calpain-inhibitor-iii and Ischemic-Attack--Transient

Stroke patients often experience a significant temporal delay between the onset of ischemia and the time to initiation of therapy. Thus, there is a need for neuroprotectants with a long therapeutic window of opportunity. The efficacy of a potent, central nervous system-penetrating calpain inhibitor (MDL 28,170) was evaluated in a temporary model of focal cerebral ischemia to determine the window of opportunity for intracellular protease inhibition.. An ex vivo brain protease inhibition assay established pharmacodynamic dosing parameters for MDL 28,170. Middle cerebral artery (MCA) occlusion was accomplished by advancing a monofilament through the internal carotid artery to the origin of the MCA. Postmortem infarct volumes were determined by quantitative image analysis of triphenyltetrazolium-stained brain sections.. Maximal inhibition of brain protease activity was observed 30 minutes after injection of MDL 28,170 with an estimated pharmacodynamic half-life of 2 hours. MDL 28,170 caused a dose-dependent reduction in infarct volume when administered 30 minutes after MCA occlusion. A window of opportunity study was conducted to determine the maximal delay between the onset of ischemia and the initiation of efficacious therapy. MDL 28,170 reduced infarct volume when therapy was delayed for 0.5, 3, 4, and 6 hours after the initiation of ischemia. The protective effect of MDL 28,170 was lost after an 8-hour delay.. These data indicate that the therapeutic window of opportunity for calpain inhibition is at least 6 hours in a reversible focal cerebral ischemia model. This protection is observed despite the lethal hypoxic and excitotoxic challenge, suggesting that calpain activation may be an obligatory, downstream event in the ischemic cell death cascade.

Topics: Animals; Brain; Calpain; Carotid Artery, Internal; Cell Death; Cerebral Arterial Diseases; Cerebral Infarction; Coloring Agents; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Half-Life; Hypoxia; Image Processing, Computer-Assisted; Ischemic Attack, Transient; Male; Neuroprotective Agents; Neurotoxins; Rats; Rats, Wistar; Tetrazolium Salts; Time Factors

Global brain ischemia and reperfusion result in the degradation of the eukaryotic initiation factor (eIF) 4G, which plays a critical role in the attachment of the mRNA to the ribosome. Because eIF-4G is a substrate of calpain, these studies were undertaken to examine whether calpain I activation during global brain ischemia contributes to the degradation of eIF-4G in vivo. Immunoblots with antibodies against calpain I and eIF-4G were prepared from rat brain postmitochondrial supernatant incubated at 37 degrees C with and without the addition of calcium and the calpain inhibitors calpastatin or MDL-28,170. Addition of calcium alone resulted in calpain I activation (as measured by autolysis of the 80-kDa subunit) and degradation of eIF-4G; this effect was blocked by either 1 micromol/L calpastatin or 10 micromol/L MDL-28,170. In rabbits subjected to 20 minutes of cardiac arrest, immunoblots of brain postmitochondrial supernatants showed that the percentage of autolyzed calpain I increased from 1.9% +/- 1.1% to 15.8% +/- 5.0% and that this was accompanied by a 68% loss of eIF-4G. MDL-28,170 pretreatment (30 mg/kg) decreased ischemia-induced calpain I autolysis 40% and almost completely blocked eIF-4G degradation. We conclude that calpain I degrades eIF-4G during global brain ischemia.

Topics: Animals; Brain; Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Eukaryotic Initiation Factor-4G; Female; Ischemic Attack, Transient; Kinetics; Male; Peptide Initiation Factors; Rabbits; Rats; Reperfusion; Subcellular Fractions

Excessive elevation of intracellular calcium and uncontrolled activation of calcium-sensitive events are believed to play a central role in ischemic neuronal damage. Calcium-activated proteolysis by calpain is a candidate to participate in this form of pathology because it is activated under ischemic conditions and its activation results in the degradation of crucial cytoskeletal and regulatory proteins. The present studies examined the effects of a cell-penetrating inhibitor of calpain on the pathological outcome after transient focal ischemia in the brain.. Twenty-five male Sprague-Dawley rats were divided into four groups: a saline-treated group, a vehicle-treated group, and two calpain inhibitor-treated groups (Cbz-Val-Phe-H; 30-mg/kg and 60-mg/kg cumulative doses). Ischemia was induced by occluding the left middle cerebral artery and both common carotid arteries for 3 hours followed by reperfusion. Animals were killed 72 hours after surgery, and quantitative measurements of infarction volumes were performed using histological techniques. Eight additional rats were killed 30 minutes after ischemia and examined for the extent of proteolysis using immunoblot techniques. A final group of 12 animals was decapitated after injection of vehicle or calpain inhibitor, and the proteolytic response was measured after 60 minutes of total ischemia.. Rats treated with Cbz-Val-Phe-H exhibited significantly smaller volumes of cerebral infarction than saline-treated or vehicle-treated control animals. Intravenous injections of cumulative doses of 30 mg/kg or 60 mg/kg of Cbz-Val-Phe-H were effective in reducing infarction, edema, and calcium-activated proteolysis. The proteolytic response to postdecapitation ischemia was also reduced by the calpain inhibitor.. These results demonstrate the neuroprotective effect of a cell-penetrating calpain inhibitor when administered systemically. The findings suggest that targeting intracellular, calcium-activated mechanisms, such as proteolysis, represents a viable therapeutic strategy for limiting neurological damage after ischemia.

Topics: Analysis of Variance; Animals; Brain Edema; Calpain; Cerebral Infarction; Dipeptides; Disease Models, Animal; Ischemic Attack, Transient; Male; Rats; Rats, Sprague-Dawley