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.

Antiplatelet and antithrombotic activity of SL65.0472, a mixed 5-HT1B/5-HT2A receptor antagonist.

The antiplatelet and antithrombotic activity of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-di-hydroquinoline-acetamide), a mixed 5-HT1B/5-HT2A receptor antagonist was investigated on 5HT-induced human platelet activation in vitro, and in rat, rabbit and canine platelet dependent thrombosis models. SL65.0472 inhibited 5-HT-induced platelet shape change in the presence of EDTA (IC50 values = 35, 69 and 225 nM in rabbit, rat and human platelet rich plasma (PRP)), and also inhibited aggregation induced in human PRP by 3-5 microM 5-HT + threshold concentrations of ADP (0.5-1 microM) or collagen (0.3 microg/ml) with mean IC50 values of 49 +/- 13 and 48 +/- 6 nM respectively. SL65.0472 inhibited thrombus formation when given both intravenously 5 min and orally 2 h prior to assembly of an arterio-venous (A-V) shunt in rats as from 0.1 and 0.3 mg/kg respectively. It was active in a rabbit A-V shunt model with significant decreases in thrombus weight as from 0.1 mg/kg i. v. and at 10 mg/kg p.o. The delay to occlusion in an electric current-induced rabbit femoral artery thrombosis model was increased by 251% (p <0.05) after 20 mg/kg p.o. SL65.0472 (30 microg/kg i.v.) virtually abolished coronary cyclic flow variations (7.2 +/- 1.0/h to 0.6 +/- 0.6/h, p <0.05) and increased minimum coronary blood flow (1.2 +/- 0.8 ml/min to 31.8 +/- 8.4 ml/min, p <0.05) in a coronary artery thrombosis model in the anaesthetised dog. Finally, SL65.0472 significantly increased the amount of blood lost after rat tail transection at 3 mg/kg p.o. Thus the anti-5-HT2A component of SL65.0472 is reflected by its ability to inhibit 5-HT-induced platelet activation, and platelet-rich thrombus formation.

Activity of a sub-cutaneously administered novel mixed micellar formulation of argatroban in rat and rabbit models of venous thrombosis.

We studied the antithrombotic activity of a mixed micellar formulation containing 14 mg/ml argatroban administered by the subcutaneous (s.c.) route in rat and rabbit models of venous thrombosis. The effects on bleeding time in the rat tail transection bleeding time test were also studied. In a tissue factor-dependent arterio-venous shunt model, argatroban treatment led to dose-dependent reduction in thrombus weight with an estimated ID50 of 1.8 mg/kg s.c. In the same model, heparin had an estimated ID50 of 179 IU/kg. The antithrombotic activity of argatroban was accompanied by increases in the thrombin and ecarin clotting times but not the aPTT, whereas heparin increased the thrombin time and aPTT but not the ecarin clotting times. Argatroban also inhibited thrombus formation in a rabbit model of thromboplastin + stasis induced thrombosis in the rabbit jugular vein with an estimated ID50 of 1 mg/kg s.c. When tested in the rat tail transection bleeding time test, the mixed micellar formulation of argatroban caused significant increases in the bleeding time as from 8 mg/kg s.c., while heparin significantly increased the bleeding time at 800 U/kg. Mixed micellar argatroban appears to have a superior safety margin to heparin in terms of antithrombotic efficacy and bleeding risk. Thus, a mixed micellar formulation of argatroban, which markedly enhances its solubility, could be useful as a potential antithrombotic agent for subcutaneous administration.

Antithrombotic activity of argatroban in experimental thrombosis in the rabbit.

