clazosentan and Disease-Models--Animal

Cerebral vasospasm and late cerebral ischemia (LCI) remain leading causes of mortality in patients experiencing a subarachnoid hemorrhage (SAH). This occurs typically 3 to 4 days after the initial bleeding and peaks at 5 to 7 days. The underlying pathophysiology is still poorly understood. Because SAH is associated with elevated levels of endothelin-1 (ET-1), focus has been on counteracting endothelin receptor activation with receptor antagonists like clazosentan, however, with poor outcome in clinical trials. We hypothesize that inhibition of intracellular transcription signaling will be an effective approach to prevent LCI. Here, we compare the effects of clazosentan versus the MEK1/2 blocker U0126 in a rat model of SAH. Although clazosentan directly inhibits the contractile responses in vivo to ET-1, it did not prevent SAH-induced upregulation of ET receptors in cerebral arteries and did not show a beneficial effect on neurologic outcome. U0126 had no vasomotor effect by itself but counteracts SAH-induced receptor upregulation in cerebral arteries and improved outcome after SAH. We suggest that because SAH induces elevated expression of several contractile receptor subtypes, it is not sufficient to block only one of these (ET receptors) but inhibition of transcriptional MEK1/2-mediated upregulation of several contractile receptors may be a viable way towards alleviating LCI.

Topics: Animals; Brain Ischemia; Butadienes; Cerebral Arteries; Dioxanes; Disease Models, Animal; Endothelin-1; Enzyme Inhibitors; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Pyridines; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptors, Endothelin; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles; Up-Regulation

Subarachnoid hemorrhage (SAH) remains a condition with suboptimal functional outcomes, especially in the young population. Pharmacotherapy has an accepted role in several aspects of the disease and an emerging role in several others. No preventive pharmacologic interventions for SAH currently exist. Antiplatelet medications as well as anticoagulation have been used to prevent thromboembolic events after endovascular coiling. However, the main focus of pharmacologic treatment of SAH is the prevention of delayed cerebral ischemia (DCI). Currently the only evidence-based medical intervention is nimodipine. Other calcium channel blockers have been evaluated without convincing efficacy. Anti-inflammatory drugs such as statins have demonstrated early potential; however, they failed to provide significant evidence for the use in preventing DCI. Similar findings have been reported for magnesium, which showed potential in experimental studies and a phase 2 trial. Clazosentane, a potent endothelin receptor antagonist, did not translate to improve functional outcomes. Various other neuroprotective agents have been used to prevent DCI; however, the results have been, at best inconclusive. The prevention of DCI and improvement in functional outcome remain the goals of pharmacotherapy after the culprit lesion has been treated in aneurysmal SAH. Therefore, further research to elucidate the exact mechanisms by which DCI is propagated is clearly needed. In this article, we review the current pharmacologic approaches that have been evaluated in SAH and highlight the areas in which further research is needed.

Topics: Adrenal Cortex Hormones; Animals; Anticoagulants; Apoptosis; Brain Ischemia; Calcium Channel Blockers; Clinical Trials as Topic; Dexamethasone; Dioxanes; Disease Models, Animal; Drugs, Chinese Herbal; Estrogens; Evidence-Based Medicine; Free Radical Scavengers; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Magnesium Sulfate; Neuroprotective Agents; Nimodipine; Platelet Aggregation Inhibitors; Pregnatrienes; Progesterone; Pyridines; Pyrimidines; Receptor, Endothelin A; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles

Delayed cerebral ischemia (DCI) is a significant cause of morbidity and mortality for patients surviving the rupture of an intracranial aneurysm. Despite an association between vasospasm and DCI, thrombosis and thromboembolism may also contribute to DCI. In this study we investigate the time course of intravascular microclot formation after experimental subarachnoid hemorrhage (SAH) and assess the effects of the following two drugs on microclot burden: mutant thrombin-activated urokinase-type plasminogen activator (scFv/uPA-T), which is bound to red blood cells for use as a thromboprophylactic agent, and clazosentan, an endothelin antagonist. In the first study, adult male C57BL/6 mice were sacrificed at 24 (n=5), 48 (n=6), 72 (n=8), and 96 (n=3) hours after SAH induced by filament perforation of the anterior cerebral artery. Sham animals (n=5) underwent filament insertion without puncture. In the second study, animals received scFv/uPA-T (n=5) 3 hours after hemorrhage, clazosentan (n=5) by bolus and subcutaneous pump after SAH just prior to skin closure, or a combination of scFv/uPA-T and clazosentan (n=4). Control (n=6) and sham (n=5) animals received saline alone. All animals were sacrificed at 48 hours and underwent intra-cardiac perfusion with 4% paraformaldehyde. The brains were then extracted and sliced coronally on a cryostat and processed for immunohistochemistry. An antibody recognizing thrombin-anti-thrombin complexes was used to detect microclots on coronal slices. Microclot burden was calculated for each animal and compared among groups. Following SAH, positive anti-thrombin staining was detected bilaterally in the following brain regions, in order of decreasing frequency: cortex; hippocampus; hypothalamus; basal ganglia. Few microclots were found in the shams. Microclot burden peaked at 48 hours and then decreased gradually. Animals receiving scFv/uPA-T and scFv/uPA-T+clazosentan had a lower microclot burden than controls, whereas animals receiving clazosentan alone had a higher microclot burden (p<0.005). The overall mortality rate in the time course study was 40%; mortality was highest among control animals in the second study. Intravascular microclots form in a delayed fashion after experimental SAH. Microclots may be safely reduced using a novel form of thromboprophylaxis provided by RBC-targeted scFv/uPA-T and represent a potential target for therapeutic intervention in the treatment of DCI.

