WO1998003191A1

WO1998003191A1 - Calpain inhibitors for the treatment of cerebral ischemia, spinal cord injury or stroke

Info

Publication number: WO1998003191A1
Application number: PCT/US1997/012672
Authority: WO
Inventors: Ronald L. Hayes; Guy L. Clifton
Original assignee: Neurotrauma Therapeutics, Inc.
Priority date: 1996-07-19
Filing date: 1997-07-18
Publication date: 1998-01-29
Also published as: AU4042397A

Abstract

Disclosed are methods relating to the treatment of brain and spinal cord injury, and strokes utilizing calpain inhibitors. In preferred embodiments, calpain 1 and 2 inhibitors are utilized in the protection agains NF protein loss, both due to depolarization and impact injury.

Description

PATENT SPECIFICATION

TITLE: CALPAIN INHIBITORS FOR THE TREATMENT OF

CEREBRAL ISCHEMIA, SPINAL CORD INJURY OR STROKE

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pharmaceutical compositions, and to medical treatment methods utilizing such compositions. In another aspect, the present invention relates to pharmaceutical compositions comprising calpain inhibitors, and to medical treatment methods utilizing such compositions. In even another aspect, the present invention relates to pharmaceutical compositions comprising peptide aldehyde type of calpain inhibitors, and to medical treatment methods utilizing such compositions. In still another aspect, the present invention relates to pharmaceutical compositions comprising calpain I and/or II inhibitor, and to medical treatment methods utilizing such compositions for brain injury, spinal cord injury, and strokes .

2. Description of the Related Art In the 1970' s, studies employing isolated axonal preparations demonstrated that excessive entry of calcium into axons resulted in significant degradation of cytoskeletal proteins. Such observations led to speculation that excessive calcium accumulation in spinal cord injury could contribute to cytoskeletal damage produced by mechanical insults to the spinal cord (Schlaepfer and Bunge, 1973) . Subsequently, calcium accumulation in central nervous tissue has been inferred in ischemia (Siesjo, 1988); Siesjo and Bengtεεon, 1989; Uematsu et al . , 1991) TBI (Fineman et al . , 1993) as well as

spinal cord injury (Balentine, 1988) . Neuronal calcium overload in these disorders may be triggered by several mechanisms, including brief (six to ten minutes) potassium depolarization (Katayama et al . , 1990; Katayama et al . ,

1991; Nilsson et al . , 1993; Szatkowski and Atwell, 1994) and excessive exposure to certain excitatory amino acids (for reviews see Rothman and Olney, 1995) .

In the 1980' s, the Ca^{^}-activated neutral protease calpain, one of many cellular proteins involved in Ca^~' signalling in mammalian cells, was purified allowing a number of in vi tro studies to determine that various cytoskeletal proteins including neurofilaments (NF) , microtubule associated protein 2 (MAP2) and spectrin were substrates for protein proteolysis.

NF proteins are the most abundant intermediate cytoskeleton proteins found in neurons (Nixon and Sihag, 1991; Liem, 1993) . They consist of three separate protein elements collectively called the NF triplet proteins, or separately called, high (NF-H) , medium (NF-M) , and low molecular weight (NF-L) neurofilament proteins, (Shaw et al . , 1986; Liem, 1993; and Nixon and Shea, 1984) . These subunits have apparent molecular weights of 200 kDa (NF200) , 150 kDa (NF150) and 68 kDa (NF68) as estimated by gel electrophoresis (Dautingy et al . , 1988). The NF68

subunit iε an assembly protein found predominantly in the NF core, while the NF150 and NF200 subunits are cross- linking proteins found in the connecting branches (Gotow et al . , 1994) .

Although their neuronal distribution partially depends on their phosphorylation state, antibodies against phosphorylated and non-phosphorylated epitopes can detect

NF-H, -M, and -L in cell bodies, axons and dendrites (Shaw et al . , 1986) . While limited proteolysis of cytoskeletal

proteins is often thought to be a housekeeping function in the central nervous system (CNS) , increased proteolysis of cytoskeletal proteins could result in derangement of neuronal structure and function. A variety of enzymes including calpainε (Zimmerman and Schlaepfer, 1982) , cathepsins (Banay-Schwartz et al . , 1987), trypsin and

chymotrypsin (Chin et al . , 1983) cleave NFs . However, only

the two known calpain isoforms, calpain I and II, require increased intracellular calcium levels for optimal activation (for reviews see Murachi, 1983; and Suzuki et al . , 1987) .

NF degradation has been reported in experimental spinal cord injury (Banik et al . , 1982) . NF-H, -M, and -L

protein levels are decreased in vivo after cerebral ischemia/hypoxia (Kaku et al . , 1993; Nakamura et al . , 1992;

Ogata et al . , 1989). Additionally, NF-H and NF-L protein

loss has been described after TBI in vivo (Posmantur

et al . , 1994) . More recently, studies of the role of calcium dependent proteolysis by calpain have been conducted primarily by investigatorε of ischemia and spinal cord injury. These studies to date have provided evidence that ischemia results in accumulation of calpain- specific breakdown products to cytoskeletal proteins such as spectrin (Siman et al . , 1988; Roberts-Lewis et al . 1993) . In addition, investigators have reported that administration of specific calpain inhibitors following global ischemia can significantly reduce contusion volume (Bartus et al . , 1994; 1995). These in vivo observations

have been corroborated by a number of in vi tro studies

showing that hypoxic-anoxic insults in various neuronal culture systems can reduce cell death and cytoskeletal loss (Arai et al . , 1991) . Calpain inhibitor II has also been reported to improve efficacy of synaptic transmission in hippocampal slices following hypoxic-anoxic insults (Arai et al . , 1990) . Increased accumulation of calpain has also been reported following spinal cord injury in vivo (Banik

et al . , 1982) . Finally, a number of investigators have speculated that calpain proteolysis could contribute to a variety of neurodegenerate diseases associated with cytoskeletal derangements including Alzheimer's disease (Sousson et al . , 1994; Nixon et al . , 1994), Huntington's disease and ALS (Migheli et al . , 1994; Nagaraj i et al . , 1994) .

Wang and Yuen, 1994, teach that "increasing evidence now suggests that excessive activation of the Ca²⁺ dependent protease calpain could play a key or contributory roll in the pathology of a variety of disorders, including cerebral ischemia, cataract, myocardial ischemia, muscular dystrophy and platelet aggregation". Wang and Yuen, further teach that "given the multiple therapeutic indications for calpain, it appears that achievement of selective calpain inhibition is an important pharmacological goal" (Wang and Yuen, 1994) .

However, the prior art teaches away from the use of non-highly selective calpain inhibitors, such as for example calpain inhibitor I and calpain inhibitor II, for use in treating traumatic brain injury, cerebral ischemia, spinal injury and stroke, for at least the following several reasons . First, historically, investigators have not believed that peptide aldehyde type of calpain inhibitors, such as calpain inhibitors I and II, were clinically useful agents. This was because it was believed that there was a strong requirement for absolute specificity, and none of these peptide aldehyde inhibitors iε highly selective (Wang and Yuen, 1994) . It was also felt that compounds inhibiting other proteases such as cathepsin, a lysosomal enzyme, could produce significant toxicity especially when administered over long time periods. Peptide aldehyde inhibitors unfortunately inhibit other cystein proteases (Wang and Yuen, 1994) .

Second, Wang and Yuen, 1994, while suggesting the use of calpain inhibitors in general for a multiplicity of uses, specifically cite Lampi et al . , who teach that calpain inhibitor I and II were not effective in cataract treatment because of their cytotoxicity .

Finally, regarding traumatic brain injury in particular, Povlishock, 1993, teaches that neurofilament degradation and dissolution mediated by increased intracellular calcium is not the pivotal event in pathology of all forms of axonal injury. Povlishock, 1993, further teaches that in the instance of traumatic brain injury, there is no evidence that a traumatically induced influx of Ca'⁺ causes neural protease activation with neurofilament degradation, lending to subsequent axonal collapse. Therefore, Povlishock teaches away from proteolysis which could be effected by calpain inhibitors as the pathology for traumatic brain injury.

Thus, in spite of these advancements in the prior art, none of these prior art references disclose or suggest the use of peptide aldehyde inhibitors for treating traumatic brain injury, cerebral ischemia, spinal injury and stroke.

SUMMARY OF THE INVENTION According to one embodiement of the present invention there is provided a method for treating cerebral ischemia, spinal cord injury or stroke in an animal. The method generally includes administering to said animal a pharmaceutically-acceptable calpain inhibitor composition, in a dose effective to improve neurological outcome or brain/tissue damage indices.

According to another embodiment of the present invention, there is provided a method for treating cerebral ischemia, spinal cord injury, or stroke in an animal. The method generally includes administering to said animal a pharmaceutically-acceptable calpain inhibitor composition, in an amount effective to reduce cytoskeletal protein loss .

For both of the above embodiments, preferred calpain inhibitors are peptide aldehyde type of calpain inhibitors .

More preferred inhibitors are calpain inhibitor I and calpain inhibitor II, with calpain inhibitor II being the most preferred. Commencement of administration of the inhibitors should occur as soon after the injurious event as possible, and will continue for at least an hour at a dose of at least 0.01 mg/kg/hr.

DETAILED DESCRIPTION OF THE INVENTION The Ca²" -activated neutral protease calpain is just one of many cellular proteins involved in Ca²⁺ signaling in mammalian cells. There are two major isoforms: calpain I (or -calpain) and calpain II (or m-calpain) .

According to the present invention, calpain inhibitors are administered as treatment for traumatic brain injury, cerebral ischemia, spinal cord injury, and stroke in mammals, especially humans.

As used herein, "traumatic brain injury" generally includes non-penetrating closed head injuries (i.e., cranium intact) and penetrating injuries (i.e., as a non- limiting example, a gun shot wound penetrating the cranium) .

The calpain inhibitors useful in the present invention are selected to provide improvement in neurological outcome or improvement as measured by indices of brain/tissue damage, following brain or spinal cord injury, or stroke.

As used herein, "improvement in neurological outcome" refers at the very least, to improvement in any dimension of emotional/affective state, memory deficits, motor performance, and higher order cognitive performance, or improvement in indices of brain/tissue damage, including asseεsments of contusion, mass legion and/or infarction.

Brain/tisεue assessments may be made non-invasively, by computerized tomograph, and/or by magnetic resonance imaging or spectroscopy . Preferably, there will be substantial performance in any dimension of emotional/affective state, memory deficits, motor performance, and higher order cognitive performance or brain/tissue indices. Most preferably, there will be improvement in the quality of life of the patient as measured, for example by the Glasgow Outcome Scale.

The calpain inhibitors of the present invention generally are selected to provide for reduction of cytoskeletal protein^" loss in an animal after such brain or spinal cord injury, or stroke. The cytoskeletal proteins of interest are generally tau protein, microtubule associated protein 2, and neurofilament proteins. Specific neurofilament protein of preferred interest include NF200, NF68 or NF150.

Calpain inhibitors useful in the practice of the present invention are generally selected from the peptide aldehyde class of inhibitors. An inhibitor is acceptable if the benefit gained from the improvement in neurological outcome, or improvement in brain/tissue indices, or the reduction of cytoskeletal protein loss, outweighs any resulting toxicity or side effect.

Peptide aldehydes suitable for use in the present invention are peptides which comprise at least two of the amino acid residues selected from the group of residues consisting of tryosine, methionine, leucine, lysine, arginine, valine and isoleucine, with one of the residues selected being in the form of an aldehyde derivative of that residue and wherein the aldehyde derivative is positioned at the C-terminal of the peptide. Preferably the aldehyde derivative is selected from the group consisting of norleucinal and methioninal . More preferably, the peptide aldehyde comprises at least one leucine residue positioned adjacent the aldehyde derivative, with the aldehyde derivative selected from the group consisting of norleucinal and methioninal . Even more preferably, the peptide aldehyde comprises at least two leucine residues positioned in the two amino acid positions nearest the aldehyde derivative, with the aldehyde derivative selected from the group consisting of norleucinal and methioninal . Preferably, the peptide aldehyde comprises in the range of about 2 to 6 amino acid residues, most preferably in the range of about 2 to 3 residues.

Non- limiting examples of inhibitors suitable for use in the present invention include calpain inhibitor I, calpain inhibitor II and MDL28170. Calpain inhibitor I is N-acetyl-leucine-leucine-norleucinal and calpain inhibitor II is N-acetyl-leucine-leucine-methioninal . The preferred calpain inhibitors for use in the present invention are calpain inhibitor I and calpain inhibitor II, with calpain inhibitor II being the most preferred.

It is to be understood that modifications and changes may be made in the structure of the inhibitor peptides of the present invention and still obtain a functional molecule with desirable characteristics. For example, it is well known in the art that substitution may be made between amino acids utilizing either a the hydropathic amino acid index or hydrophilicity values to obtain a biologically equivalent peptide. Exemplary substitutions which are well known to those of skill in the art include: arginine and lysine; and valine, leucine and isoleucine.

