Postischemic Administration of AK275, a Calpain Inhibitor, Provides Substantial Protection against Focal Ischemic Brain Damage

Abstract

Summary: Experiments were conducted to determine whether a potent, reversible calpain inhibitor could re duce the cortical ischemic brain damage associated with focal ischemia in the rat. AK275 (Z-Leu-Abu-CONH CH 2 CH 3 ), the active isomer of the diastereomeric mix ture, CX275, was employed in conjunction with a novel method of perfusing drug directly onto the infarcted cor tical surface. This protocol reduced or eliminated numer ous, nonspecific pharmacokinetic, hemodynamic, and other potentially confounding variables that might com plicate interpretation of any drug effect. Focal ischemia was induced using a variation of the middle cerebral arIschemic episodes in the brain initiate a time related series of extracellular and intraneuronal events that lead to delayed degeneration of vulner able neurons (Zivin and Choi, 1991). With global ischemia (e. g. , cardiac arrest, drowning), substan tial neuronal death can take several days (Ginsberg and Busto, 1989), whereas with focal ischemia (e. g. , stroke), significant cell death typically occurs within hours (Selman et aI. , 1990; Buchan et aI. , 1992). This delayed cell death in response to isch emia is believed to provide a "therapeutic window 537 tery occlusion method. These studies demonstrated a re liable and robust neuroprotective effect of AK275 over the concentration range of 10 to 200 fLM (perfused supra cortically at 4 fLl/h for 21 h). Moreover, a 75% reduction in infarct volume was observed when initiation of drug treatment was delayed for 3 h postocclusion. Our data further support an important role of calpain in ischemia induced neuropathology and suggest that calpain inhibi tors may provide a unique and potentially powerful means of treating stroke and other ischemic brain inci dents.

Time-Related Neuronal Changes following Middle Cerebral Artery Occlusion: Implications for Therapeutic Intervention and the Role of Calpain

“…Independent support for an important role of cal pain in focal ischemia has recently come from studies reporting significant reductions in neo cortical infarct volume when relatively selective calpain inhibitors were administered (Bartus et al , 1994a,b;Hong et al , 1994). One study, in particu lar, supports the potential advantages of cal pain in hibition via a 75% reduction in infarct volume when the inhibitor was administered several hours after the occlusion (Bartus et al , 1994a). Collectively, the pathogenetic and pharmacologic studies pub-Ii shed to date provide mutually corroborating sup port for the hypothesis that calpin-induced proteol ysis plays an important role in focal ischemia and that selective inhibitors of calpain may provide a novel and effective means of protecting the brain from ischemia-related neuronal death.…”

“…It has thus been argued that the accumulation of nu merous, irreversible changes to these multiple sub strate proteins must eventually be lethal to the cell (Siman et al , 1984;Lynch et al , 1986;Seubert et aI. , 1987;Bartus, 1990;Bartus et al , 1994aBartus et al , ,b, 1995.…”

Calpain inhibitor AK295 attenuates motor and cognitive deficits following experimental brain injury in the rat.

“…In models of cerebral ischemia, treatment with putative calpain inhibitors has afforded significant neuroprotection, supporting a role for calciuminduced proteolysis by calpain in the pathogenesis of acute ischemic injury (30)(31)(32)(33)(34)(35). Recently, ketoamide calpain inhibitors have been shown to be neuroprotective even when administered after initiation of an ischemic event (30,31). Prolonged calpain activation was described after experimental brain injury in regions that correlate with neuronal degeneration and cell death (36) (31).…”

“…Indeed, evidence accumulated over the past decade suggests that calpain proteolysis contributes to cytotoxicity in many forms of neurodegeneration, including ischemic brain damage, Alzheimer disease, spinal cord injury, and neural muscular degeneration (26,29). In models of cerebral ischemia, treatment with putative calpain inhibitors has afforded significant neuroprotection, supporting a role for calciuminduced proteolysis by calpain in the pathogenesis of acute ischemic injury (30)(31)(32)(33)(34)(35). Recently, ketoamide calpain inhibitors have been shown to be neuroprotective even when administered after initiation of an ischemic event (30,31).…”

Pre, intra and post-ischemic hypothermic neuroprotection in temporary focal cerebral ischemia in rats: morphometric analysis

“…The ischemic area of each section was obtained by the difference between the total area and the sum of all measures in blue and red areas, of both hemispheres. The partial ischemic volume, for 60 stained sections, was calculated of each animal using measurements of ischemic areas, the thickness of each section and the number of sections using Bartustechnique 19 and corrected when necessary according to Chang technique 20 . The approximate ischemic volume was obtained by the partial volume x 1,000/60.…”