Antithrombotic effect of LB-30057 (CI-1028), a new synthetic thrombin inhibitor, in a rabbit model of thrombosis: comparison with inogatran.

LB-30057 (CI-1028) is a novel, orally bioavailable, direct thrombin inhibitor with a Ki of 0.38 nM against human thrombin. The effects of LB-30057 on thrombus formation and hemostasis were evaluated in a veno-venous shunt model of thrombosis in rabbits, and compared with inogatran, another direct inhibitor of thrombin. Each compound was studied at 5 or 6 different doses with 5 or 6 rabbits in each group. After administration as a bolus i.v. injection followed by continuous infusion, both LB-30057 and inogatran dose-dependently inhibited thrombus formation, which was measured as an increase in time to occlusion (TTO) and a decrease in thrombus weight. Both compounds also improved vena caval blood flow and reduced the overall incidence of thrombotic occlusion. LB-30057 significantly prolonged TTO from 23 +/- 4 min (before dose) to 110 +/- 10 min at the highest dose (0.7 mg/kg + 47 microg/kg/min) (p < 0.001), and reduced thrombus weight from 57 +/- 2 mg to 15 +/- 5 mg (p < 0.001). Occlusive thrombus formed in only one of six rabbits that received the highest dose of LB-30057 (vs. 13/13 in the control group, p < 0.01). At the dose that produced the maximum antithrombotic effect (0.7 mg/kg + 47 microg/kg/min), LB-30057 increased aPTT and bleeding time approximately 2-and 2.5-fold above baseline, respectively. On a gravimetric basis, LB-30057 and inogatran displayed comparable in vivo antithrombotic efficacy. When compared to equally effective anti thrombotic doses of inogatran, LB-30057 caused less prolongation in aPTT, had no effect on PT, and tended to have less of effect on bleeding time. These results indicate that LB-30057 is an effective antithrombotic compound and it appears to have a better benefit/risk profile than inogatran in this experimental model.

A novel model of venous thrombosis in the vena cava of rabbits.

The objective of this study was to develop and validate a new experimental model of venous thrombosis in the rabbit. A 3-cm length of siliconized PE tubing was used as a veno-venous shunt inserted into the abdominal vena cava of anesthetized rabbits. The PE tubing contained six cotton threads which helped to restrict blood flow through the tubing and served as a foreign, thrombogenic surface upon which a thrombus could develop. By continuously measuring blood flow through the vena cava, the rate of thrombus development can be monitored until zero flow is achieved indicating that a completely occlusive thrombus is present. The shunt can be removed making it possible to weigh the thrombus and/or determine its composition. A second shunt can be placed in the vena cava to make a second determination of time to occlusion and thrombus weight, using the data from the first shunt as an internal control standard for comparison. Reproducibility of the technique was demonstrated in a control group (n = 7) in which two successive shunts were used without an antithrombotic intervention. In studies with the first and second shunts, time to occlusion averaged 20.6+/-5.2 min and 20.2+/-5.7 min (pNS), respectively. The net thrombus weights (less the wet weight of the cotton threads) were 49.0+/-3.5 mg and 47.0+/-3.3 mg (pNS). Histologic examination of the thrombi indicated that they were largely composed of fibrin and red blood cells, consistent with the characteristics of venous thrombi. The low molecular weight heparin (LMWH) enoxaparin was used as an antithrombotic intervention to validate the model. Dose-dependent changes in time to occlusion and thrombus weight were achieved which paralleled alterations in coagulation parameters (thrombin time and activated partial thromboplastin time) and bleeding time determined with an ear bleeding technique. The veno-venous shunt model is easy to use, reproducible, and responds appropriately to an antithrombotic intervention, indicating that it should be useful for experimental evaluation of antithrombotic agents designed for venous thromboembolic disorders.

Effects of endothelin ETA receptor antagonism with PD 156707 on hemodynamics and renal vascular resistance in rabbits.

The objective of this study was to determine the in vivo effectiveness of the selective endothelin ETA receptor antagonist PD 156707 (sodium 2-benzo[1,3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4,5-trimet hoxy- benzyl)-but-2-enoate). Effectiveness was defined by the ability of the compound to block increases in renal vascular resistance and mean arterial blood pressure induced by an intravenous bolus of 0.3 nmol/kg of human endothelin-1 in pentobarbital anesthetized rabbits. Different groups of rabbits received hour long intravenous infusions of PD 156707 at doses of 0.003, 0.01, 0.03 or 0.3 mg/kg per h. During baseline conditions, mean arterial blood pressure, heart rate, renal blood flow, and renal vascular resistance were similar among the groups. The intravenous bolus of endothelin-1 significantly decreased mean arterial blood pressure (82 +/- 3 mmHg to 65 +/- 3 mmHg, P < 0.05) and increased renal vascular resistance (2.8 +/- 0.3 mmHg/ml per min to 9.2 +/- 1.1 mmHg/ml per min, P < 0.05) in untreated control animals. At doses of 0.3 and 0.03 mg/kg per h, PD 156707 virtually abolished endothelin-1 induced increases in renal vascular resistance, but did not affect the endothelin-1 induced decrease in mean arterial blood pressure. At 0.01 and 0.003 mg/kg per h, PD 156707 also inhibited endothelin-1 induced increases in renal vascular resistance but the effects were less striking, leading to the conclusion that the minimum effective intravenous dose of the compound in rabbits is in the range of 0.01-0.03 mg/kg per h. The results of this study demonstrate that PD 156707 is an extremely potent and highly selective endothelin ETA receptor antagonist. In addition, this study demonstrates the utility of renal vascular resistance as an in vivo bioassay for evaluating the selective vascular effects of endothelin receptor antagonists in this species.