The time for administration of the calpain inhibitor is important. Generally, in the practice of the present invention, the calpain inhibitor should be administered as soon after the "event" (i.e., traumatic brain injury, cerebral ischaemia, spinal cord injury or stroke) as possible, with the likelihood of success for improvement in neurological outcome, or likelihood for improvements in brain/tissue indices, or likelihood to provide for reduction of cytoskeletal protein loss, all decreasing the longer after the event commencement of calpain inhibitor administration occurs.

The administration of calpain inhibitors to the cerebrovasculature is also within the scope of practice of the present invention. The cerebrovasculature can be a target of calcium-activated proteolysis under pathological circumstances. As in other cell types, elevated intracellular calcium is a critical trigger of cellular pathology in endothelial and vascular smooth muscle cells. One pathological condition in which calcium-activated proteolysis appears to play a crucial role iε cerebral vasospasm following a subarachnoid hemorrhage. Cerebral vasospasm is a spastic narrowing of large cerebral vessels in the vicinity of a subarachnoid blood clot, and this phenomenon represents the primary cause of mortality and morbidity after subarachnoid hemorrhage from a ruptured aneurysm. Vasospasm is thought to arise from the release of spasmogenic substances, such as hemoglobin, from lysed blood cells in the subarachnoid clot. One major cellular response observed under these conditions is an elevation of intracellular calcium in the cells of the vessel wall. Recent studies indicate that calpain is activated in the wallε of spastic ^"arteries and that this proteolytic response plays an important role in the response of the injured vessel. In experimental models of subarachnoid hemorrhage, a reduction in the native form of calpain and an elevation in the autolyzed form of calpain have been demonstrated in the wall of the basilar artery. The concentrations of calpastatin, an endogenous inhibitor of calpain, are reduced in spastic arteries, while the levels of proteolytic fragments of spectrin are increased. These findings indicate that calpain is functionally activated, and that its endogenous regulatory mechanisms are attenuated in spactic cerebral vessels after subarachnoid hemorrhage .

The exact manner in which calpain-mediated proteolysis contributes to cerebral vasospasm is unknown, but multiple mechanisms are likely to be involved. Calpain substrates found in vascular smooth muscle and endothelial cells include structural and regulatory proteins that participate in the maintenance of cerebrovascular function. One substrate that has received a great deal of attention in the context of cerebral vasospasm is protein kinase C (PKC) . Stimulation of PKC in normal vessels elicits a strong and long-lasting constriction. During vasospasm, PKC activity is enhanced, and kinase inhibitors capable of blocking PKC activity can attenuate the vasoεpastic response. It is conceivable that a limited proteolysis of PKC by calpain contributes to this constrictor response. Limited proteolysis of PKC converts the kinase to an activated form with a reduced requirement for calcium. This partial proteolysiε diεεociates the catalytic and regulatory εubunitε of PKC, an effect that would enhance the activity of the kinase and facilitate vasoconstriction. Consistent with thiε idea are observations that an inhibitor of the catalytic subunit of PKC attenuates cerebral vasoεpasm, while an inhibitor of the regulatory subunit of PKC is ineffective. The concept that a limited proteolysis of PKC by calpain contributes to cerebral vasospasm is an attractive hypothesis. Calpain inhibition appears to be a potential therapeutic procedure for reducing PKC activation in this spastic response.

Other calpain substrates in vascular smooth muscle that participate in the control of vascular tone include myosin light-chain kinase (MLCK) , calponin, and caldesmon. To date, there is no direct evidence regarding the proteolysis of these substrates during vasospasm. However, an effect of calpain on any of these substrates could significantly modify vascular tone. For instance, partial proteolysis of MLCK reduces its requirements for calcium and calmodulin, an effect that can enhance its kinase activity. Increased phoshporylation of MLCK leads to increased contractile force and vasoconstriction. In this way, calpain-mediated proteolysis of MLCK could contribute directly to the increased vascular tone observed with vasospasm. The other vasomotor-related substrates for calpain, i.e., calponin and caldesmon, are thin filament- aεεociated proteinε that inhibit actomyoεin ATPase; these proteins are apparently involved in maintaining smooth muscle in a relaxed state. The functional impact of calpain on these proteins is unclear,- however, a substantial proteolytic response could attenuate their function. If this were to occur, calpain could serve to remove the endogeous relazant influences of calponin and caldesmon, an effect that would accentuate vasoconstriction and, thus, calpain inhibition would result in the amelioration of the such proteolytic reεponseε . Finally, calpain-mediated proteolysis may contribute to structural modifications observed after subarachnoid hemorrhage. Both endothelial and smooth muscle cells undergo severe structural changes during cerebral vasospasm. Proteolysis of cytoskeletal substrates such as spectrin could directly contribute to the structural compromise of vascular wall constituents during vasospamε . The numerous substrates for calpain in endothelial and smooth muscle cells make calcium-activated proteolysis a likely candidate to participate in vascular injury triggered by calcium.

The activation of calpain in spastic cerebral arteries and its potential to modify vascular tone raise the possibility that targeting this mechanism would likely be of therapeutic value af er subarachnoid hemorrhage . Recent studies indicate that calpain inhibitors can indeed attenuate cerebral vasospasm after subarachnoid hemorrhage . Topical application of calpain inhibitors to the basilar artery is effective in blocking the development of vasospasm and in reducing the intensity of an established vasoεpastic response. Thus, the ability to administer calpain inhibitors directly to the site of a subarachnoid hemorrhage to ameliorate blood-induces vasospasm would provide a useful adjunct to the surgical treatments.

Generally, in the practice of the present invention, administration of the calpain inhibitors will commence within 24 hours of the event. Preferably, commencement of the administration of the calpain inhibitors will begin within 12 hours of the event, more preferably within 6 hours of the event, even more preferably within 3 hours of the event, and still more preferably within 1 hour of the event, and most preferably within 30 minuteε of the event.

To provide treatment as rapidly as possible, the inventors anticipate that calpain inhibitors of the present invention will be made available where there is a high likelihood of brain injury, spinal cord injury or strokes. For example calpain inhibitors may be provided to emergency response crews, in emergency rooms, in battlefield medical kits, at potentially dangerous sporting events, to police, and the like .

The calpain inhibitors of the present invention should be administered at a dose range that is suitable to provide improvement in neurological outcome, and/or improvement in brain/tissue indices, and/or to provide for reduction of cytoskeletal protein loss, that outweighs any side effects. It must also be understood, that the dose range is also a function of the route of administration. Generally, the calpain inhibitor dose range suitable for use in the present invention will be at least 0.01 mg/kg/hr. Preferably, the dose range is in the range of about 0.01 mg/kg/hr to about 20 mg/kg/hr. The calpain inhibitor dose range suitable for use in the present invention is more preferably in the range of about 0.1 mg/kg/hr to about 10 mg/kg/hr, even more preferably in the range of about 0.15 mg/kg/hr to about 7 mg/kg/hr, and most preferably in the range of about 0.2 mg/kg/hr to about 4 mg/kg/hr . The calpain inhibitors of the present invention should be administered for a duration treatment time that is suitable to provide improvement in neurological outcome, and/or improvement in brain/tisεue indiceε, and/or to provide for reduction of cytoεkeletal protein loεε, that outweighs any side effects. Of course, the duration treatment time will be dependent upon the dose rate and route of administration. Generally, the calpain inhibitors of the present invention will be administered for a duration treatment time of at least about 1 hour. In the practice of the present invention, the duration treatment time is preferably at least about 12 hours , more preferably at least about 24 hours, even more preferably at least about 48 hours, and most preferably at least about 72 hours . In the practice of the present invention, the calpain inhibitors may be administered via any suitable route of administration. Examples of non-limiting routes of administration include oral, intravenous, intraarterial, parenteral or intraperitoneal administration. The preferred route of administration is intervenous or interarterial administration, with the most preferred being intervenous administration. For orally administration, the calpain inhibitors of the present invention may be administered with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compresεed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active calpain inhibitor compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active calpain inhibitor compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

For administered parenterally or intraperitoneally, solutions of the active calpain inhibitor can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose . Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms of the calpain inhibitor suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

The administration of calpain inhibitors of the present invention may be utilized in combination with the application of hypothermia. U.S. Patent No. 5,486,204, issued January 23, 1996, to Clifton, for Method of Treating A Non-Penetrating Head Wound With Hypothermia", is herein incorporated by reference.

For hypothermia treatment, prior to or during the cooling process, the calpain inhibitors of the present invention are administered to the patient. Cooling is generally accomplished utilizing a cooling blanket, for example set at 5°C. The rate of cooling is generally selected to minimize injury to the patient, and is generally in the range of about 0.25°C/hr to about l°C/hr, preferably about 0.4°C/hr. The cooling process continues until the patient is εufficiently cooled to a holding temperature below normal body temperature εuitable to provide an improvement in neurological outcome. Generally, this holding temperature will be lesε than about 35 °C, preferably in the range of about 30°C to about 35°C, and more preferably in the range of about 32°C to about 33°C. The patient iε maintained at this holding temperature for at least about 1 hour, preferably at least about 12 hours, more preferably at least about 24 hours, and moεt preferably at leaεt about 48 hours. Subsequently, the patient is then gradually warmed to normal body temperature at a warming rate suitable to minimize damage to the body. This warming rate is generally at least about 0.75°C/4hrε, preferably in the range of about 0.75°C/4hrs to about 1.50°C/4hrs, more preferably in the range of about 1.14°C/4hrs to about 1.33°C/4hrs about, and most preferably at about l°C/4hrs. Calpain inhibitors are continued while the patient is maintained at the holding temperature, and during warming. Once the patient reaches 35°C, the calpain inhibitors are discontinued. A muscle and relaxant may be administered along with the calpain inhibitors, during cooling, maintaning at the holding temperature and during warming.

It must be understood that the present invention may be utilized along with any other acceptable medical procedures, pharmalogical agents or treatments.

EXAMPLES The following examples are provided merely to illustrate embodiments of the invention, and are not meant to limit the scope of the claims in any way. Those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EX.AMPLE 1 CALPAIN INHIBITORS PROTECT AGAINST DEPOLARIZATION

INDUCED NF PROTEIN LOSS OF SEPTO-HIPPOCAMPAL

NEURONS IN CULTURE

This Example examines the effect of a six min depolarization insult with 60 mM KCl and 1.8 mM, 2.8 mM, or 5.8 mM extracellular CaCl, on high (NF-H), medium (NF-M) , and low (NF-L) molecular weight NF proteins in primary septo-hippocampal cultures. One day after injury, Western blot analyses revealed losses of all three NF proteins. Increasing the extracellular calcium concentrations from 1.8 mM to 5.8 mM CaCl_ produced increased losεes of all three NF proteins to approximately 80% of control values in the absence of cell death. Calcium dependent losses of the NF proteins were associated with calcium dependent increases in calpain 1 mediated BDP to -spectrin. Calpain inhibitorε 1 and 2, applied immediately after depolarization and made available to cultures for twenty-four hours, reduced losses of all three NF proteins to approximately 14% of control values. The protective effects of calpain inhibitorε 1 and 2 were influenced by different levels of extracellular calcium. Qualitative immunohistochemical evaluations and Western blot data confirmed protection of NF losε by calpain inhibitors 1 and 2. These data indicate calpain inhibitors may represent a viable therapeutic strategy for preserving the cytoskeletal structure of injured neurons. Using Western blot analysis and immunohistochemistry a εix min potassium depolarization, similar to depolarization produced by experimental TBI and cerebral ischemia in vivo, produced losseε of NF-H, NF-M and NF-L proteinε of primary septo-hippocampal neurons in vi tro,

that were associated with calpain 1 mediated BPD of -spectrin. Losεes were calcium dependent, but not associated with cell death. Furthermore, calpain inhibitors 1 and 2, which inhibit calpainε, cathepsins, and to a leεεer extent chymotrypsin, strongly protected against losεes of all three NF proteinε even when administered after brief depolarization in vi tro .

A. MATERIALS AND METHODS

Materials

Standard reagents, including antibodies against NF-H, -M, -L, and GFAP were obtained from Sigma. Cell culture media were obtained from Gibco-BRL. Calpain inhibitors 1 and 2 were purchased from Boehringer-Mannheim. Gel electrophoresis and Western blotting reagents were purchased from Biorad.

2. Cell cultures Eighteen days old rat fetuses were removed from deeply anaesthetized dams. Hippocampi and septi were dissected in Ca²⁺/Mg²⁺-free Hanks balanced salt solution (HBSS) . After rinsing, cells were disεociated by trituration through the narrowed bore of a flame-constricted Pasteur pipette, resuspended in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum and distributed at a density of approximately 2.18 x 10⁵ cells/well to 16 mm poly-L-lyεine coated plaεtic culture dishes. Cultures were maintained in a humidified C0₂ incubator at 37°C. After 5 days of culture the media was changed to DMEM plus B18 supplement (Brewer and Cotman, 1989) . Subsequent media changes were carried out three timeε a week.