Topics: Analysis of Variance; Animals; Cerebral Arteries; Dioxanes; Disease Models, Animal; Disease Progression; Drug Delivery Systems; Erythrocytes; Fibrinolytic Agents; Glycophorins; Male; Mice; Mice, Inbred C57BL; Pyridines; Pyrimidines; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles; Thrombosis; Time Factors; Urokinase-Type Plasminogen Activator

Endothelin receptor antagonists such as clazosentan decrease large-artery vasospasm after experimental and clinical subarachnoid hemorrhage. We used clazosentan to gain insight into the pathophysiology of subarachnoid hemorrhage by determining if decreasing vasospasm is associated with alleviation of other secondary complications of subarachnoid hemorrhage such as oxidative stress, endothelial nitric oxide synthase dysfunction, microthromboembolism, and neuronal injury.. Mice were subjected to subarachnoid hemorrhage by injection of blood into the chiasmatic cistern. They were treated with clazosentan or vehicle by continuous intraperitoneal infusion for 48 hours. Middle cerebral artery vasospasm, superoxide anion radical, peroxynitrite, microthromboemboli, endothelial nitric oxide synthase uncoupling, cerebral blood flow, neuronal injury, and mortality were assessed.. Clazosentan preserved cerebral blood flow, alleviated vasospasm, and decreased mortality but did not affect superoxide anion radical, peroxynitrite, or microthromboemboli in the brain. Endothelial nitric oxide synthase uncoupling and neuronal injury also were not reduced by clazosentan.. This study shows large-artery vasospasm is pathophysiologically independent of some other effects of subarachnoid hemorrhage. The findings have implications for development of treatments for this disease.

Topics: Animals; Apoptosis; Dioxanes; Disease Models, Animal; Endothelin A Receptor Antagonists; Mice; Mice, Transgenic; Nitric Oxide Synthase Type III; Oxidative Stress; Pyridines; Pyrimidines; Regional Blood Flow; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles; Thromboembolism; Vasospasm, Intracranial

One of the major complications after subarachnoid hemorrhage (SAH) is angiographic vasospasm in the large arteries at the base of the brain. However, a clinical trial of clazosentan demonstrated a 65% relative risk reduction in angiographic vasospasm but no effect on mortality or clinical outcome, raising questions about the role of angiographic vasospasm played in outcome after SAH. The purpose of this study was to determine if reducing or reversing angiographic vasospasm with clazosentan reduced other secondary complications such as microthromboembolism, loss of long-term potentiation (LTP) and neuronal cell death in a rat model of SAH. SAH in rats was created by injection of 300 μl non-heparinized autologous blood into the pre-chiasmatic cistern. Clazosentan, 10mg/kg bolus, or vehicle control was administered 1h after SAH intravenously, followed by a continuous infusion (1mg/kg/h) into the jugular vein using an osmotic pump. Rats treated with clazosentan had less large-artery vasospsam compared to vehicle-treated controls. However, clazosentan did not prevent the formation of microthromboemboli, neuronal cell death and degeneration and loss of LTP, suggesting there is a dissociation between large-artery angiographic vasospasm and other secondary complications of SAH. This result suggests that alleviation of angiographic vasospasm alone may not be sufficient to prevent other secondary complications or that off-target drug effects after systemic administration of clazosentan counteract the beneficial effects on angiographic vasospasm.

Topics: Analysis of Variance; Animals; Caspase 3; Chi-Square Distribution; Dioxanes; Disease Models, Animal; Electric Stimulation; Fibrinogen; Fluoresceins; Hippocampus; In Situ Nick-End Labeling; In Vitro Techniques; Long-Term Potentiation; Male; Organic Chemicals; Phosphopyruvate Hydratase; Pyridines; Pyrimidines; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles; Vasospasm, Intracranial

The purpose of this study was to test the efficacy of a novel endothelin receptor A antagonist on blood flow and behavioral outcome given 30 minutes following traumatic brain injury.. Male Sprague-Dawley rats (400-450 g) were used in this study. All animals were scanned for initial blood flow using arterial spin labeling magnetic resonance imaging (n = 72 total). Half were subjected to traumatic brain injury using a weight acceleration impact device (n = 36 total). Sham operated animals were used as control (n = 36 total). Thirty minutes following traumatic brain injury, animals were given one intravenous injection of vehicle (0·9% saline) or 1·0 mg/kg clazosentan, a novel endothelin receptor A antagonist, for a total of four groups. At 4, 24, and 48 hours post-traumatic brain injury, blood flow determination continued. On the second day post-traumatic brain injury/sham operation, behavioral testing commenced using a radial arm maze to assess cognitive function.. Our results indicate that 1·0 mg/kg clazosentan was effective in ameliorating hypoperfusion seen after traumatic brain injury. Saline had no effect. Furthermore, clazosentan treatment was effective in significantly improving behavioral outcome following traumatic brain injury.. Collectively, these results indicate that clazosentan, given at 30 minutes post-traumatic brain injury, is effective in improving outcome following injury.

Topics: Animals; Behavior, Animal; Brain Injuries; Cerebrovascular Circulation; Cerebrovascular Disorders; Cognition Disorders; Dioxanes; Disease Models, Animal; Endothelin A Receptor Antagonists; Male; Maze Learning; Pyridines; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Sulfonamides; Tetrazoles; Vasodilator Agents