Disodium disuccinate astaxanthin prevents carotid artery rethrombosis and ex vivo platelet activation.

BACKGROUND/AIMS: The disodium disuccinate derivative of astaxanthin (DDA) is a carotenoid antioxidant under development for the treatment of ischemic cardiovascular events. Recent evidence suggests that reactive oxygen species (ROS) play an important role in platelet activation. This study seeks to investigate the effects of a reactive oxygen species quencher, DDA, in a canine model of carotid artery thrombosis. METHODS: After formation of an occlusive carotid thrombus, dogs were administered recombinant tissue plasminogen activator intra-arterially to achieve thrombolysis in the presence of either 0.9% NaCl solution or DDA (10-50 mg/kg i.v. infusion). Ex vivo platelet aggregation and tongue bleeding times were measured before and after drug administration. Residual thrombus mass was analyzed at the end of each experiment. RESULTS: The data indicated a dose- dependent reduction in the incidence of carotid artery rethrombosis. In addition, platelet aggregation and thrombus weights were dose-dependently inhibited by DDA. No change was recorded in tongue bleeding time among the treatment groups. CONCLUSIONS: The data demonstrate that at the doses used in this study, DDA significantly reduced the incidence of secondary thrombosis while maintaining normal hemostasis. The results suggest that upon further study, DDA may one day find utility in revascularization procedures.

Preclinical evaluation of S18886 in an experimental model of coronary arterial thrombosis.

The specific thromboxane receptor antagonist, S18886, was evaluated for prevention of coronary arterial thrombosis and myocardial ischemia-reperfusion in anesthetized canines. For the primary thrombosis study in left circumflex (LCX) coronary artery, 26 dogs were randomized to receive either vehicle (n = 7) or intravenous S18886 (0.3 mg/kg, n = 6; 1.0 mg/kg, n = 6; and 3.0 mg/kg, n = 7). The respective times to occlusion after S18886 were as follows: 56.8 +/- 9.3, 83.5 +/- 14.9, and 92.4 +/- 15.7 minutes compared to 43.3 +/- 8.2 minutes after vehicle. S18886 caused a minimal increase in tongue bleeding time and a significant decrease in ex vivo platelet aggregation to arachidonic acid or U46619. Another 37 dogs were randomized to receive placebo (n = 12), clopidogrel 1.0 mg/kg p.o. QDX3 (n = 9), clopidogrel + S18886 0.3 (n = 9) or 1.0 (n = 7) mg/kg intravenous. Clopidogrel produced a 50% reduction in adenosine diphosphate-induced platelet aggregation and a slight increase in the time to occlusion. However, clopidogrel + S18886 1.0 mg/kg prevented occlusive thrombus formation in most of the coronary vessels over 6 hours. S18886 did not alter myocardial infarct size in the ischemia-reperfusion model. In conclusion, S18886 alone caused a dose-dependent prolongation in the time to primary occlusive coronary artery thrombosis, whereas S18886 + clopidogrel displayed effective in preventing occlusive thrombus formation with only a moderate increase of tongue-bleeding time.

Effect of sodium/hydrogen exchange inhibition on myocardial infarct size after coronary artery thrombosis and thrombolysis.