3. Brief depolarization injury

By day 10 in vi tro, astrocyte formed a confluent monolayer beneath morphologically mature neurons as judged by process formation and the ability to sequester microtubule associated protein 2 (MAP2) and tau. At that point, depolarization injury was performed by replacing normal media containing 5.3 mM KCl and 1.8 M CaCl₂ with medium containing 60 mM KCl and 1.8, 2.8, or 5.S ml. CaCl^ . The selection for dose ranges of extracellular calcium was chosen to provide reliable differences in calcium dependent NF loss. After 6 minutes, depolarization media were replaced with normal media. Control culture were exposed for 6 minute to normal media containing 5.3 mM KCl and 1.8 mM CaCl₂. Separate studies revealed no differences in NF immunoreactivity, or live and dead cell numbers among control cultures treated for 6 minutes with 5.3 mM KCl and 1.8 mM CaCl₂, 2.8 mM CaCl₂ or 5.8 mM CaCl, . 4. Calpain inhibitors

N-acetyl-Leu-Leu-norleucinal (calpain inhibitor 1) and N-acetyl-Leu-Leu-methioninal (calpain inhibitor 2) were prepared as 50 mM stock solutions in ethanol, diluted in DMEM plus B18 supplement and added in a final concentration of 5, 25, 50, 100, 150 μM (calpain inhibitor 1) or 5 , 25, 37.5, 75, 100 μM (calpain inhibitor 2) immediately after brief depolarization injury and were available to the cultures for 24 hours. The selection of dose ranges for calpain inhibitor 1 and 2 was based on preliminary studies and the manufacturer's recommendation.

Equivalent volumes of vehicle (ethanol) added to depolarized culture had no effect on NF losε . None of the calpain inhibitor 1 and 2 concentrationε exhibited toxic or trophic effects on control cultures, quantified by trypan blue staining and Western blot analysiε.

5. Sample Preparation

Sampleε were prepared using a modification of the method of Taft et al . (1992). Twenty-four hours after depolarization, media were removed, cultures were rinsed two times with sterile PBS, and 100 μl of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 1% Nonidet P-40™ (NP-40) , 0.5% SDS, and 0.02% sodium azide. To restrict protease activity and cytoskeletal protein degradation during sample procesεing, 2 mM EGTA, 1 mM EDTA, 100 μg/ml PMSF, 1 μg/ml aprotinin, and 0.1 mM leupeptin were added to culture wells. Lysates from 3 identical culture wells were pooled for each sample.

6. SDS-PAGE, Immunoblotting and Quantification

The amount of protein in samples was determined using BCA reagents (Pierce) with albumin standards. Protein-balanced samples were prepared for polyacrylamide gel electrophoresis in two-fold loading buffer containing 0.25 M Tris (pH 6.8), 0.2 M DDT, 8% SDS, 0.02% Bromophenol Blue, and 24% glycerol in distilled water. Samples were heated at 95°C for 5 minutes. Proteins were resolved in a vertical electrophoresiε chamber uεing a 4% acrylamide εtacking gel over a 7.5% acrylamide resolving gel. Gelε were run at constant voltage (200 V) for approximately 1 hour. 20 μg of sample protein was resolved in each lane. Following separation, proteins were immediately transferred to a nitrocellulose membrane using Western blotting (Towbin et al . , 1979) . Lateral transfer was employed using a transfer buffer made up of 0.192 M glycine and 0.025 M Tris (pH 8.3) with 10% methanol at a constant voltage of 100 V for 1 hour at 4°C. Blots were immediately blocked for immunolabeling by overnight incubation using 3% non-fat milk in 20 mM Tris HC1, 0.15 M NaCl, and 0.005% Tween-20™ at 4°C. Monoclonal antibodies specific for individual NF proteins were used for immunolabeling. Antibodies binding NF proteins were NR4 , recognizing phosphorylated and non-phoεphorylated NF-L, NN18 recognizing phosphorylated and non-phosphorylated NF-M, and N52 recognizing phosphorylated and non-phosphorylated NF-H (Shaw et al . ,

1986) . A polyclonal antibody recognizing glial fibrillary acidic protein (GFAP) , an astrocytic marker, was used to assess glial alterations and specificity of protein changes. Antibody 38, a polyclonal antibody that recognizes a calpain 1 mediated BDP of -spectrin (Roberts-Lewis et al . , 1994) was obtained from Cephalon

Inc . Following incubation with primary antibodies (dilution 1:1000 for NF and spectrin antibodies; and 1:8000 for GFAP antibody) for 2 hours, blots were incubated with a secondary antibody linked to horseradiεh peroxidase (dilution 1:12500) for 1 hour. Enhanced chemiluminescence (ECL; Amersham) reagents were used to visualize the immunolabeling on X-ray film. Semiquantitative evaluation of protein levels was performed via computer-assisted

2-dimenεional densitometric scanning (Biorad Model GS-670) . Data acquired in arbitrary densitometric units were transformed to percentages of the densitometric levels observed on scans from non-depolarized control cultures visualized on the same blot.

7. I unohistochemistry

Cultures were fixed with 4% formalin, permeabilized with 0.1% Triton X-100^® and blocked with 1% normal serum for 20 minutes. Cultures were then incubated for 2 hours with primary antibodies N52 (NF-H) and NN18 (NF-M) at dilutions used for immunolabeling of Western blots. In initial immunohistochemical experiments, however, NR4 labeling was determined to be too weak to be reliably examined in cultures. Peroxidase-conjugated secondary antibodies were applied at a concentration of 1:200 for l hour. Staining was visualized with DAB (1 mg/ml) and 0.002% H₂0₂. Wells without primary antibody did not stain.

8. Assessment of cell numbers and cell viability

Control and depolarized cultures were stained 24 hours after depolarization insult with fluorescein diacetate (FDA 11.5 μg/ml), which labels live cellε and with propidium iodine (PI 3.2 μg/ml) which stains dead cells. Both compounds were dissolved in phosphate buffer and left in contact with the cells for 10 minuteε . No fixation waε done before treatment. Cells were then observed with a fluorescence microscope. Cell loss was calculated for five wells in each εtudy as a percentage of total cell number.

9. Statistical Analysis

Except as noted, data from Western blots and cell counts were evaluated by analysis of variance (ANOVA) with a post-hoc Tukey-test. Values given are mean ± standard error of the mean (SEM) . Differences were considered significant when p < 0.05.

B . RESULTS 1. NF-L, -M, and -H losses after six minute

depolarization insult

Twenty-four hours after a six min depolarization with 60 mM KCl and 1.8 mM CaCl₂ NF-L immunoreactivity decreased 18% (17.63±1.41%) , NF-M immunoreactivity 12% (11.86±1.91%) and NF-H immunoreactivity 18% (17.80±1.47%) of control values .

Increasing the extracellular calcium concentration to

2.8 mM CaCl₂ produced increased losses of all three NF proteins. NF-L immunoreactivity decreased 63%

(62.93±3.59%) , NF-M immunoreactivity 39% (39.07±4.43%) and

NF-H immunoreactivity 51% (50.56±2.77%) of control values.

The losses of all three NF proteins were statistically greater after depolarization with 2.8 mM CaCl, as compared to 1.8 mM CaCl₂. (2.8 mM CaCl₂ vs. 1.8 mM CaCl_: p<0.001 for

NF-L; p<0.01 for NF-M; and p<0.001 for NF-H).

Depolarization in the presence 5.8 mM CaCl caused even greater decreases in NF-L, -M, and -H immunoreactivity. NF-L immunoreactivity declined 83% (82.73±3.66%) , NF-M immunoreactivity 71% (70.50±2.74%) , and NF-H immunoreactivity 66% (65.66+3.00%) of control values. The losses of all three NF proteins were statiεtically greater than in the preεence of 2.8 mM CaCl₂. (5.8 mM CaCl vs. 2.8 mM CaCl₂ p<0.01 for NF-L; p<0.01 for NF-M; and p<0.05 for

NF-H) .

Glial fibrillary acidic protein (GFAP) levels were unchanged after depolarization with 60 M KCl and 2.8 mM CaCl₂ or 5.8 M CaCl, . Coomassie blue staining confirmed that the same protein amount was loaded in each lane.

2. Depolarization causes loss of cytoskeletal

proteins in the absence of cell death

Brief depolarization insults with 60 mM KCl and 2.8 mM CaCl₂ or 5.8 mM CaCl, did not increase the numbers of dead cells 24 hours after the depolarization injury, as compared to non-depolarized controls. Depolarization injury with 60 mM KCl and 2.8 mM CaCl, or with 60 mM KCl and 5.8 mM CaCl₂ produced 3% (3.33±1.68%) and 4% (3.67±1.76%) dead cells, respectively, versus 2% (2.47+1.51%) dead cells in control cultures (p=0.87) .

3. Calpain 1 activation after depolarization

To further assess the possibility that depolarization with 60 mM KCl and 2.8 mM CaCl₂ or 5.8 mM CaCl, resulted in increased calpain activation, Western blots were stained with a polyclonal antibody against calpain 1 mediated BDP of -spectrin. Depolarization with 60 mM KCl and 5.8 mM CaCl, caused εignificantly more BDP than after depolarization with 60 mM KCl and 2.8 mM CaCl,, (p<0.001;

Studentε t-test; FIG. 2A and FIG. 2B) . These results indicate that calpain 1 activation is increased by this paradigm, since increased extracellular CaCl_? produced increased calpain 1 mediated spectrin BDP. 4. Effects of calpain inhibitor 1 and 2 on NF

protein loss after depolarization in the presence

of 2.8 mM and 5.8 mM extracellular CaCl₂

Tables 1 and 2 summarize the results of studies systematically examining the effects of administration of varying doseε of calpain inhibitor 1 and 2 following depolarization at 2.8 mM and 5.8 mM extracellular calcium.

TABLE 1

EFFECTS OF CALPAIN INHIBITORS 1 AND 2 ON NF PROTEIN LOSS AFTER DEPOLARIZATION WITH 60 mM KCl + 2.8 mM CaCl₂

Cl (μM) NF-L NF-M NF-H

0 62.93 + 3. .59 39.07 ± 4.43 50.56 ± 2.77

5 58.43 + 3. .72 33.36 ± 1.42 42.63 ± 2.52

25 45.76 + 2, .83* 25.36 + 1.40* 36.90 ± 1.91*

5500 4411..2266 +± 33.. .3355**** 19.53 ± 3.40** 26.50 ±

2.95***

100 49.60 + 2 .92 29.96 ± 2.91 39.66 ± 3.22

150 50.07 + 2 .32 29.70 ± 2.60 42.73 ± 2.06

C2 (μM) NF-L NF-M NF-H 0 62.93 ± 3.59 39.07 ± 4.43 50.56 ± 2.77

5 61.92 ± 4.33 40.93 ± 5.08 53.76 ± 4.28

25 58.83 ± 4.02 40.63 ± 3.42 52.16 ± 3.90

37.5 63.50 ± 4.31 39.16 ± 4.39 50. 0 ± 2.94

75 61.53 ± 2.77 40.33 ± 3.17 52.93 ± 6.13 100 60.86 ± 5.36 45.63 ± 5.74 50.83 ± 3.34

Values are means (±SEM) of 3 studies. *p<0.05; **p<0.01; ***p<0.001 as compared to depolarized cultures without calpain inhibitor l (Cl) or calpain inhibitor 2 (C2) . (ANOVA followed by post -hoc Tukey test)

TABLE 2

EFFECTS OF CALPAIN INHIBITORS 1 AND 2 ON NF PROTEIN LOSS AFTER DEPOLARIZATION WITH 60 mM KCl + 5.8 mM CaCl₂

Cl NF-L NF-M NF-H (μM)

0 82.73 ± 3.66 70.50 ± 2.74 65.66 ± 3.00

5 64.93 ± 2.10** 52.40 ± 1.65** 43.40 ± 2.75*** 25 51.43 + 1.98*** 40.63 + 2.39*** 40.36 ± 2.61***

50 38.46 ± 1.51*** 36.76 + 2.05*** 34.43 ± 2.13***

100 43.26 ± 1.47*** 42.20 ± 3.87*** 43.10 ± 4.25***

150 44.70 ± 3.87*** 54.30 ± 1.84* 54.86 ± 2.29*

C2 NF-L NF-M NF-H

(μM)

0 82.73 ± 3.66 70.50 ± 2.74 65.66 + 3.00

5 54.00 ± 1.86*** 40.06 ± 2.30*** 31.63 ± 2.08***

25 41.33 + 2.36*** 36.23 ± 2.93*** 19.53 ± 3.12*** 37.5 18.10 ± 2.41*** 17.86 ± 2.00*** 13.50 ± 2.37***

75 51.10 ± 2.65*** 38.83 ± 3.55*** 35.40 ± 2.57***

100 52.76 ± 1.61*** 43.73 + 1.56*** 45.20 ± 2.78***

Values are means (±SEM) of 3 studies. *p<0.05,- **p<0.01; ***p<0.001 as compared to depolarized cultures without calpain inhibitor 1 (Cl) or calpain inhibitor 2 (C2) . (ANOVA followed by post-hoc Tukey test) Two concentrations, 25 μM and 50 μM of calpain inhibitor 1 protected against NF loss after depolarization in the presence of 2.8 mM CaCl_;.. 50 μM calpain inhibitor 1 was most effective and reduced NF-L loss to 415 (41.26±3.35%) , NF-M loss to 20% (19.53±3.40%) , and NF-H losε to 27% (26.50±2.95%) . 25 μM calpain inhibitor 1 revealed εmaller but still significant protective effects against losses of all three NF proteins. NF-L losε was reduced to 46% (45.76+2.83%), NF-M losε to 25% (25.36±1.40) and NF-H loss to 37% (36.90+1.191%). Calpain inhibitor 2 was ineffective against NF protein losε after depolarization with 60 mM KCl and 2.8 mM CaCl₂.

