytic strategies have a number of important shortcomings including resistance to recanalization and development of acute reocclusion. The purpose of this study was to investigate whether the lysis of venous thrombi by streptokinase could be enhanced by SR 46349, a novel 5-HT2 receptor antagonist, or clopidogrel, an analogue of ticlopidine. The activity of these compounds was evaluated by following the lysis of radiolabelled fibrin under a continuous infusion of streptokinase (4,000 IU kg-1 h-1 over 4 h). Streptokinase alone induced 42% thrombolysis when compared to saline. The i.v. co-administration of SR 46349 or clopidogrel (10 mg/kg) enhanced significantly streptokinase-induced thrombolysis. Thrombolysis measured by [125I]-fibrinogen lysis increased to 65 and 59% respectively. This efficacy was achieved without additional prolongation of the template bleeding time observed with streptokinase alone. Thus, the concomitant use of a 5-HT2 receptor antagonist or an anti-ADP agent during streptokinase therapy may facilitate clot lysis.

A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII.

We have constructed new B domain deletion derivatives of human factor VIII (FVIII) by manipulating the cDNA using recombinant DNA techniques. One of these new derivatives, FVIII delta II, in which amino acids 771(pro)-1666(asp) have been deleted, no longer contains the protease cleavage site at amino acid position 1648(arg)-1649(glu) known to be involved in the initial step of FVIII processing. We have expressed this molecule in both baby hamster kidney (BHK) 21 cells using the vaccinia virus (VV) expression system and have established Chinese hamster ovary (CHO) derived permanent cell lines expressing either recombinant (r)FVIII or FVIII delta II. The characteristics of FVIII delta II have been compared to those of rFVIII and/or plasma derived (pd) FVIII. FVIII delta II has the following properties: (i) it exhibits FVIII procoagulant activity; (ii) it is expressed at 5-fold higher levels than is the complete molecule in comparable systems; (iii) it migrates for the most part as a single major band on SDS-PAGE, in contrast to the complete molecule; (iv) it is activated to a greater extent by thrombin than is either rFVIII or pdFVIII; and (v) it retains the ability to bind von Willebrand factor (vWf).