calpain-inhibitor-iii and Brain-Injuries

Traumatic brain injury (TBI) is a leading cause of death and morbidity with no effective therapeutic treatments for secondary injury. Preclinical drug evaluation in rodent models of TBI is a lengthy process. In this regard, the zebrafish has numerous advantages to address the technical and time-dependent obstacles associated with drug evaluation. We developed a reproducible brain injury using glutamate excitoxicity in zebrafish larvae, a known initiator of delayed cell death in TBI. Glutamate challenge resulted in dose-dependent lethality over an 84-h observation period. We report significant decrease in locomotion (p < 0.0001) and mean velocity (p < 0.001) with 10 μM glutamate application as measured through automated 96-well plate behavioral analysis. Application of the NMDA receptor antagonist MK-801 (400 nM) or the calpain inhibitor, MDL-28170 (20 μM), resulted in significant recovery of locomotor function. A secA5-YFP transgenic line was used to visualize the localization of cell death due to glutamate exposure in vivo using confocal fluorescence microscopy. Our results indicate that zebrafish larvae exhibit responses to excitotoxic injury and pharmacotherapeutic intervention with pathophysiological relevance to mammalian excitotoxic brain injury. This system has potential to be applied as a high-throughput drug screening model to quickly identify candidate lead compounds for further evaluation.

Topics: Animals; Brain Injuries; Dipeptides; Dizocilpine Maleate; High-Throughput Screening Assays; Larva; Motor Activity; Neuroprotective Agents; Zebrafish

The cytoskeletal and neuronal protective effects of early treatment with the blood-brain barrier- and cell-permeable calpain inhibitor MDL-28170 was examined in the controlled cortical impact (CCI) traumatic brain injury (TBI) model in male CF-1 mice. This was preceded by a dose-response and pharmacodynamic evaluation of IV or IP doses of MDL-28170 with regard to ex vivo inhibition of calpain 2 activity in harvested brain homogenates. From these data, we tested the effects of an optimized MDL-28170 dosing regimen on calpain-mediated degradation of the neuronal cytoskeletal protein α-spectrin in cortical or hippocampal tissue of mice 24 h after CCI-TBI (1.0 mm depth, 3.5 m/sec velocity). With treatment initiated at 15 min post-TBI, α-spectrin degradation was significantly reduced by 40% in hippocampus and 44% in cortex. This effect was still observed with a 1-h but not a 3-h post-TBI delay. The cytoskeletal protection is most likely taking place in neurons surrounding the area of mainly necrotic degeneration, since MDL-28170 did not reduce hemispheric lesion volume as measured by the aminocupric silver staining method. This lack of effect on lesion volume has been seen with other calpain inhibitors, which suggests that pharmacological calpain inhibition by itself, while able to reduce axonal injury, may not be able to produce a measurable reduction in lesion volume. This is in contrast to certain other neuroprotective mechanistic approaches such as the mitochondrial protectant cyclosporine A, which produces at least a partial decrease in lesion volume in the same model. Accordingly, the combination of a calpain inhibitor with a compound such as cyclosporine A may be needed to achieve the optimal degree of post-TBI neuroprotection.

Topics: Analysis of Variance; Animals; Blood-Brain Barrier; Blotting, Western; Brain Injuries; Calpain; Cerebral Cortex; Dipeptides; Dose-Response Relationship, Drug; Hippocampus; Male; Mice; Nerve Degeneration; Neurons; Spectrin

Calpains are Ca(2+)-activated enzymes which cleave cytoskeletal and other proteins, contributing to neuronal damage in conditions of pathological intracellular Ca(2+) elevation, including stroke. However, the consequences of calpain overactivation have typically been observed hours after insult. To identify the earliest events attributable to calpain activation, and thus potentially isolate calpain substrates involved in acute neuronal damage, we dynamically recorded the effects of calpain inhibition in an in vitro model of stroke. Extracellular DC potentials and fEPSPs were monitored together with changes of light transmittance (as a measure of cell and mitochondrial swelling) and Rh 123 fluorescence (to monitor mitochondrial membrane potential; DeltaPsi(m)) in hippocampal slices obtained from P12-P17 rats. No differences were observed in the latencies of fEPSP disruption or onset of extracellular DC shifts associated with hypoxic spreading depression (HSD) evoked by oxygen-glucose deprivation (OGD) under control conditions or in the presence of calpain inhibitor III (MDL 28170). However, a significant difference was observed in transmitted light signals during OGD with calpain inhibition. Given the potential contribution of mitochondrial swelling to changes in light transmittance, these experiments were also conducted in the presence of cyclosporin A to block opening of the mitochondrial permeability transition pore (MPTP). Our results indicate that differences in OGD-induced changes of light transmittance in the presence of MDL 28170 are not likely the result of MPTP blockade or changes in dendritic beading. We propose that calpain inhibition may alter changes in light transmittance by limiting conformational changes of mitochondria.