All concentrations of calpain inhibitor 1 protected against loss of NF proteins after depolarization with 5.8 mM CaCl₂. 50 μM of calpain inhibitor 1 revealed the highest protection against losε of all three NF proteinε. NF-L loεε was reduced to 38% (38.46±1.51%) , NF-M loss decreased to 37% (36.76±2.05%) , and NF-H loss decreased to 34%

(34.43±2.13%) . All concentrationε of calpain inhibitor 2 alεo protected against NF loss after depolarization with 5.8 mM CaCl₂. Administration of 37.5 μM calpain inhibitor 2 provided greatest protection against losε of all three NF proteins. NF-L protein loεε decreased to 18% (18.10±2.41%) , NF-M loss to 18% ( 17.85±2.00%) , and NF-H loss to 14% (13.50±2.37%) . The graphical presentation of calpain inhibitor 1 and 2 data after depolarization with 5.8 mM CaCl_? suggested a u- shaped dose response curve. The comparison of the highest protective concentrations for calpain inhibitor 1 (50 μM) and calpain inhibitor 2 (37.5 μM) , revealed that calpain inhibitor 2 produced greater protection than calpain inhibitor 1 from losses of all three NF proteins. (Calpain inhibitor 2 vs .

1 p<0.01 for NF-L; p<0.01 for NF-M; and p<0.01 for NF-H; Students- -test) .

5. Immnohistochemistry

Neuronal immunolabeling showed morphological correlates of the Western blot data. Immunohistochemical studies with NN18 (NF-M) produced strong labeling of cell soma and neurites in depolarized and control cultures. Fine neurites appeared to be lost after depolarization with 2.8 mM CaCl₂ and 5.8 M CaCl, . Both calpain inhibitors, applied immediately after depolarization with 60 mM KCl and 2.8 mM CaCl₂ or 5.8 mM CaCl protected against losses of fine neurites. For .example, 37.5 μM calpain inhibitor 2 revealed high protection against loss of fine neurites compared to depolarized cultures without calpain inhibitor 2. Immunolabeling with N52 (NF-H) produced similar labeling patterns to NN18.

C. DISCUSSION The present study haε provided evidence that NF protein loεs following a six min potassium depolarization was accompanied by appearance of calpain l mediated BDP of α-spectrin. Losses of the high-, medium-, and low molecular weight NF proteins and appearance of spectrin BDP in vitro were calcium dependent and not due to cell death.

Furthermore, it was shown that calpain inhibitors 1 and 2 provided strong protection against depolarization induced loss of the NF triplet proteinε.

The loss of NF proteins in this Example is at least partially attributable to overactivation of calpain. The calcium dependency of NF protein loss shown in this Example suggests that the intracellular, calcium dependent protease, calpain, rather than cathepεinε, trypsin and/or chymotrypsin are primarily responsible for losses of these cytoskeletal proteinε in the in vi tro syεtem.

Furthermore, it waε εhown that brief depolarization resulted in a calcium dependent appearance of calpain 1 mediated BDPs of α-spectrin. In this Example significant protection by calpain inhibitors 1 and 2 against neuronal cytoskeletal loss was detected.

Immunohistochemical studies revealed losses of fine neurites 24 hours after depolarization. Although it is impractical to definitively characterize neuritic processes as dendritic, axonal or both in high density cultures, other lines of evidence suggest that dendritic loεε may occur. Appearance of the spectrin BDPs indicated that calpain 1 activation occurred after depolarization.

The present Example provides evidence that calpain inhibitors 1 and 2 can reduce depolarization- induced loss of the NF triplet proteins in vi tro . Since NFε, together with microtubule polypeptides and spectrin, are components of three major cytoskeletal syεtemε of neurons, these data support the hypothesiε that these inhibitors are potential agents for protecting cytoskeletal integrity following experimental brain injury in vivo and in vi tro .

In this Example, calpain inhibitorε 1 and 2 provided marked protection against losses of all three NF proteinε even after experimental neuronal injury in vi tro, supporting the therapeutic potential of these agents. Both calpain inhibitors 1 and 2 strongly protected against losseε of all three NF proteins after depolarization injury with 60 mM KCl and 5.8 mM CaCl₂.

EXAMPLE 2 CALPAIN INHIBITORS REDUCE DEPOLARIZATION INDUCED

LOSS OF TAU PROTEIN IN PRIMARY SEPTO-HIPPOCAMPAL

CULTURES

This Example studies the effects of a six min potassium depolarization injury produced by 60 mM KCl and 1.8 mM or 5.8 mM extracellular CaCl₂ on tau protein levels in primary rat septo-hippocampal cultures .

One day after injury, Western blot analyses revealed a calcium dependent loss of tau protein of approximately 50% of control values. Loss of tau protein was associated with calpain 1 mediated BDP to α-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization injury and available to cultures for twenty-four hours reduced depolarization induced degradation of tau protein to approximately 35% or 25% of control values, respectively. These data indicate calpain inhibitors l and 2 represent a viable strategy for preserving the cytoskeletal structured of injured neurons.

1. MATERIALS AND METHODS

Primary septo-hippocampal cells (ratio 1:1) were prepared from 18-day old rat fetuses and distributed at a density of 2.18 x 10⁵ cells/well to 16 mm poly-L-lysine coated plastic culture dishes and kept in a humidified C0₂ incubator. After five days in media with serum, cultures were maintained in B18 media (Brewer and Cot an, 1989) . After ten days in culture, astrocyteε formed a confluent monolayer beneath morphologically mature neurons as judged by procesε formation and the ability to εequester microtubule asεociated protein 2 and tau. At that point a six minute depolarization injury was performed by replacing normal media containing 5.3 mM KCl and 1.8 mM CaCl_? with media containing 60 mM KCl and 1.8 mM CaCl₂ or 5.8 mM CaCl₂. Control cultures were exposed for six minutes to media containing 5.3 mM KCl and CaCl_.-. concentrations identical to that of depolarized cultures. At the end of six min, depolarization media was replaced with normal media (5.3 mM KCl and 1.8 mM CaCl₂) .

2. RESULTS

Twenty-four hours after depolarization with 60 mM KCl and 1.8 mM CaCl₂, tau immunoreactivity (phosphorylated and non-phosphorylated tau quantified by Western blot analysis) decreased 10% (10.45+2.90%) as compared to non-depolarized control cultures. Increasing the extracellular calcium concentration produced an increased loss of tau protein. Tau immunoreactivity declined 51% (50.71±2.84%) of control values after depolarization with 60 mM KCl and 5.8 mM CaCl . The loss of tau protein was significantly greater after depolarization with 5.8 mM CaCl₂ as compared to 1.8 mM CaCl₂ (p<0.001, Student's t-test) .

To further investigate if decreases in tau protein levels were neuron specific, glial specific or both, purified cultures of septo-hippocampal astrocytes prepared by the method of McCarthy and de Vellis (1980) were depolarized using a protocol identical to that used in mixed (glial-neuronal) septo-hippocampal cultures. Western blot analyses of purified cultures of astrocytes did not produce positive tau immunoreactivity after either depolarization or control treatment. These data suggest that astrocytic tau did not contribute to depolarization induced changes in tau immunoreactivity observed in mixed glial -neuronal cultures. Moreover, levels of glial fibrillary acidic protein (GFAP) , an astrocytic marker, were unchanged in mixed septo-hippocampal cultures after depolarization (FIG. 3B) . Coomassie blue staining confirmed that the same protein amount was loaded in each lane (FIG. 3C) .

To assess the possibility that brief depolarization in the presence of 5.8 mM CaCl₂ produced increased calpain activation, Western blots were stained with a polyclonal antibody (dilution 1:1000) against calpain 1-mediated BDP of α-spectrin (Roberts-Lewi et al . , 1994). Although

calpain 1 mediated BDP of α-spectrin were undetectable in control cultures, they were readily apparent after depolarization in the presence of 5.8 mM CaCl₂.

Since overactivation of calpains may be implicated in the pathology of TBI and cerebral ischemia, inhibition of proteases including calpains may be an attractive therapeutic strategy for treating injuries of the central nervous system. Calpain inhibitors l and 2 were applied immediately after depolarization injury of septo-hippocampal neurons in vi tro with 5.8 mM CaCl₂ at final concentrations of 5, 25, 50, 100 and 150 μM for calpain inhibitor 1 and of 5, 25, 37.5, 75 and 100 μM for calpain inhibitor 2. The inhibitors were available to the cultures for 24 hours. None of the calpain inhibitor 1 and 2 concentrations exhibited toxic or trophic effects on control cultures quantified by trypan blue staining and Western blot analysiε . After depolarization with 5.8 mM CaCl 25 μM and 50 μM calpain inhibitor 1 gave similar protective effects reducing tau protein loss to about 35% (to 34.67±2.88% and 33.83+3.30% relative to controls for 25 μM and 50 μM respectively. Calpain inhibitor 2 gave significant protection against loss of tau protein after depolarization injury with 5.8 mM CaCl₂ at three concentrations. 25 μM reduced tau protein loss to 35% (35.34±2.91%) . 75 μM reduced tau protein loss to 38% (37.99±2.22%) . 37.5 μM calpain inhibitor 2 provided greatest protection against tau degradation, and reduced tau protein losε to 22% (22.24±2.10%) . At their optimal concentrations, calpain inhibitor 2 (37.5 μM) produced greater protection than calpain inhibitor 1 (50 μM) against loεs of tau protein (p<0.01; Student's t-test) .

As shown in this Example, brief depolarization injury of septo-hippocampal neurons in vi tro, similar to acute depolarization produced by experimental cerebral ischemia and TBI in vivo, causes a calcium dependent loss of

phosphorylation independent tau protein. Significant protection by calpain inhibitors 1 and 2 against degradation of tau protein was detected.

The present Example demonstrates that calpain inhibitors 1 and 2 can reduce losε of tau protein after brief potaεεium depolarization of CNS cellε in vi tro . Since microtubule polypeptides together with NFs and spectrin are components of the three major cytoskeletal systems of neurons, the data further support the use of calpain inhbitors 1 and 2 as agents for protecting cytoskeletal integrity following experimental neuronal injury in vivo and in vi tro .

EXAMPLE 3 CYTOSKELETAL DERANGEMENTS OF CORTICAL

NEURONALPROCESSES THREE HOURS AFTER TBI

This Example studied use of antibodies against NF68 (Sigma NR4) , NF200 (Sigma N52) , and MAP2 (Sigma AP-20) to demonstrate the cytoskeletal derangements in a widely-used model of experimental mechanical brain injury, lateral cortical impact injury (Dixon et al . , 1991) . Antibodies were specifically selected to examine axons as well as dendrites. The decision to focus on cortical regions 3 hours post-TBI was based on previous Western data showing MAP2, NF68 and NF200 loss in cortical homogenates but not in hippocampal samples at this early time point (Posmantur et al . , 1994). Cortical impact injury produced profound alterations in NF68, NF200, and MAP2 immunofluorescence including reduction in labeling of neuronal cell bodies and dendritic fragmentation at 3 hours post-TBI. Changes observed in immunofluorescence were evident in apical, basal, and arborized dendrites within (overlaying subarachnoid hemorrhage) and beyond areas of cortical contusion. Confocal microscopy revealed loss of NF asεembly state associated with immunofluorescence alterations. Little evidence of axonal involvement was detected at 3 hours post-TBI. These studies suggeεt that derangements of NF68, NF200 and MAP2 as early as 3 hours after lateral impact injury occur preferentially in dendrites rather than axonε within and beyond areas of contusion .

A . METHODS

1. Treatment Groups and Experimental Procedures This Example employed a controlled cortical impact device at a magnitude sufficient to produce cortical contusions similar to those seen after severe human injury (Dixon et al . , 1991). Cortical impact models utilize a pneumatic piston to deform a specifiable volume of expoεed cortex over a range of impact velocities . The magnitude of cortical impact injury in thiε study (6 m/s) produces over disruption of the overlying vasculature from approximately -1.5 to -4.2 Bregma. In addition thiε magnitude of impact is associated with significant motor and spatial memory deficits (Ham et al . , 1992).