This study examines the cardioprotective effects of Na+/H+ exchange inhibition with BIIB-722 or ischemic preconditioning after occlusive thrombus formation and subsequent thrombolysis for reperfusion. Coronary artery thrombosis was induced by vessel wall electrolytic injury. Thrombotic occlusion was maintained for 60 or 90 min in 4 different groups: (1) control; (2) Na+/H+ exchange inhibitor, BIIB-722 (3 mg/kg) before occlusion; (3) BIIB-722 (0.75 mg/kg) before reperfusion; (4) ischemic preconditioning (4 x 5 min). Thrombolysis with intracoronary recombinant tissue plasminogen activator produced reperfusion in 6.3 +/- 1.4 min (average for 68 dogs). After restoration of blood flow, vessel patency was maintained for 4 h with the glycoprotein IIb/IIIa receptor antagonist, BIBU 52ZW. BIIB-722, administered before (26.9 +/- 3.6%) or after (22.0 +/- 2.3%) 60-min ischemia or preconditioning (18.4 +/- 2.8%), produced comparable and significant reductions in infarct size (percent of area at risk) compared to controls (47.2 +/- 2.0%). After 90 min of ischemia, BIIB-722 administered before occlusion (37.3 +/- 1.1%) and ischemic preconditioning (35.0 +/- 4.8%) provided significant cardioprotection compared to control (45.9 +/- 1.8%). BIIB-722 was not cardioprotective when administered during occlusion (48.0 +/- 2.4%). The results indicate that Na+/H+ exchange inhibition and preconditioning provide a comparable degree of cardioprotection against 60 min of regional ischemia. However, when the regional ischemic period is extended to 90 min, the degree of cardioprotection is markedly reduced. Further studies incorporating clinically relevant events such as thrombosis and thrombolysis are required before one can conclude that Na+/H+ exchange inhibition is effective against more prolonged myocardial ischemia.

Microvascular permeabilization and cardiomyocyte injury provoked by myocardial contrast echocardiography in a canine model.

OBJECTIVES: The aim of this research was to evaluate the potential for myocardial contrast echocardiography (MCE) to provoke microscale bioeffects in a canine model. BACKGROUND: Myocardial contrast echocardiography induces bioeffects in rat hearts, but translation of such results to larger animal models is uncertain. METHODS: Dogs were anesthetized and prepared for open- (n = 22) or closed- (n = 6) chest MCE. Evans blue dye was injected intravenously as an indicator of microvascular leakage, and propidium iodide was used to stain for irreversibly injured myocytes in frozen sections. The contrast agent (Definity, Bristol-Myers Squibb Medical Imaging Inc., North Billerica, Massachusetts) was diluted in saline and infused intravenously at 2 microl/kg/min. Myocardial contrast echocardiography in a short-axis (open-chest) or modified four-chamber view (closed-chest) with 1:4 end systolic electrocardiogram triggering was performed at 1.5 MHz for 10 min in a single imaging plane. RESULTS: Petechiae and leakage of Evans blue were observed in the ultrasound scan plane within the anterior left ventricle. For 1.2 MPa and 2.2 MPa, open- or closed-chest MCE, Evans blue content in tissue within the scan plane was significantly greater than in tissue outside this plane. Counts of propidium-iodide-stained nuclei for 2.2 MPa open-chest MCE were also significantly greater inside than outside the scan plane. CONCLUSIONS: In a canine model, MCE induces myocardial injury comparable to that seen in the rodent model.

The antithrombotic effect of melagatran in combination with clopidogrel and/or aspirin (carotid artery primary thrombosis study).

Melagatran with aspirin and/or clopidogrel was evaluated for prevention of arterial thrombosis in a model of vessel wall injury. Thirty-five dogs were randomized to receive placebo (n=14), aspirin (7 to 8 mg/kg, p.o. q12 h for three doses with the last dose administered 12 hours before surgery, n=7), clopidogrel (1 mg/kg p.o. QDx3, n=7), or aspirin+clopidogrel (n=7). The right carotid artery (RCA) was the control vessel, whereas the left carotid artery (LCA) was subjected to injury after administration of Melagatran (0.033 mg/kg i.v.+0.1 mg/kg/h). Clopidogrel, but not aspirin pretreatment, increased time (135.6+/-13.5 vs. 116.1+/-27.8 minutes) to RCA thrombosis versus placebo (88.1+/-10.5 minutes). Melagatran prolonged time to occlusion (min) in the LCA (192.4+/-10.9) versus the placebo-treated RCA (88.1+/-10.5). Addition of Melagatran plus aspirin or clopidogrel prevented formation of occlusive thrombosis, in all LCAs. A two-fold increase in tongue bleeding time was observed after aspirin+Melagatran (178.6+/-14.7 to 347.1+/-87.3 seconds) or clopidogrel+Melagatran (279.9+/-97.3 to 437.1+/-142.5 seconds). However, the combination of aspirin and clopidogrel prevented occlusive thrombosis in the RCA and the subsequent addition of Melagatran did not further increase bleeding time. The combination of Melagatran+aspirin or clopidogrel can reduce formation of occlusive arterial thrombosis without eliciting a significant increase in bleeding-time.

