Background and PurposeThis research was performed to determine whether a selective inhibitor of the calcium-dependent protease, calpain, could reduce ischemia-associated brain damage when peripherally administered after a vascular occlusion.Methods A variation of the rat middle cerebral artery occlusion model was used. A range of doses of AK295 (a novel calpain inhibitor synthesized for this purpose) was continuously infused through the internal carotid artery, beginning 1.25 hours from the initiation of the occlusion. Rats were killed at 21 hours, and the infarct volume was quantified.Results Postocdusion (1.25-hour) infusion of the calpain inhibitor AK295 elicited a dose-dependent neuroprotective effect after focal ischemia. The highest dose tested (3 mg/kg

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.

Peptidyl alpha-keto amides have been synthesized and tested as inhibitors of the cysteine protease calpain. A stereospecific synthesis was devised in which Cbz-dipeptidyl-alpha-hydroxy amides were oxidized with TEMPO/hypochlorite to the corresponding alpha-keto amides. This oxidation was accomplished in good yields and without epimerization of the chiral center adjacent to the ketone. The potent inhibition of porcine calpain I by the L,L diastereomers, combined with the poor inhibition by the L,D diastereomers, established the requirement for the all-L stereochemistry of the active inhibitor. The early lead inhibitors were very hydrophobic and, therefore, poorly soluble in aqueous solutions. Using the stereospecific route, new compounds were prepared with polar groups at the C- and N-termini. These modifications resulted in more soluble inhibitors that were still potent inhibitors of calpain. Studies of the stability of these alpha-keto amides showed that absolute stereochemistry can be maintained in acidic and unbuffered environments but general base-catalyzed epimerization of the chiral center adjacent to the ketone occurred rapidly. The alpha-hydroxy precursors were inactive as inhibitors of calpain, which supports the hypothesis that the alpha-keto compounds reversibly form an enzyme-bound tetrahedral species that results from the nucleophilic addition of the catalytic thiol of calpain to the electrophilic ketone of the inhibitor.

No abstract

Calpains are cytosolic, neutral proteases that normally exist in an inactive or quiescent state. They require higher than normal levels of calcium for activation which, once accomplished, lead to irreversible proteolysis of numerous cytoskeletal, membrane-associated and regulatory proteins. Because of these characteristics, calpain is gaining attention as a potentially important pathogenic variable in ischemic neuronal death. This manuscript explores this hypothesis by briefly reviewing current support for the role played by calpain in ischemic neurodegeneration, and then discussing a series of recently published studies which: 1. offer further evidence for the hypothesis, and 2. provide direct support for the idea that selective inhibition of calpain can greatly limit the neuronal damage that would normally occur following both global as well as focal brain ischemia. Thus, the data reviewed in this manuscript support the ideas that unregulated activation and proteolysis of intraneuronal calpain plays a significant role in the brain damage that occurs following an ischemic event and that delivering selective and membrane permeant calpain inhibitors to ischemic tissue may provide a powerfully effective therapeutic means of limiting neuronal damage.