Thirty-male Sprague-Dawley rats (250-350 g) were used in this Example. Treatment groups (n=8-10/group) included naive, sham-injured and injured rats at 3 hours post-TBI. Sham-injured animals underwent identical surgical procedures but did not receive cortical impact injury. To induce cortical impact injury, rats were intubated and anesthetized with 2% isofluorane in a 2:1 mixture of N₂0/0_? . Core body temperature was monitored continuously by a rectal thermistor probe and maintained at 37-38°C. Two 7-mm diameter craniotomies were performed adjacent to the central εuture, midway between lambda and bregma. The dura waε kept intact. Injury was produced by impacting the right (i.e. ipsilateral) exposed cortex with a 6 mm

diameter tip at a rate of 6 m/sec and a 2 mm compresεion (Miyazaki et al . , 1992) to assist in standardizing injury

levels . Impact velocity is directly measured by the linear velocity differential transformer (LVDT) that produces an analog signal recorded by a PC based data acquisition system for analyseε of time/diεplacement parameterε of the impactor. Immediately after injury the anesthetic gases were discontinued in order to minimize the anesthetic effects on the acute neurological assessments. Following TBI animals were immediately assessed for recovery of reflexes (Dixon et al . , 1991). Animalε which recovered righting reεponse in 5 minutes or lesε or omitted from the Example .

2. Histopathological Assessment

Animalε from all treatment groupε were given a lethal injection of phenobarbital (0.5 cc) prior to perfuεion. Rats were transcardially perfused through the left ventricle (120 ml of 0.9% saline and 200 ml of 10% buffered formalin) 3 hours after injury and were sliced coronally at 3 μm intervals . Paraffin sections were used to obtain thin sections and optimal hematoxylin and eosin (H&E) staining. Brain sections were processed through graded alcohols and xylenes prior to embedding in paraffin. Sections were cut at 4-5 μm on a microtome, from +0.2 to -3.8 Bregma, mounted on glass slideε and stained with H&E. 3. Immunofluorescent Histoc emistry

Fixative solutions for the selected antibodies included 4% paraformaldehyde for anti-NF200 (Sigma N52) and 4.2% formalin for anti-NF68 (Sigma NR4) and anti-MAP2 (Sigma AP-20) immunolabeling. The brain waε then removed and incubated in 30% εucrose overnight for adequate cryoptection. The brain waε then groεεly sectioned, frozen, and mounted in a Hacker-Bright cryostat. Coronal sections of 30-40 μm thickness were cut at -15°C and immediately placed into wells containing PBS (136 mM NaCl, 81 mM KCl and 1.6 mM Na_?HP0₄ and 14 mM KH₂P0₄, pH 7.4) .

The entire immunolabeling procesε waε performed in 24 -well culture plates. Sections were first incubated in 3% horse serum at 4°C for two hours. Primary antibodies (Sigma NR4 , N52, and AP20) in blocker εolution (Tween- PBS:10 mM NaP0₄ [pH 7.5], 0.9% NaCl, Tween-20^®, 1% antifoam A (Sigma A-5758) and 5% non-fat dry milk (Carnation, Inc. were incubated for 3 hourε at 25°C. The tiεεue slices were then washed with blocker solutionε 3 x 10 minuteε . Secondary antibody (anti-mouεe IgG) linked to a εpecific fluorophore was applied for two hours. Fluorescein (Vector labs) was used to detect NF68, as well as MAP2 , and rhodamine (Boehringer-Mannheim) was used to detect NF200. The tissue εliceε were then washed 3 timeε in PBS εolution, mounted on εubbed εlides (Fisher 12-550-15) , and allowed to dry. Dried sections were then coverεlipped with Elvanol^®

(Dupont) and stored in the dark prior to clasεical immunofluorescence and/or confocal microscopy (Molecular Dynamics) examination. Control sections without primary antibodies did not stain.

4. Confocal Microscopy Confocal microscopy was performed to better examine NF infrastructure following TBI. NF200 (Sigma N52) immunofluorescence was selected because the N52 antibody does not detect proteolytic fragments and thus reflects a more accurate depiction of NF assembly state. Confocal microscopy was performed using a Nikon-Diaphot inverted microscope and Molecular Dynamics laser scanning confocal system, incorporating a mercury lamp light source. The data acquisition and analysis employed a Silicon Graphics Indigo computer station. The internal and external pyramidal cell layers in ipsilateral and contralateral cortical tissue were examined. Pyramidal cells and their dendritic processes are of a sufficient size that visualization of intracellular morphology (i.e., NFs) was within the limits of confocal microscopy. Ten to fifteen representative sections were selected from naive, sham-injured and injured animals after preliminary asseεsment by classical immunofluorescence. All tissue sections were serially scanned at 600X. Confocal photomicrographs represent an individual section from a set of serial sections (n=20-25) through the entire z-plane of a single neuron and its processes. Serial sectioning through an entire neuron ensures that changes observed with immunofluorescence are not due to microscopy artifactε that can result when neuronal structures are not within a single focal plane.

5. Binding Characteristics of Primary Antibodies

All three antibodies were selected to minimize labeling alterations due to change sin phosphorylation state which may occur after TBI. NR4 (anti-NF68) is targeted against the α-helical domain common to all NF protein at a site independent of phosphorylation state, thus detecting total NF 68 protein including low molecular weight (MW) NF fragments in somatodendritic and axonal regions (Shaw, 1986) . N52 (anti-NF200) is targeted against the carboxyl terminal at a site independent of phosphorylation state and detects total protein (Shaw, 1986) . However, N52 does not retain antigenicity to the low molecular weight α-helical containing proteolytic fragments. AP-20 (anti-MAP2) detectε MAP2 only in neuronal somato-dendritic regions at a site independent of phosphorylation state and does not detect lower molecular weight proteolytic fragments.

6. Hematoxylin and Eosin Examination of

Histopathology of Cortical Impact Injury

Sham rat brains showed no discernable histological alterations in either cortical hemisphere. The injured brain was characterized by focal subpial and intracortical acute hemorrhage. Ipsilateral superficial cortical contusions overlay an area of cortical damage manifesting large numbers (80-90%) of dark, markedly shrunken neurons in an area approximately 3-4 mm in maximum transverse direction (at -3.4 Bregma). This central area showed a gradual transition to normal cortex on either side with decreasing numbers of dark, shrunken neurons. There was also a vacuolar appearance to the neuropil in the deeper cortex. The contralateral cortex showed a well-defined area corresponding to a contrecoup lesion in an area 1-2 mm wide approximately 0.5-1 mm below the pial surface. High power (520X) H&E staining revealed triangular neurons with darkened eosinophilic cytoplasm and pyknotic nuclei. Histopathological changes rostral to cortical impact (at + 0.2 Bregma) revealed pallor and dark shrunken neurons only in the superficial ipsilateral and contralateral cortical layers (1-3), with the most remarkable pathology occurring ipεilateral to the side of the injury. No remarkable changes were observed in pyramidal neuronal layer 5. Similar histopathological changes were also observed caudally to the site of impact within ipsilateral and contralateral cortices.

7. Sham Injury Did Not Affect NF68, NF200, and MAP2

Immunofluorescence

All sham animals (n=6) were compared with naive rats to determine the effect of animal surgery (craniotomies) in the absence of TBI. Ipsilateral and contralateral cortical tissueε were examined rostrocaudally from +0.2 mm Bregma to -3.8 Bregma. Naive NF68, NF200, and MAP2 immunofluorescence labeling in cortex did not differ from sham- injured animals at 3 hours post-TBI. Cortical immunolabeling in uninjured rats revealed prominent pyramidal neurons in layers 3 and 5 with long extending apical dendrites. Pyramidal neurons from naive and sham- injured cortices possess long extending apical and basal dendrites arranged in a linear fashion. Layer 1 revealed massive dendritic arborization. Some coronal sectionε uεing NF68 displayed extensive axonal labeling extending from the cortical layers into the corpus callosum.

8. TBI Produced Derangements in NF200

Immunofluorescence Labeling of Cortical Neurons

TBI produced prominent alterations in the labeling pattern of cortical neurons detected by anti-NF200 (Sigma N52) . A fragmented pattern of apical dendrites and loss of fine procesεes waε evident throughout the cortical layers in the ipεilateral cortex. Fragmented apical dendrites were also detected in a well defined focal area contralateral to the site of injury, 1-2 mm wide. Normal cortical labeling was re-established lateral to the affected area in the ipsilateral and contralateral corticeε. In addition, there was a prominent loss of fine dendritic procesεeε within layer 1 in comparison to sham-injured animals. The dramatic reduction of NF200 immunoreactivity was observed in the area of contusion from -1.5 Bregma through -3.8 Bregma rostrocaudally, corresponding to regions showing morphopathology characteristic of injured neurons. Changes in NF200 immunoreactivity rostrocaudally were characterized by a disappearance of the overt distinction between cortical pyramidal layerε 3 and 5. NF200 immunoreactivity loεs 3 hours post-TBI was not apparent in white matter tracts including the corpus callosum and internal capsule.

Confocal microscopy of anti-NF200 immunofluorescence revealed that fragmented apical dendrites contained varying degrees of NF disaεsembly. In contrast, sham- injured pyramidal neurons contained NFε showing defined, linear characteristics throughout their apical dendrites . Changes in NF assembly were found in injured neurons within the cortex ipsilateral and contralateral to the site of impact. By using microscopy to scan through the z-plane of an entire neuron with several sectionε (n=30) , changes observed with classical immunofluorescence could be further verified. These data also indicate that NF loss could occur in focal regions along dendrites and that, in some instances, the plasmalemma may still be intact, even when disaεsembly of NFs is quite apparent.

9. MAP2 immunoreactivity loss was detected within

injured cortex

TBI produced a loss of MAP2 immunoreactivity in cortical neuronal processes using anti-MAP2 (AP-20) , aε compared to εham-injured animalε. Loεses were observed through the rostrocaudal extent studied: -1.5 Bregma to -3.8 Bregma. This fragmented pattern in apical dendrites and fine processes was similar to that detected with anti-NF200 (N52) antigenicity . A smaller focal area contralateral to the injured site also manifested dendritic fragmentation .

10. NF68 Immunofluorescence Revealed Less Dramatic

Derangements in Apical Dendrites than Detected

with NF200 Immunofluorescence

NF68 (Sigma NR4) immunoreactivity 3 hours post-TBI was altered but not dramatically as observed with anti-NF200. Losses of NF68 immunoreactivity after injury resulted in the disappearance of the pyramidal neuronal lamina, especially in layer 3, in contrast to the clear laminar organization seen in sham- injured animalε. Anti-NF68 at low power (160X) demonstrated the appearance of a more continuous plasmalemma and far fewer fragmented- like apical dendrites than seen with anti-NF200 following injury. The appearance of vacuoles was a feature of dendritic alterations 3 hours post-TBI that was not apparent in sham-injured animals. Higher magnification (512X) revealed small breaks, an increase in immunoreactivity adjacent to the plasmalemma, and an absence of immunolabeling in focal areas within cortical apical dendrites. In the contralateral injured cortex, NF68 immunoreactivity revealed limited loss compared to that seen with NF200 immunofluorescence. Broken and vacuolized neuronal processeε were found throughout the roεtrocaudal extent of tissue studied (+0.2 Bregma to -3.8 Bregma). Furthermore, fine dendritic arborization within layer 1 was dramatically reduced in ipsilateral injured cortex relative to εham-injured cortex. Minimal alterationε in axonal NF68 immunoreactivity alterationε were observed in white matter tracts such aε the corpus callosum following TBI, as compared to sham injured controls. In particular, there was an absence of retraction bills characterized by focal NF68 immunoreactivity which have been previously described in injured white matter tracts at later time points after injury (Yaghmai and Povlishock, 1992) . 11. Alterations in MAP2, NF200, and NF68

immunolabeling were found in brain areas rostral

of cortical contusion

MAP2 immunofluorescence alterations (i.e., fragmented

apical dendrites) were observed rostral to cortical impact (at +0.2 Bregma) as compare to sham injured animals following TBI. Importantly, changeε in MAP2 immunofluoreεcence could be detected in areaε (i.e., apical

dendrites emanating from layer 5) not demonstrating any remarkable pathology with H&E. Alterations in dendritic NF200 and NF68 (at +0.2 Bregma) immunoreactivity were also seen in these regions. Alterations in MAP2 and NF200 immunoreactivity were more dramatic than those seen with NF68. No changes in NF200, NF68, MAP2 immunofluorescence as well as H&E staining were detected 3-4 mm lateral to the middle longitudinal fissure (MLF) in both ipsilateral and contralateral cortices (+0.2 Bregma through -3.8 Bregma).

C. DISCUSSION

This Example is a syεtematic immunohiεtochemical examination of derangements in cytoskeletal proteins following TBI in rats. In contrast to a substantial literature emphasizing the contribution of diffuse axonal injury to pathology of TBI, the data presented here indicate that preferential dendritic rather than axonal damage occurs within three hours post TBI. Although neuronal alterations seen with NF68, NF200 and MAP2 immunofluorescence were predominantly associated with pathological changes detected by H&E staining, significant dendritic pathology extended beyond focal contusion sites. The magnitude of NF200 and MAP2 loss detected by immunofluorescence was greater than that of NF68. Furthermore, confocal microscopy revealed varying degrees of NF200 disassembly within injured apical dendrites.