Prevention of carotid artery thrombosis after oral administration of the glycoprotein IIb/IIIa antagonist CRL42796.

This study investigates the effect of the glycoprotein IIb/IIIa receptor antagonist CRL42796 in a canine model of carotid artery thrombosis. Both carotid arteries developed occlusive thrombosis in each of the five control animals (time to occlusion: right carotid artery, 92.6 minutes; left carotid artery, 89.0 minutes). A single oral dose of CRL42796 (3 mg/kg) prevented occlusive thrombosis in 4 of 6 vessels and increased time to thrombosis, albeit not significantly (right carotid artery, 134.1 minutes; left carotid artery, 145.0 minutes). When the initial dose of CRL42796 was followed by a second oral dose (3 mg/kg) 2 hours later, 10 of 10 carotid arteries remained patent throughout the period of electrolytic injury. CRL42796 reduced thrombus weight in both treatment protocols. Ex vivo platelet aggregation with arachidonic acid (AA) or adenosine diphosphate (ADP) was reduced at 120, 240, and 360 minutes after two doses of CRL42796. A single oral dose reduced ADP-induced responses at 240 and 360 minutes, but significant effects were not observed with AA. Bleeding time increased 360 minutes after two oral doses of CRL42796, but not at 120 minutes. Bleeding time was unchanged with the single dose of CRL42796. The results demonstrate that oral administration of CRL42796 prevents carotid artery thrombosis in response to deep vessel wall injury and may have potential value to be characterized in extended preclinical and clinical study.

Glycoprotein IIb/IIIa receptor antagonist (2S)-2-[(2-Naphthyl-sulfonyl)amino]-3-[[2-([4-(4-piperidinyl)-2-[2-(4-piperidinyl)ethyl] butanoyl]amino)acetyl]amino]propanoic acid dihydrochloride (CRL42796), in combination with aspirin and/or enoxaparin, prevents coronary artery rethrombosis after successful thrombolytic treatment by recombinant tissue plasminogen activator.

The antithrombotic effect of the glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist (2S)-2-[(2-naphthyl-sulfonyl)amino]-3-[[2-([4-(4-piperidinyl)-2-[2-(4-piperidinyl)ethyl] butanoyl]amino)acetyl]amino] propanoic acid dihydrochloride (CRL42796), administered alone, or in combination with aspirin, and/or enoxaparin, was examined in a canine left circumflex (LCX) coronary artery rethrombosis model. The electrolytic induction of arterial thrombosis was followed by intracoronary recombinant tissue plasminogen activator administration to achieve thrombolysis, and the adjunctive therapy was initiated 15 min earlier and maintained for 4 h. Thirty-five purpose-bred beagle dogs were randomized to receive one of the following treatments: group 0 (n = 6, placebo); group 1 (n = 6, CRL42796 15 microg/kg i.v. loading dose followed by 0.31 microg/kg/min i.v. infusion), group 2 (n = 6, aspirin 7 mg/kg, administered orally, at -47, -23, -17 h before entry into the experimental protocol); group 3 (n = 6, aspirin + CRL42796); group 4 (n = 6, aspirin + enoxaparin 0.6 microg/kg i.v. loading dose followed by 6.0 microg/kg/min i.v. infusion); and group 5 (n = 5, aspirin + CRL42796 + enoxaparin). The incidence of LCX reocclusion was as follows: group 0, 6/6; group 1, 3/6; group 2, 5/6; group 3, 2/6; group 4, 2/6; and group 5, 0/5. Aspirin pretreatment increased the tongue-bleeding time, whereas the addition of CRL42796 or enoxaparin did not prolong bleeding time to a further degree. However, the combination of the three drugs did increase bleeding time significantly, from 173.9 +/- 19.8 to 620.0 +/- 98.7 s. In conclusion, low-dose CRL42796 together with aspirin and enoxaparin prevented coronary artery rethrombosis, although bleeding time was prolonged. The latter may be of concern in the clinical use of combination therapy.