Heat stress increases cardiac HSP72i but fails to reduce myocardial infarct size in rabbits 24 hours later.

Recent reports suggest that delayed myocardial protection ("second window of preconditioning") occurs 24 hours after brief ischemic or thermal stress. In order to test this hypothesis, we subjected New Zealand White rabbits to a heating regimen (42 degrees C for 15-20 minutes). Twenty four hours later, the effect of heat stress on infarct size was determined by conducting a 30 minute ischemia/3 hour reperfusion protocol. In a separate group of rabbits, Western blot analysis was used to verify that the heating regimen increased expression of HSP72i. The size of the region at risk was delineated by infusion of Unisperse blue and infarcted myocardium was identified by incubation of left ventricular slices in triphenyl tetrazolium chloride. In contrast to expectations, induction of HSP72i with thermal stress was not effective in limiting infarct size in rabbits 24 hours later, calling into question the concept that heat stress induces delayed or "second window" myocardial protection.

Myocardial infarct size is smaller in dogs with pacing-induced heart failure.

OBJECTIVE: Determine if ischemic tolerance is reduced in the setting of experimental heart failure (HF). METHODS: Dogs were paced for 3 weeks at 240 BPM to induce heart failure which was confirmed with hemodynamic and echocardiographic measurements. The pacemaker was turned off 30 min prior to the ischemia study. Normal (n = 9) and HF dogs (n = 12) were anesthetized with sodium pentobarbital, instrumented for cardiovascular assessment through a left lateral thoracotomy, and myocardial blood flow was measured with radioactive microspheres. The left circumflex (LCX) artery was occluded for 90 min followed by 3 h of reperfusion. Infarct size was determined with triphenyl tetrazolium chloride staining. RESULTS: Two-dimensional echocardiograms were obtained before and after 3 weeks of pacing in the HF group. Ejection fraction was reduced from 67 +/- 1 to 32 +/- 2% (P < 0.001) and left ventricular end-diastolic volume (LVEDV) increased from 29 +/- 4 ml before pacing to 47 +/- 5 ml (P < 0.001). HF dogs were characterized by a smaller peak positive dP/dt (1110 +/- 72 vs. 2546 +/- 41 mmHg/s, P < 0.01), a greater LV end-diastolic pressure (34 +/- 3 vs. 9 +/- 2 mmHg, P < 0.01), and lower LV end-systolic pressure (99 +/- 5 vs. 130 +/- 5 mmHg, P < 0.05) compared to control dogs. Heart rate was not significantly different between the two groups throughout the experiment. More HF dogs died from ventricular fibrillation (4/12) than control dogs (1/9), but this difference was not statistically significant (P > 0.2). The LCX occlusion produced a comparable decrease in blood flow in HF and normal dogs (0.08 +/- 0.01 vs. 0.09 +/- 0.01 ml/min/g), but infarct size as a percentage of the region at risk was smaller in HF dogs compared to normal dogs (21 +/- 4 vs. 45 +/- 4%, P < 0.01). Region at risk size was also smaller in HF versus normal dogs (29 +/- 3 vs. 40 +/- 2%, P < 0.05). Accordingly, a subgroup analysis of 6 HF and 5 control dogs with similar RAR sizes (35 +/- 2% vs. 37 +/- 2%) was performed and it also demonstrated that infarct size in HF dogs was smaller than in control dogs (19 +/- 5 vs. 40 +/- 4%, P < 0.01), suggesting that disparities in risk region size did not explain the differences in infarct size. CONCLUSION: Infarct size produced by a standardized ischemia-reperfusion protocol was smaller in dogs with pacing-induced HF. The reduced extent of infarction could not be attributed to differences in collateral blood flow or the size of the region at risk. Although the hearts in HF dogs were dilated, LV systolic blood pressure and the strength of contraction were lower than controls potentially reducing myocardial oxygen demand and explaining the smaller infarct size in HF dogs. Other mechanisms, however, cannot be discounted. Thus, ischemic tolerance is not reduced and may be augmented in dogs with pacing-induced heart failure.