1. NF200 Immunofluorescence Revealed Preferential

Loss in Somatodendritic Neuronal Regions NF200 immunolabeling revealed the appearance of prominent fragmentation of dendritic processes and loss of somal labeling primarily in cortical layers 1 and 3-5 ipsilateral and contralateral to the site of injury. Furthermore, confocal microscopic analyseε of fragmented apical dendriteε imaged with N52 contained varying degrees of NF200 disassembly. Posεible explanations for observed differences in immunolabeling patterns post-TBI include the binding characteriεticε of the selected antibodies, the role of the tertiary structure of the individual subunits, and potential post-translational variations such as phosphorylation state. Anti-NF200 (Sigma N52) is selective against the highly repeated KSP (lysine-serine-proline) segment of the carboxyl terminal. Consequently, N52 does not retain antigenicity to the low molecular weight (MW) fragments containing the amino α-helical domain common to all NF proteins. These low MW fragments have been reported after calpain mediated proteolysis in vivo (Schlaepfer

et al . , 1985; Posmantur et al . , 1994) and in vitro Kamakura et al . , 1985) . Thus, the appearance of fragmented apical dendrites observed with claεsical and confocal microscopy after TBI could represent the inability of the N52 antibody to detect the low MW α-helical containing NF fragments and/or physically broken dendrites. In addition, the tertiary structure of the NF200 subunit in assembled NFs may contribute to its susceptibility after injury. The NF200 subunit possesses high accessibility to protease action as a consequence of its extended carboxyl terminal which cross links adjacent proteins including NF and microtubules (Nixon and Sihag, 1991) . Finally, the phosphorylation state of the NF200 subunit can modulate protein degradation. NF200 in dendrites, in contrast to axons, is predominantly unphosphorylated (Gotow and Tanaska, 1994) thus making it more vulnerable to proteolysis. For example, dephosphorylation of NF200 and MAP2, haε been shown to increase suεceptibility to calpain proteolysis in vitro (Pant, 1988; Johnson and Foley, 1993).

2. MAP2 Immunofluorescence Independently Documents

Dendritic Loss

MAP2 is a cytoskeleton protein restricted to somato-dendritic neuronal domainε . MAP2 (AP-20) immunofluorescence revealed fragmented dendritic processes throughout injured ipsilateral and contralateral cortical neuronal layers similar to fragmentation patterns detected with anti-NF200 (N52) . The rostrocaudal extent of MAP2 loεε waε also εimilar to that observed with NF200 (N52) . AP-20 is a monoclonal antibody that recognizes the subunit independent of phosphorylation state but does not retain antigenicity to proteolytic fragments. The similarities of NF200 and MAP2 immunofluorescence are conεiεtent with the binding characteristics of the antibodies.

3. NF68 Immunofluorescence 3 hours Post-TBI Revealed

Focal Losses in Immunofluorescence within

Neuronal Processes

Anti-NF68 (Sigma NR4) immunofluorescence showed less fragmentation of apical dendrites, as compared to NF200, especially in cortical layers 1, and 3-5 ipsilateral to the side of injury. NF68 immunofluorescence in the contralateral cortex, although showing some limited breaks in neuronal processes manifested considerably less derangement than seen in the ipsilateral cortex. The binding characteristics of immunofluorescence labeling of

NF68 with NR4 provides complementary data to NF200 immunoreactivity. For instance, a unique morphological feature detected with NR4 (anti-NF68) immunofluorescence was the presence of regular spaced vacuoles within apical dendrites. Further, there was increased immunofluorescence along the plasmalemma. This may be due to NR4 ' ε ability to detect the α-helical domain of all NF subunitε (NF68, NF150, and NF200) that are retained by low molecular weight proteolytic fragmentε . Thus, the increased immunoreactivity found adjacent to the plasmalemma could have been produced by immunopositive degraded or disassembled NF subunits. This finding is consistent with previous electron microscopy findings demonstrating the aggregation of disassembled and/or degraded microtubules and NFs along the axolemma within injured peripheral nerve (Schlaepfer and Bunge, 1973). The vacuolization observed with NR4 (anti-NF68) appears to correspond to the fragmentation pattern detected with N52 (anti-NF200) in apical dendrites.

4. Comparison of Immunofluorescence and H&E

Morphopathology at three hours post-TBI

The rostrocaudal extent of morphopathological changeε detected with H&E generally, but not exclusively, corresponded with alterations in NF200, NF68, and MAP2 immunofluorescence. The colocalization of H&E morphopathology and immunofluorescence alterations in siteε of contusion occurred in cortical layers 1 and 3-5. However, immunofluorescence studies detected significant dendritic derangements beyond areas of cortical contusion ( +0.2 Bregma) not associated with prominent H&E morphopathology . 5. Possible Mechanisms of Cytoskeletal Protein Loss

Although the precise mechanisms mediating losε of cytoεkeletal proteins following TBI are unknown, excitotoxic losε of calcium homeostasis and pathologic activation of calcium-dependent proteaseε may be important causes of cytoskeletal degradation. Western blot analyses detected the presence of BDP of lower molecular weight NF protei(NF68) potentially asεociated with pathological activation of calpain (Posmantur et al . , 1994; Saatman

et al . , 1994; Saatman and Mclntoεh, 1994) . Since cortical

impact injury may produce iεchemia in the cortex ipεilateral to the site of injury, focal ischemia could have also contributed to possible loss of calcium homeostasis and calpain activation in this Example. The presence of focal ischemia ipsilateral to the site of injury could have contributed to the greater losε of NF68, NF200 and MAP2 in the ipsilateral cortex as compared to the cortex contralateral to the site of cortical impact.

6. Acute Cytos eleton Derangements Occur Primarily

In Dendrites Rather Than Axons

While experimental brain ischemia and epilepsy in vivo

have also described preferential dendritic, rather than axonal damage this Example provides first evidence that within 3 hours TBI in vivo can produce significant cytoskeletal derangements in dendrites in the absence of marked axonal changes . The occurrence of early extensive dendritic damage might be due to the electrophysiological properties of axons and dendrites. Dendritic procesεes (apical dendrites) propagate neuronal transmission by low voltage calcium channels and consequently may be more likely to open after TBI. Thus, the electrophysiological properties of dendrites can potentially yield immediate focal alterations in intracellular Ca' homeostasis at sites of ion entry. In contrast, axons within white matter tracts ( i . e . , corpus callosum) propagate neuronal

transmission by opening and closing of NA⁺ and K⁺ channels, suggesting axons are less likely to experience large, immediate changes in calcium homeostasis after neuronal excitation. In addition, electrophysiological studies examining long term potentiation (LTP) following TBI Also support impaired dendritic functioning as inferred by decreased efficacy of synaptic transmission.

Although extensive dendritic damage waε obεerved at 3 hourε poεt-TBI, subtle and/or more extensive axonal injury might be observed at earlier or later time points with other microscopic techniques and/or use of different antibodies. Past research has documented evolutionary axonal changes that culminate in the production of axonal swelling and retraction balls over a period of days. Recent studies have establiεhed axolemma permeability changes within 3 hours following TBI (Pettus et al . , 1994) .

Theεe εtudieε using electron microscopy observed the passage of injected peroxidase from the extracellular to intracellular compartment which waε associated with ultrastructural changes such as the initiation of NF misalignment. Nevertheless, for this Example, claεεical macroscopic axonal changes described at later time points following injury (i.e., retraction balls) were not yet

evident at 3 hours post-TBI using NR4 (anti-NF68) , and N52 (anti-NF200) .

C. DISCUSSION

These combined immunohistochemical and histopathological studies have confirmed that within 3 hours TBI can produce significant dendritic morphopathology, an observation consistent with NF68, NF200, and MAP2 protein losε detected by Western blotting following lateral cortical impact injury. These cytoskeletal derangements observed with qualitative light immunofluorescence and confocal microscopy occurred focally along dendriteε reεulting in varying degrees of NF disassembly. Remarkable changes in NF immunofluorescence were not evident in axons . Furthermore, immunofluorescence alterations were not exclusively restricted to brain regions demonstrating H&E pathology suggeεting that cytoskeleton derangements can occur in areas not necesεarily undergoing acute neuronal cell death. Therefore, cytoskeletal derangements may not solely be a function of the contusion, but also may reflect more global, neuronal responses to injury. The degree of dendritic losε observed in diffuse procesε injury (DPI) suggeεtε profound effects on neuronal connectivity and synaptic efficacy that may contribute to neurobehavioral deficits .

EXAMPLE 4 A CALPAIN INHIBITOR ATTENUATES CORTICAL

CYTOSKELETAL PROTEIN LOSS AFTER EXPERIMENTAL TBI

IN THE RAT

This Example studies the ability of a calpain inhibitor to reduce losses of NF200 and NF68 proteins after TBI in the rat. The efficacy of calpain inhibition therapy to reduce the accumulation of calpain 1 mediated spectrin BDP following TBI was also studied. Twenty- four hours after unilateral cortical impact injury, Western blot analyses revealed NF200 decreases ipsilateral and contralateral to the injury site of 65% and 36% of levelε obεerved in naive, uninjured rat corticeε, reεpectively .

NF68 protein levels decreased by 35% of naive levels only in the ipsilateral cortex. Calpain inhibitor 2, administered ten minutes after injury via continuous

intraarterial adminiεtration into the right external carotid artery for 24 hourε, εignificantly reduced NF200 losses to 17% and 3% of naive levels in the ipsilateral and contralateral cortices, respectively. Calpain inhibitor treatment abolished NF68 losε in the ipsilateral cortex and waε accompanied by a reduction of putative calpain mediated NF68 BDPs. Calpain 1-mediated BDPs to brain α-spectrin were detectable in ipsi- and contralateral cortical tissue 24 hours following TBI. Calpain inhibitor 2 significantly reduced the amount of these BDPs in both cortical hemispheres . Qualitative immunofluorescence studies of NF200 and NF68 confirmed Western blot data, demonstrating morphological preεervation of neuronal structure throughout cortical regions of the traumatically-injured brain. In addition, histopathological studies employing hematoxylin and eosin staining also revealed preservation of neuronal somata in areas of contusion. These εurprising data demonstrate that calpain inhibitors represent viable therapeutic compositions for preserving the cytoskeletal structure of injured neurons and treating TBI in ligand animals . This Example employed a cortical impact model of TBI

(Dixon et al . , 1991) that reproduceε a number of features of severe TBI in humans . Examination of cytoskeletal proteins was reεtricted to cortical areaε since no alterations in NF protein levels have been observed in hippoca pal sa pleε following lateral cortical impact injury (Posmantur et al . , 1994).

Using Western blot analyseε, the present invention demonstrates that a systemically-administered calpain inhibitor (calpain inhibitor 2) protects against cortical NF protein losε and reduces calpain-mediated spectrin BDP following experimental TBI in vivo . In addition, histopathological studies, using immunohistochemical and H&E staining techniques provided evidence that calpain inhibitor 2 dramatically preserves neuronal structure throughout the traumatically injured brain. It is proposed that calpain inhibitors might be a viable strategy for reducing cytoskeletal protein loss after TBI in vivo .

A. MATERIAL AND METHODS

1. Rat Model of TBI

For induction of a severe magnitude of TBI, a controlled cortical impact device as described previously

(Dixon et al . , 1991) was employed. Briefly, rats were intubated and anesthetized with 2% Halothane in a 2:1 mixture of N₂0/0₂. Bilateral craniotomies (expanded only on the right cortex ipsilateral to the injury site) were performed adjacent to the central suture, midway between lambda and bregma. The dura was kept intact over both hemispheres. Injury was induced by impacting the right cortex (ipsilateral cortex) with a 6 mm diameter tip at the rate of 6 m/sec and a 2 mm compresεion. Velocity waε measured directly by the linear velocity displacement transducer (LVDT; Shaevitz Model 500 HR) which produces an analog signal that was recorded by a PC-based data acquisition system (R.C. Electronics) for analysis of time/diεplacement parameterε of the impactor. Sham- injured animals underwent identical surgical procedures including craniotomy on both hemispheres, but did not receive impact injury. However, expanded craniotomy was only performed on the (right) ipεilateral cortex, identical to the injured (right) ipεilateral cortex. Naive ratε were not expoεed to any injury related surgical procedures. Following cortical impact, animals were extubated and immediately assessed for recovery of reflexes (Dixon et al . , 1991) .

Five groups (n = 8-10 per group) of male Sprague- Dawley rats (250-350 g) were used in this study. Naive (N) , animals received no drug, while vehicle and surgery Sham- injured animals were treated with calpain inhibitor 2 (SD) ,• Sham- injured animals were treated with vehicle (SV) ; Injured animals treated with vehicle (IV); Injured animals treated with calpain inhibitor 2 (ID) .

