Hypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model. Hypoxic-ischemic (H-I)1 encephalopathy in the prenatal and perinatal period is a major cause of morbidity and mortality and often results in cognitive impairment, seizures, and motor impairment leading to cerebral palsy (1, 2). Many studies of neonatal H-I brain injury have utilized the well characterized Levine model in which unilateral carotid ligation is followed by exposure to hypoxia in postnatal day (P) 7 rats (3-5). This model of H-I results in a reproducible pattern of hemispheric injury ipsilateral, but not contralateral, to the carotid ligation (5-7). There are prominent features of both apoptosis and necrosis when this model is performed in neonatal rats and mice (1, 8 -11). Inhibition of caspases utilizing a pan-caspase inhibitor partially protects against brain injury after neonatal H-I injury in this model (12), and similar inhibitors have been shown to partially protect against ischemic injury in adult models (13-16). Previously utilized peptide-based caspase inhibitors (e.g. Boc-D-fmk, z-VAD-fmk, z-DEVD-fmk) required relatively large doses in vivo for their protective effects, and at high concentrations, their effects are more likely to be less selective. Thus, although these studies suggest a role for caspases, the specific caspases and other proteases, which contribute to brain injury after neonatal H-I, have not been clarified.Caspases are a family of cysteine asp...

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[structure: see text] The total synthesis of the polyether antibiotic ionomycin, a calcium ionophore, is described. The synthesis demonstrates the utility of ring-opening methodologies as applied to the synthesis of polypropionate and deoxypolypropionate subunits, which are found in two of the four fragments in the synthesis.

1 Caspases, key enzymes in the apoptosis pathway, have been detected in the brain of HD patients and in animal models of the disease. In the present study, we investigated the neuroprotective properties of a new, reversible, caspase-3-specific inhibitor,, in a rat malonate model of HD. 2 Pharmacokinetic and autoradiography studies after intrastriatal (i.str.) injection of 1.5 nmol of M826 or its tritiated analogue [ 3 H]M826 indicated that the compound diffused within the entire striatum. The elimination half-life (T 1/2 ) of M826 in the rat striatum was 3 h. 3 I.str. injection of 1.5 nmol of M826 10 min after malonate infusion induced a significant reduction (66%) in the number of neurones expressing active caspase-3 in the ipsilateral striatum. 4 Inhibition of active caspase-3 translated into a significant but moderate reduction (39%) of the lesion volume, and of cell death (24%), 24 h after injury. The efficacy of M826 at inhibiting cell death was comparable to that of the noncompetitive NMDA receptor antagonist MK801. 5 These data provide in vivo proof-of-concept of the neuroprotective effects of reversible caspase-3 inhibitors in a model of malonate-induced striatal injury in the adult rat.

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.