Prevention of experimental carotid and coronary artery thrombosis by the glycoprotein IIb/IIIa receptor antagonist CRL42796.

1. The antithrombotic effect of the glycoprotein IIb/IIIa receptor antagonist, CRL42796, was examined in canine models of carotid and coronary artery thrombosis. 2. In the carotid artery thrombosis model, occlusion occurred in all control vessels (time to thrombosis 47.6+/-8.9 min). After treatment with low dose CRL42796 (15 microg kg(-1) loading dose +0.31 microg kg(-1) min(-1) i.v.), two of five vessels occluded. Time to thrombosis increased significantly to 155.2+/-23.1 min. When the drug infusion was increased (0.69 microg kg(-1) min(-1)), each of five vessels remained patent. 3. Ex vivo platelet aggregation in response to arachidonic acid (AA) and ADP was examined in platelet rich plasma (PRP) prepared from citrate or heparin anticoagulated blood. CRL42796 reduced platelet reactivity at low and high doses in PRP from citrate anticoagulated blood. However, in PRP from heparin anticoagulated blood, only the higher infusion dose produced a significant reduction in ex vivo platelet responses. 4. A combination of oral aspirin (4.6 mg kg(-1) -41, -17 h) and the low infusion dose of CRL42796 did not produce an additional benefit beyond that provided by CRL42796 alone. 5. Coronary artery thrombosis was inhibited in four of five vessels treated with the lower infusion dose of CRL42796 and in five of five vessels treated with the higher infusion. Time to thrombosis increased with both doses (Control, 90.8+/-10.4 min; low dose, 165.8+/-14.2 min; high dose, >180.0+/-0 min). 6. The results indicate that CRL42796 is an effective in vivo antithrombotic agent against experimentally-induced carotid and coronary artery thrombosis.

Intimatan prevents arterial and venous thrombosis in a canine model of deep vessel wall injury.

Resistance of fibrin-bound thrombin to inactivation by the heparin/antithrombin III complex is considered a limitation in the use of heparin as an antithrombotic agent. Intimatan (dermatan 4,6-di-O-sulfate) is a heparin cofactor II agonist that inhibits both free and bound forms of thrombin. The present study examines the hypothesis that Intimatan prevents thrombotic occlusion in response to vascular wall injury in a canine model of carotid artery/jugular vein thrombosis. The left carotid artery and right jugular vein served as vehicle-treated control vessels, whereas the right carotid artery and left jugular vein were subjected to electrolytic injury after administration of Intimatan (9 mg/kg bolus + 300 microg/kg/min infusion, i.v.) or dalteparin (Fragmin) (400 IU/kg, s.c.). Intimatan significantly increased time to carotid artery (226.0 +/- 14.0 min) and jugular vein (240.0 +/- 0.0 min) thrombosis, compared with control vessels (carotid artery, 87.1 +/- 7.9 min; jugular vein, 60.6 +/- 7.4 min). Vessel patency was maintained in eight of eight jugular veins and seven of eight carotid arteries during treatment with Intimatan. Dalteparin significantly increased time to carotid artery thrombosis (122.1 +/- 17.5 min) compared with control (64.3 +/- 8.2 min), but did not change the time to thrombosis in the jugular vein. Only one carotid artery remained patent at the end of the dalteparin protocol. The two drugs produced minimal increases in bleeding times, and Intimatan increased the activated partial thromboplastin time above that observed with dalteparin. The results demonstrate that Intimatan is effective in preventing occlusive arterial and venous thrombosis in an experimental model of deep vascular wall injury.