2. Calpain Inhibitor Administration Ten minutes following cortical impact injury, animals received either vehicle (saline plus ethanol (pH 6.78) at a final ethanol concentration of 0.03%) or N-Acetyl-Leu- Leu-Methioninal (Calpain inhibitor 2; Boehringer-Mannheim) . Calpain inhibitor 2 was disεolved in ethanol and diluted in εaline to a final concentration of 150 μM (pH=6.78; final concentration of ethanol: 0.03%) . The animalε were prepared for intraarterial drug administration, as previously described (Bartus, et al . , 1994). Briefly, the right

internal (ICA) and external (ECA) carotid arteries were exposed. The ECA was ligated approximately 4 mm from the carotid bifurcation. Administration of the drug occurred through a PE-10 tubing, which was inserted approximately 3 mm into the right external carotid artery. Animals were attached to a continuous-drive syringe pump (Razel, Stamford, CT) , to allow continuous, intra-arterial infusion of vehicle or drug to a freely moving animal. A priming dose of 9 ml/hr was infused for the first ten minutes followed by a continuous slower infusion of 0.7 ml/hr which persisted until the rats were sacrificed 24 hours after cortical impact injury. Total volume each rat received was approximately 18 ml. Vehicle treated animals received the same volume at a perfusion rate identical to anti-protease treated animals .

Body temperature was regularly monitored ( via rectal probe) and maintained normothermic throughout and following cortical impact injury procedure. If an animal temperature fell below 36°C or rose above 38°C the animal was eliminated from the study. No significant differences in body temperature were observed between rats perfused with vehicle or calpain inhibitor 2.

3. Sample Preparation

Before sacrifice all animals were given a lethal dose of phenobarbital (0.5 cc) . Both cortices were selected for this study because NF protein loss at this time has been described previously in cortical tissue ipεilateral and contralateral to the injury εite following unilateral cortical impact injury at thiε timepoint (Poεmantur et al . ,

1994) . Excision of the parietal cortices beneath the craniotomieε extended approximately 4 mm laterally, approximately 7 mm rostrocaudally, and to a depth extending to the white matter. Samples were prepared using a modification of the method of Taft et al . , (1993). All

tissue was frozen immediately in liquid N₂. The microdisεected tissue was homogenized at 4°C in an ice cold homogenization buffer containing 20 mM PIPES (pH 7.1) 2 mM EGTA, 1 M EDT2A, 1 mM dithiothreitol, 0.3 mM phenyl ethyl- sulfonylflouride (PMSF) and 0.1 mM leupeptin. Chelators and proteases and subsequent artifactual degradation of spectrin and NF proteins in vi tro .

4. SDS-PAGE, Immunoblotting and Quantification

The amount of protein in samples was determined using BCA^® reagents (Pierce) with albumin standards. Protein- balanced samples were prepared for polyacrylamide gel electrophoresiε in two-fold loading buffer containing 0.025 M Tris (pH 6.8), 0.2 M DtT, 8% SDS, 0.02% Bromophenol Blue, and 24% glycerol in distilled water. Samples were heated at 95°C for 5 min. Proteins were resolved in a vertical electrophoresis chamber using a 4% acrylamide stacking gel over a 6% acrylamide resolving gel. Gels were run at a constant current (120 A) for approximately 1 hour. 80 μg of sample protein was resolved in each lane . Following separation, proteins were immediately transferred to a nitrocelluse membrane using Western blotting (Towbin et al . , 1979). Lateral transfer was employed using a transfer buffer made up of 0.192 M glycine and 0.025 M Tris (pH 8.3) with 10% methanol at a constant voltage of 100 V for 3 hours at 4°C. Blots were immediately blocked for immunolabeling by overnight incubation using 3% non-fat milk in 20 mM Tris HCL, 0.15 M NaCl, and 0.005% Tween-20^® at 4°C. Coomassie blue and Ponceau Red staining were routinely performed to confirm that equal amounts of protein were loaded in each lane.

Monoclonal antibodies specific for individual NF proteins were used for immunolabeling. .Antibodies binding NF proteins were NR4, recognizing phosphorylated and non- phosphorylated NF68, and N52 recognizing phosphorylated and non-phosphorylated NF200. In contrast to N52 (NF200) , NR4 (NF68) detects low molecular weight BDP to NF proteins. Antibody 38, a polyclonal antibody that recognizes a calpain 1 mediated BDP of α-spectrin was obtained from Cephalon, Inc. Following incubation with primary antibodies (dilution 1:1000 for NF and spectrin antibodieε) for 2 hourε, blots were incubated with a secondary antibody linked to horseradish peroxidaεe (dilution 1:12500) for 1 hour. Enhanced chemiluminescence (ECL; Amersham) reagents were used to visualize the immunolabeling on X-ray film. Semiquantitative evaluation of protein levelε waε performed via computer-aεεisted 2-dimensional denεitometric scanning (Biorad Model GS-670) . Data acquired in arbitrary densitometric units were transformed to percentages of the denεitometric levelε obεerved on scans from naive animals visualized on the same blot. 5. Immunohistochemistry

Prior to perfusion all animals were given a lethal dose of phenobarbital (0.5 ml) . Transcardial perfusion was administered through the left ventricle (120 ml of 0.9% saline and 200 ml of prepared fixative) of the heart 24 hourε following injury. The fixative solutions for the selected antibodies was 4.2% formalin for anti-NF200 (Sigma N52) and for anti-NF68 (Sigma NR4) immunolabeling. The brain was then grossly sectioned, frozen, and mounted in a Hacker-Bright cryostat . Coronal sectionε of 30-40 μm thickneεε were cut at -15°C and immediately placed into wells containing PBS (136 mM NaCl, 81 mM KCl and 1.6 mM NaHPO,, and 14 M KH₂P0₄, pH7.4) .

The entire immunolabeling process was performed in 24 -well culture plates. Sections were first incubated in 3% horse serum at 4°C for two hours. Primary antibodies (Sigma NR4 and N52) were incubated for 3 hours at 25°C in blocker solution: Tween-PBS : 10 mM NaP0₄ (pH 7.5), 0.9% NaCl, Tween-20^®, 0.1% antifoam A (Sigma A-5758) and 5% non- fat dry milk (Carnation) . The tissue sliceε were then waεhed with blocker εolution 3 x 10 minuteε . Secondary antibody (anti-mouse IgG, 1:1000) linked to a specific fluorophore was applied for two hourε. Fluoreεcein (Vector labs) waε used to detect NF200 and NF68. The tisεue slices were then washed 3 times in PBS solution, mounted on subbed slides with Elvanol (Dupont) and stored in the dark prior to immunofluorescence examination. Sections without primary antibodies did not εtain. 6. Histopathological Assessment

Animals were given a lethal injection of phenobarbital

(0.5 μL) prior to perfusion. Rats were transcardially perfused through the left ventricle (120 ml of 0.9% saline and 200 ml of 10% buffered formalin) 24 hours after injury and were sliced coronally at 3 micron intervals. Paraffin sections were used to obtain thin sectionε and optimal hematoxylin and eosin (H&E) staining. Brain sectionε were processed through graded alcohols and xylenes prior to embedding in paraffin. Sections were cut at 4-5 microns on a microtome, from +0.2 to -3.8 Bregma, mounted on glass slideε and stained with hematoxylin and eosin staining.

7. Statistical Analyses Semiquantitative evaluation of protein levels detected by Western blotting was performed via computer-asεiεted 2- dimenεional denεitometric εcanning (Biorad Model GS-670) . Data acquired in arbitrary denεitometric unitε were transformed to percentages of the densitometric levels observed on scans from naive animals visualized on the same blot. Except as noted, data form Western blots were evaluated by analysis of variance (ANOVA) with a post-hoc Tukey-test-HSD. Values given are mean ± standard error of mean (SEM) . Differences were considered significant when p<0.05. In addition the above parametric analyses were also performed using logarithmic transformed data to allow for greater homoscedastic varianceε . Differences were alεo conεidered significant when p<0.05.

B. RESULTS

Animals receiving vehicle only, manifested similar NF68 and NF200 loss following TBI to animals not receiving the vehicle. Additionally, injured animals treated with vehicle revealed similar amounts of NFε and spectrin BDPs in comparison to animals that did not receive the vehicle.

Statistical analyses of untransformed data and logarithmic transformed data revealed identical findings for immunoreactivity to NF68, NF200, and calpain 1 specific

BDP to α-spectrin. No differences in significance between groups was noted with transformed data that was not observed with untransformed data using one way ANOVA and post-hoc Tukey teεt-HSD analyses (p<0.05). All valueε expresεed below incorporate untransformed data, expressed as percentage of data obtained from naive animals .

1. Calpain inhibition treatment reduces NF68 loss

and putative calpain mediated BDP to NF68 in

ipsilateral cortex after TBI

Twenty-four hours after injury, NF68 protein levels in ipsilateral cortex obtained from traumatized animals receiving vehicle only (IV) decreased by 35% (34.75±2.70%) of naive levels (<0.001; compared to naive). In injured animals receiving calpain inhibitor 2 (ID) , NF68 immunoreactivity decreased only by 5% (4.99±3.49%) of naive levels. Animals receiving calpain inhibitor 2 (ID) had significantly less NF68 protein loss as compared to animals receiving vehicle (IV;+++p≤0.001) . There was no

statistical significance in NF68 immunoreactivity between sham-injured animals and ID animals twenty-four hours following cortical impact injury. Additionally, sham injury groups (receiving vehicle [SV] or calpain inhibitor 2 [SD] ) showed no statistically significant NF68 loss as compared to naive animals.

The presence of low molecular weight immunopositive bands at 56 kDa and 52 kDa, suggestive of calpain-mediated BDP to NF68, were significantly reduced in ID animals compared to IV animalε (p≤O.OOl; Students t-test) . No significant difference in the amount of NF68 BDP was observed between injured, drug treated animals (ID), sham- injured (SD and SV) and naive animals.

NF68 levels in the contralateral cortex did not differ

between naive, sham-injured animals groups (SD and SV) as well as from the injury groups (IV and ID) twenty four hours after injury. Further, no lower molecular-weight immunopositive BDP were detectable in the contralateral cortical samples .

2. Calpain inhibition treatment reduces ipsilateral

and contralateral NF200 loss after TBI

Cortical impact injury resulted in reduction of NF200 protein levels form ipsilateral cortex. Ipsilateral NF200 protein levels from IV animals decreased by 65%

(64.72±4.24%) of naive levels twenty-four hours after TBI

(p≤O.OOl). NF200 protein levels form ID animals decreased only by 17% (17.29±2.67%) of naive levels, which was significantly lower as compared to animals only treated with vehicle (IV; +++p<0.001). Similar to NF68, no

differences in NF200 protein levels were observed between naive and sham-injured animal groups. However, in contrast to NF68 protein levels of NF200 from animals treated with calpain inhibitor 2 did not return to levels detected in naive animals (*p <0.05) .

NF200 immunoreactivity in the contralateral cortex waε decreased by 36% (35.82+9.09%) in injured, vehicle treated animals (IV) of naive levelε (p<0.001). In contrast NF200

immunoreactivity form animals receiving calpain inhibition treatment (ID), decreased by only 3% (3.00 ± 3.36%) of naive levels. Injured animals receiving vehicle (IV;+++p<0.001) . There was no statistical difference in NF200 immunoreactivity between injured, drug treated animalε, sham injured animals and naive animals.

3. Calpain inhibitor treatment reduces calpain 1

mediated spectrin BDP in ipsilateral and

contralateral cortices 24 hours after TBI

In the ipsilateral cortex injured animals treated with vehicle (IV) only, manifested an increase of calpain l mediated spectrin BDPε to 8600% of naive levelε. In injured animals receiving calpain inhibitor 2 (ID), the amount of εpectrin BDPε waε reduced to 3900%. The amount of spectrin BDPs was significantly lower in ID animals as compared to IV animals (+++p<0.0001. ) . While the levels of spectrin BDPs in animalε treated with calpain inhibitor 2 were reduced to levelε comparable to levelε from sham injured animals, there was still a statistically reliable difference between ID animals and naive animals (**p<0.01) .

In contrast to NF 68, sham- injury cauεed the appearance of εpectrin BDPε. Sham injury groupε (SD and SV) manifested significant higher amounts of spectrin BDPs as compared to naive animals (p<0.01 for SD and SV respectively), and there was no significant difference between sham injured animalε treated with calpain inhibitor 2 (SD) or vehicle (SV) . In the contralateral cortex IV animals manifested an increase in spectrin BDPs of 1200% of naive levels. Samples from ID animals revealed significantly lower spectrin BDPs levels as compared to samples from IV animals (+++p<0.001) . The amount of spectrin BDPs from ID animals

was not significantly different from those of naive animals. Similar to the ipsilateral cortex, sham injured groups (SD and SV) manifeεted εignificant increases in the amounts of spectrin BDPs (p<0.05 for SD and p≤O.Ol for SV)

aε compared to naive animals. However εha injured, receiving calpain inhibitor 2 (SD) , had significantly lesε spectrin BDPs as compared to εham injured animalε receiving vehicle (SV; p<0.05). There was no significant difference

between SD and ID treated animals .