Topics: Animals; Animals, Newborn; Brain Injuries; Calpain; Cortical Spreading Depression; Cysteine Proteinase Inhibitors; Dipeptides; Edema; Electric Stimulation; Excitatory Postsynaptic Potentials; Glucose; Hippocampus; Hypoxia; Membrane Potential, Mitochondrial; Rats; Rats, Wistar; Tissue Culture Techniques

It is known that calpain activation is involved in human traumatic brain injury (TBI) and that calpain inhibition can have neuroprotective effects on both gray matter and white matter injury of TBI models. However, the role of calpain activation in the corpus callosum remains unclear and requires elucidation given its potential clinical relevance. We evaluated the neuroprotective effects of calpain inhibitor MDL-28170 on corpus callosum function and structural destruction using a fluid percussion injury (FPI) model. The therapeutic time window for a single administration of MDL-28170 was up to 4 h post injury in protecting the corpus callosum structural integrity, and up to 30 min in protecting the axonal function evaluated 1 day following injury. When given 30 min prior injury, MDL-28170 showed neuroprotective effects that lasted up to 7 days. However, 30 min post injury administration of the drug afforded neuroprotection only up to 3 days. In contrast, two additional reinforcement injections at 24 and 48 h in addition to 30 min post FPI significantly protected both axonal function and structural integrity that lasted 14 days following FPI. Our data indicated that calpain inhibitor MDL-28170 is an effective neuroprotectant for axonal injury in corpus callosum following FPI with a therapeutic time window up to 4 hours. Although delayed treatment (2 or 4 h post FPI) was effective in protecting the axonal structure, the axons saved may not be as functional as normal fibers. Multiple drug administrations may be necessary for achieving a persisting effectiveness of this compound.

Topics: Action Potentials; Animals; Axons; Brain Injuries; Calpain; Corpus Callosum; Cysteine Proteinase Inhibitors; Diffuse Axonal Injury; Dipeptides; Disease Models, Animal; Drug Administration Schedule; Male; Neural Conduction; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Time Factors; Treatment Outcome; Wallerian Degeneration

Novel benzoylalanine-derived ketoamides were prepared and evaluated for calpain I inhibition. Derivatives carrying vinylbenzyl amino residues in the P(2)-P(3) region inhibited calpain in nanomolar concentrations and thus represent a novel class of nonpeptidic calpain inhibitors. Selected examples exhibited an improved pharmacokinetic profile including improved water-solubility and metabolic stability. In particular, these calpain inhibitors showed oral bioavailability in rats as demonstrated by N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethylaminomethylphenyl)ethen-1-yl]benzamide (5d). The closely related derivative N-(1-carbamoyl-1-oxohex-1-yl)-2-[E-2-(4-dimethylaminomethylphenyl)-ethen-1-yl]benzamide (5b) was evaluated for neuroprotective efficacy after experimental traumatic brain injury in a fluid percussion model in rats. When administered after injury, 5b reduced the number of damaged neurons by 41%, and this result would be in line with the suggested neuroprotective efficacy of calpain inhibition.

Topics: Administration, Oral; Alanine; Animals; Benzene Derivatives; Biological Availability; Blood Platelets; Brain Injuries; Calpain; Cell Survival; Cysteine Proteinase Inhibitors; Dentate Gyrus; Humans; In Vitro Techniques; Male; Neuroprotective Agents; Proto-Oncogene Proteins pp60(c-src); Rats; Rats, Sprague-Dawley; Solubility; Stereoisomerism; Structure-Activity Relationship; Vinyl Compounds; Water

Traumatic brain injury (TBI) evokes diffuse (traumatic) axonal injury (TAI), which contributes to morbidity and mortality. Damaged axons display progressive alterations gradually evolving to axonal disconnection. In severe TAI, the tensile forces of injury lead to a focal influx of Ca2+, initiating a series of proteolytic processes wherein the cysteine proteases, calpain and caspase modify the axonal cytoskeleton, causing irreversible damage over time postinjury. Although several studies have demonstrated that the systemic administration of calpain inhibitors reduces the extent of ischemic and traumatic contusional injury a direct beneficial effect on TAI has not been established to date. The current study was initiated to address this issue in an impact acceleration rat-TBI model in order to provide further evidence on the contribution of calpain-mediated proteolytic processes in the pathogenesis of TAI, while further supporting the utility of calpain-inhibitors. A single tail vein bolus injection of 30 mg/kg MDL-28170 was administered to Wistar rats 30 min preinjury. After injury the rats were allowed to survive 120 min when they were perfused with aldehydes. Brains were processed for immunohistochemical localization of damaged axonal profiles displaying either amyloid precursor protein (APP)- or RMO-14-immunoreactivity (IR), both considered markers of specific features of TAI. Digital data acquisition and statistical analysis demonstrated that preinjury administration of MDL-28170 significantly reduced the mean number of damaged RMO-14- as well as APP-IR axonal profiles in the brainstem fiber tracts analyzed. These results further underscore the role of calpain-mediated proteolytic processes in the pathogenesis of DAI and support the potential use of cell permeable calpain-inhibitors as a rational therapeutic approach in TBI.

Topics: Amyloid beta-Protein Precursor; Animals; Axons; Brain; Brain Injuries; Cysteine Proteinase Inhibitors; Dipeptides; Image Processing, Computer-Assisted; Immunohistochemistry; Models, Animal; Neurofilament Proteins; Rats; Rats, Wistar