Argatroban was evaluated in three models of thrombosis in the rabbit: the Wessler model (thromboplastin plus stasis of the left jugular vein), an arteriovenous shunt model, and a model of arterial thrombosis induced by endothelial and intimal damage of the left femoral artery. Calcium heparin was used as a comparator. Both substances inhibited thrombus formation in the Wessler model with ID50 values of 0.32 and 0.16 mg/kg intravenous bolus for argatroban and heparin respectively, with similar changes in thrombin time (4 to 5 times control) and activated partial thromboplastin time (APTT) (1.6 to 2.1 times control) for both substances at antithrombotic doses. The ID50 values of both substances were 2.4 micrograms/kg/min (argatroban) and 0.5 microgram/kg/min (heparin). When they were administered by continuous infusion, no significant effects on the APTT were noted. In the arteriovenous shunt, the ID50 values for argatroban and heparin (respectively) were 0.16 and 0.07 mg/kg intravenous bolus, and 4.5 and 2.8 micrograms/kg/min intravenous infusion. Vessel clamping followed immediately by electrical stimulation (5 mA direct current, 5 minutes) of the left femoral artery leads to the formation of an occlusive thrombus approximately 30 minutes after clamping. Argatroban infused for 60 minutes before the vascular lesion and throughout the 90 minute observation period led to a dose-dependent delay in arterial occlusion with significant effects seen at 5 micrograms/kg/min with five of eight animals showing normal femoral blood flow at 90 minutes postlesion at 20 micrograms/kg/min; no significant increases (Dunnett's test) in APTT were noted with argatroban. Heparin was without effect even at 40 micrograms/kg/min, despite an eight-fold increase in APTT at 20 micrograms/kg/min and values of more than 300 seconds at 40 micrograms/kg/min. Thus, in models of arterial but not venous thrombosis, argatroban is a more potent antithrombotic agent than heparin on a weight basis, with antithrombotic effects at a much lower degree of systemic anticoagulation.

Antithrombotic actions of argatroban in rat models of venous, 'mixed' and arterial thrombosis, and its effects on the tail transection bleeding time.

1. The antithrombotic action of argatroban, a synthetic thrombin inhibitor, was studied in three models of thrombosis in the rat, and in the tail transection bleeding time test. Heparin was studied as a reference anticoagulant. 2. In the model of venous thrombosis induced by thromboplastin followed by stasis of the abdominal vena cava, argatroban had an ED50 of 125 micrograms kg-1, when administered as an i.v. bolus 5 min prior to the thromboplastin injection: the ED50 of heparin was 42 micrograms kg-1, where ED50 is the dose which reduces the weight of the thrombus by 50% compared with that of the control animals. When the two compounds were administered by continuous i.v. infusion, argatroban (ED50 = 1.5 micrograms kg-1 min-1) had the same potency as heparin (ED50 = 1.2 micrograms kg-1 min-1). 3. Argatroban was active in the arterio-venous shunt model with an ED50 of 0.6 mg kg-1 when the compound was given as a bolus. The ED50 of heparin was 0.04 mg kg-1 under the same conditions. The two compounds had ED50 values of 6 micrograms kg-1 min-1 (argatroban) and 3 micrograms kg-1 min-1 (heparin), when administered by continuous i.v. infusion. 4. When tested against occlusive arterial thrombus formation by electrical stimulation of the left carotid artery, both compounds given as either an i.v. bolus or a continuous infusion led to dose-dependent increases in the duration of post-lesion vessel patency. Heparin bolus was more active than argatroban on a weight basis, in that 2 mg kg-1 gave a similar increase in the time to occlusion as 8 mg kg-1 argatroban. As in the other models, when given as continuous infusions, argatroban (111% increase in time to occlusion at 20 tg kg-1, min-1) had similar activity to that of heparin (180% increase at 25 jg kg-1 min-1) on a weight basis. Hoever, the antithrombotic effects of argatroban were accompanied by only moderate changes in the coagulation parameters (thrombin time and activated partial thromboplastin time, APTT), whereas, even at a subthreshold dose of heparin (12.5 pg kg-1 min-1), both the thrombin time and the APTT were greater than 150 s.5. Infusions of both compounds caused dose-dependent increases in the tail transection bleeding time,with the dose of argatroban that doubles the bleeding time (11 I g kg-1 min-1) being five times greater than that of heparin (EDI, = 2.2 fig kg-1 min-1).6. These data show that, when administered as an intravenous infusion, argatroban is a potent antithrombotic agent in rat models of venous 'mixed' and arterial thrombosis, this effect can be obtained with a lower degree of systemic anticoagulation than with heparin in the arterial model, and argatroban has a lower haemorrhagic potential than that of heparin.