4. NF200 and NF68 immunofluorescence following

administration of vehicle and/or calpain

inhibitor 2

Ipsilateral and contralateral cortical tissueε were examined rostrocaudally from +0.2 mm Bregma to -3.8 mm Bregma. NF200 immunofluorescence in naive and sham cortex revealed similar labeling patterns with prominent labeling of the cortical neuronal pyramidal layers 3 and 5 including apical dendrites, neuronal somata and axons. TBI in injured, vehicle treated animals produced a loss in the labeling pattern of neurons throughout cortical layers 1-5 in the ipsilateral cortex. The reduction of NF200 immunoreactivity was observed from -1.5 mm Bregma through -3.8 mm Bregma rostrocaudally, corresponding to regions showing morphopathology characteristic of injured neurons. Losε of NF200 immunolabeling waε also detected in a well defined area in the contralateral cortex, 1-2 mm wide. NF200 immunofluorescence in injured-drug treated animalε 24 hrs after TBI revealed a marked protection of neuronal labeling in areas of contusion in both ipsilateral and contralateral cortices. Particularly, there was clear protection of apical dendrites extending from pyramidal neuronal layers 3 and 5 as well as preεervation somal labeling. In addition, in contraεt to naive injured, vehicle treated animals manifested a prominent losε of NF200 immunofluorescence among fine dendritic processes within layer 1. Injured animals, receiving calpain inhibitor 2, showed a preservation of fine dendritic processes within the superficial cortical layers. Further, in contrast to injured vehicle treated animals, animals receiving calpain inhibitor 2 showed marked preservation of neuronal processes even in areas of direct impact (-3.4 mm Bregma) and tissue deformation.

NF68 immunoreactivity in naive animals (examined rostrocaudally from +0.2 mm Bregma to -3.8 mm Bregma) revealed a laminar organization between cortical layers 3 and 5, including neuronal somata and long extending apical dendrites. NF68 immunofluorescence in injured vehicle treated animals revealed derangements similar to NF200. A clear loss of neuronal somata and neuronal processes (i.e.

dendrites and axonε) waε obεerved in the area of contuεion (from -1.5 mm Bregma to -3.8 mm Bregma) from cortical layer 1 to layers 5 and 6. Losseε of NF68 immunoreactivity included the disappearance of the disappearance of the pyramidal neuronal lamina, especially in layer 3. NF68 immunoreactivity in injured, drug treated animals demonstrated protection of neuronal εomata and neuronal processes in cortical pyramidal layers 3 and 5. Alternations in axonal NF68 immunoreactivity ( i . e . axonal swelling, breaks, and retraction balls) were also observed in white matter tracts εuch aε the corpus callosum in injured, vehicle treated animals. These axonal changes were not detected in naive controls.

NF68 immunoreactivity in injured, drug treated animals revealed less pronounced axonal alterations including axonal swelling, breaks, and retraction balls, than εeen in injured, vehicle treated animalε.

An examination of hematoxylin and eosin (H&E) staining following calpain inhibitor 2 administration documented protection of neuronal somata in areas of contusion. Naive (-2.8 mm Bregma and -3.4 mm Bregma) and sham rat brains εhowed no discernable histological alterations in either cortical or hemisphere. H&E changes observed in injured vehicle treated cortex occurred in an area approximately 3- 4 mm in a maximum tranεverse direction (-2.8 mm Bregma; -3.4 mm Bregma, site of direct impact) ipsilateral to the site of injury. Injured vehicle cortex waε characterized by focal εubpial and intracortical hemorrhage. Ipεilateral superficial cortical contuεion overlaid an area of cortical damage manifesting large number (80-90%) of dark, shrunken, and pyknotic neurons in cortical layers 1-4, characteristic of impending cell death. Additionally, there was a vacuolar appearance of the neuropil in the deeper cortical layers 5 and 6. The contralateral cortex showed a well defined area corresponding to a contrecoup lesion in area 1-2 mm wide approximately 0.5 - 1.0 mm below the surface. In injured-drug treated animals at -3.4 mm Bregma there was an attenuation of H&E pathology in a transverse direction, limited to 1-2 mm, in the ipsilateral cortex and contralateral cortex. In injured drug treated animals slightly rostral of direct impact (-2.8 mm Bregma) the greatest reduction in histological alterations were detected. H&E changes were limited in a transverse direction to only 1 mm as well as observed in the cortical lamina. Neuronal damage was limited to layers 1-3, with a clear preservation of deeper pyramidal layers 5 and 6.

C. DISCUSSION This Example provides evidence that a systematic administration of a calpain inhibitor initiated after TBI in vivo reduces NF 200 and 68 protein loss, key proteins of the neuronal cytoskeleton. Western blotting studies showed that calpain inhibitor 2 protected against cortical NF loεεes in both hemispheres, ipsilaterally and contralaterally to the site of impact. Protective effects against NF degradation were accompanied by reduction of putative calpain mediated BDPs to NF68. In addition, the accumulation of calpain mediated BDPs to spectrin was significantly attenuated in ipsi- and contralateral cortices following calpain inhibition treatment. Immunohistochemical inveεtigation, using antibodies against NF68 and NF200 confirmed western blotting reεultε and demonstrated dramatic protection of neuronal structure among neuronal somata, axons, and dendrites after calpain inhibitor administration. H&E staining also indicated that administration of calpain inhibitor 2 reduced contusion size in the cortex ipεilateral to the injury εite. 1. Cytoskeletal Protein Loss After TBI Provides

Evidence For Calpain Involvement

In the contralateral cortex, TBI produced significant losses of NF200, while NF68 levels were unchanged. This, however, does not exclude calpain induced proteolysis of NF200, aε it has been reported previously that NF200 has a higher susceptibility to calpain induced proteolysis than NF68 in vivo and in vi tro (Kamakura et al . , 1985) . Further, calpain mediated BDPs of spectrin could be also detected in the contralateral cortex following TBI. Differential susceptibility of cytoskeletal subunits to calpain proteolysis may be a potential cause of the appearance of a sham surgery effect with both ipsilateral and contralateral cortices not seen with NF68 and NF200 immunoreactivity.

2. Calpain Inhibitor 2 Reduces Cortical Cytoskeletal

Protein Loss 24 Hours After TBI

Calpain inhibitor 2 administration effectively reduced

NF68, NF200 protein loss following TBI. As thiε inhibitor εignificantly reduced putative calpain mediated BDP to NF and spectrin, calpain inhibitor 2 administration protects, at least in part, against calpain mediated proteolysis of NF and spectrin proteins.

In contrast to NF BDPs, sham surgery resulted in the appearance of BDP to brain spectrin. These results are not unexpected since sham injury (i.e. craniotomy) is an invasive εurgical procedure even though it does not produce neuronal cell death. These reεults are consistent with a previous report that a sublethal ischemic insult can lead to an accumulation of calpain mediated spectrin BDPs

(Roberts-Lewis et al . , 1994). Furthermore, spectrin may be more vulnerable to calpain proteolysis than NFs . Although sham injured animals receiving calpain inhibitor 2 had significant less εpectrin BDPs in the contralateral cortex than sham-injured animals, it is not clear why calpain inhibitor 2 had no effect on reduction of spectrin BDPε in the ipsilateral cortex of sham injured animals.

3. Anti-CalPain Administration Preserves Neuronal Structure Following TBI

Immunohistochemical studies provided important morphological confirmation of semiquantitative Western blots of NF68 and NF200. Three major immunohistochemical observations were consistently noted among injured drug animals 24 hours after TBI using NF68 and NF200 immunofluorescence. The moεt clear observation was the preservation of apical dendrites from cortical pyramidal layers throughout the rostral - caudal extent of both ipsilateral and contralateral cortices. Secondly, in contrast to injured, vehicle treated animals, retention of cortical somal labeling was noted. Finally, there was a reduction in the appearance of retraction balls in the cortex at the site of impact and in white matter tracts such as the corpus callosum and subcortical white matter. Theεe data indicate that calpain inhibitor 2 adminiεtration not only protectε against NF loss but also preserves the cortical neuronal cytoskeletal structure of neuronal somata, dendriteε and axonε .

Furthermore, H&E staining has demonstrated that calpain inhibitor 2 administered 10 minutes after experimental TBI in vivo reduced the extent of cell death

in the area of direct contusion. This protection was characterized by a preservation of neuronal somata throughout cortical pyramidal layers 3-6 perpendicular to the pial surface, as well as, transversely away from areas of impact .

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application waε εpecifically and individually indicated to be incorporated herein by reference.

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While the illustrative embodiments of the invention have been described with particularity, it will be understood that various other modifications will be apparent to and can be readily made by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is not intended that the scope of the claims appended hereto be limited to the examples and descriptions set forth herein but rather that the claims be construed as encompassing all the features of patentable novelty which reside in the present invention, including all features which would be treated as equivalents thereof by those skilled in the art to which this invention pertains.

Claims

I CLAIM :

l. A method for an injury comprising cerebral ischemia, spinal cord injury or stroke in an animal, the method comprising administering to said animal a pharmaceutically- acceptable calpain inhibitor composition, in a dose effective to improve neurological outcome from the injury.

2. The method of claim 1, wherein the inhibitor is administered within 24 hours of the injury, wherein the dose is at least about 0.01 mg/kg/hr, and wherein the calpain is administered for at least about 1 hour.

3. The method of claim 1, wherein the inhibitor is administered within 3 hours of the injury, wherein the dose iε in the range of about 0.01 mg/kg/hr to about 20 mg/kg/hr, and wherein the inhibitor is administered for at least about 12 hours .

4. The method of claim 3, wherein the calpain inhibitor comprises at least one selected from the group consisting of calpain inhibitor I and calpain inhibitor II.

5. The method of claim 3 , wherein the animal is maintained at a temperature below about 35°C during administration of the inhibitor.

6. The method of claim 1, wherein the inhibitor comprises a peptide aldehyde comprising at least two amino acid residues selected from the group of residues consisting of tryosine, methionine, leucine, lysine, arginine, valine and isoleucine, with one of the residues selected being in the form of an aldehyde derivative of that residue positioned at the C-terminal of the peptide.

7. The method of claim 6, wherein the inhibitor is administered within 24 hours of the injury, wherein the dose is at least about 0.01 mg/kg/hr, and wherein the calpain is administered for at least about 1 hour.

8. The method of claim 6, wherein the inhibitor is administered within 3 hours of the injury, wherein the dose is in the range of about 0.01 mg/kg/hr to about 20 mg/kg/hr, and wherein the inhibitor is administered for at least about 12 hours .

9. The method of claim 8, wherein the calpain inhibitor comprises at least one selected from the group consisting of calpain inhibitor I and calpain inhibitor II.

10. The method of claim 8 , wherein the animal is maintained at a temperature below about 35°C during administration of the inhibitor.

11. The method of claim 6, wherein the aldehyde derivative comprises norleucinal or methional .

12. The method of claim 11, wherein the inhibitor is administered within 24 hours of the injury, wherein the dose is at least about 0.01 mg/kg/hr, and wherein the calpain is administered for at least about 1 hour.

13. The method of claim 11, wherein the inhibitor is administered within 3 hours of the injury, wherein the dose is in the range of about 0.01 mg/kg/hr to about 20 mg/kg/hr, and wherein the inhibitor is administered for at least about 12 hours .

14. The method of claim 11, wherein the animal is maintained at a temperature below about 35°C during administration of the inhibitor.

15. The method of claim 1, wherein the calpain inhibitor comprises at least one selected from the group consisting of calpain inhibitor I and calpain inhibitor II.

16. The method of claim 15, wherein the inhibitor is administered within 24 hours of the injury, wherein the dose is at least about 0.01 mg/kg/hr, and wherein the calpain is administered for at least about 1 hour.

17. The method of claim 15 wherein the inhibitor is administered within 12 hours of the injury, wherein the dose is in the range of about 0.01 mg/kg/hr to about 20 mg/kg/hr, and wherein the inhibitor is administered for at least about 12 hours .

18. A method for treating an injury in an animal comprising cerebral ischemia, spinal cord injury or stroke, the method comprising administering to said animal a pharmaceutically-acceptable calpain inhibitor composition, in an amount effective to reduce cytoskeletal protein loss .

19. The method of claim 18, wherein said cytoskeletal protein compires at least one of tau protein, neurofilament protein and microtubule associated protein 2.

20. The method of claim 18, wherein the inhibitor comprises a peptide aldehyde comprising at least two amino acid residues selected from the group of residues consisting of tryosine, methionine, leucine, lysine, arginine, valine and isoleucine, with one of the residues selected being in the form of an aldehyde derivative of that residue positioned at the C-terminal of the peptide.

21. The method of claim 20, wherein the aldehyde derivative comprises norleucinal or methional .

22. The method of claim 20, wherein the inhibitor comprises calpain inhibitor I or calpain inhibitor II.

23. The method of claim 19, wherein said cytoskeletal protein is NF200, NF68, or NF150 protein.