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WO1998003190A1 - Inhibiteurs de calpaine destines au traitement des lesions traumatiques du cerveau - Google Patents

Inhibiteurs de calpaine destines au traitement des lesions traumatiques du cerveau Download PDF

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Publication number
WO1998003190A1
WO1998003190A1 PCT/US1997/012668 US9712668W WO9803190A1 WO 1998003190 A1 WO1998003190 A1 WO 1998003190A1 US 9712668 W US9712668 W US 9712668W WO 9803190 A1 WO9803190 A1 WO 9803190A1
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Prior art keywords
calpain
inhibitor
injury
hours
administered
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Ronald L. Hayes
Guy L. Clifton
Rand Postmantur
Andreas Kampfl
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NEUROTRAUMA THERAPEUTICS Inc
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NEUROTRAUMA THERAPEUTICS Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans

Definitions

  • TITLE CALPAIN INHIBITORS FOR THE TREATMENT OF
  • the present invention relates to pharmaceutical compositions, and to medical treatment methods utilizing such compositions.
  • the present invention relates to pharmaceutical compositions comprising calpain inhibitors, and to medical treatment methods utilizing such compositions.
  • the present invention relates to pharmaceutical compositions comprising peptide aldehyde type of calpain inhibitors, and to medical treatment methods utilizing such compositions.
  • 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
  • cytoskeletal proteins including neurofilaments (NF) , microtubule associated protein 2 (MAP2) and spectrin were substrates for protein proteolysis.
  • NF neurofilaments
  • MAP2 microtubule associated protein 2
  • 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).
  • NF-H high
  • NF-M medium
  • NF-L low molecular weight
  • NF200 200 kDa
  • NF150 150 kDa
  • NF68 68 kDa
  • NF150 and NF200 subunits are cross- linking proteins found in the connecting branches (Gotow et al . , 1994) .
  • NF-H, -M, and -L in cell bodies, axons and dendrites (Shaw et al . , 1986).
  • CNS central nervous system
  • increased proteolysis of cytoskeletal proteins could result in derangement of neuronal structure and function.
  • a variety of enzymes including calpains (Zimmerman and Schlaepfer, 1982) , cathepsins (Banay-Schwartz et al . , 1987), trypsin and
  • chymotrypsin (Chin et al . , 1983) cleave NFs .
  • Chin et al . , 1983 cleave NFs .
  • 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; Naka ura et al . , 1992;
  • 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; Nagaraji et al . , 1994) .
  • peptide aldehyde type of calpain inhibitors such as calpain inhibitors I and II
  • 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 is 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) .
  • a method for treating traumatic brain injury in an animal 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 .
  • a method for treating traumatic brain injury in an animal generally includes administering to said animal a pharmaceutically- acceptable calpain inhibitor composition, in an amount effective to reduce cytoskeletal protein loss.
  • 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 Ca 2+ -activated neutral protease calpain is just one of many cellular proteins involved in Ca" signalling in mammalian cells. There are two major isoforms: calpain I (or ⁇ -calpain) and calpain II (or m-calpain) .
  • calpain inhibitors are administered as treatment for traumatic brain injury, cerebral ischemia, spinal cord injury, and stroke in mammals, especially humans
  • 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 pentrating 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.
  • improved 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 acessments of contusion, mass legion and/or infarction.
  • Brain/tissue acessments may be made non-invasively, by computerized tomograph, and/or by magnetic resonance imaging or spectroscopy.
  • 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.
  • the aldehyde derivative is selected from the group consisting of norleucinal and methioninal .
  • 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 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.
  • 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.
  • the time for administration of the calpain inhibitor is important.
  • 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 calpain inhibitors will commence within 24 hours of the event.
  • commencement of the administration of the calpain inhibitors will begin within 12 hours of the event, more preferaby 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 minutes of the event.
  • calpain inhibitors of the present invention will be made available where there is a high likelihood of brain injury, spinal cord injury or strokes.
  • 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/tissue indices, and/or to provide for reduction of cytoskeletal protein loss, that outweighs any side effects.
  • the duration treatment time will be dependent upon the dose rate and route of administration.
  • the calpain inhibitors of the present invention will be administered for a duration treatment time of at least about 1 hour.
  • 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 .
  • the calpain inhibitors may be administered via any suitable route of administration.
  • 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.
  • 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 compressed into tablets, or they may be incorporated directly with the food of the diet.
  • 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.
  • 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 glycol ⁇ , 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.
  • 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.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • 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.
  • 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.
  • 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.
  • this holding temperature will be less 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 is 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 most preferably at least 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/4hrs, preferably in the range of about 0.75°C/4hrs to about 1.50°C/4hrs, more preferably in the range of about l.l4°C/4hrs to about l.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 .
  • 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.
  • 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 losses 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 inhibitors 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 inhibitors 1 and 2 were influenced by different levels of extracellular calcium.
  • Qualitative immunohistochemical evaluations and Western blot data confirmed protection of NF loss 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 immunohi ⁇ tochemistry a six min potassium depolarization, similar to depolarization produced by experimental TBI and cerebral ischemia in vivo, produced losses of NF-H, NF-M and NF-L
  • calpain inhibitors 1 and 2 which inhibit calpain ⁇ , cathepsins, and to a lesser extent chymotrypsin, strongly protected against losses of all three NF proteins even when administered after brief depolarization in vi tro .
  • 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.
  • rat fetuses were removed from deeply anaesthetized dams . Hippocampi and septi were dissected in Ca :'+ /Mg"-free Hanks balanced salt solution (HBSS) . After rinsing, cells were dissociated 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 10 r ' cell ⁇ /well to 16 mm poly-L-lysine coated plastic culture dishes . Cultures were maintained in a humidified C0 2 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 times a week.
  • DMEM Dulbecco's modified Eagle's medium
  • a ⁇ trocyte formed a confluent monolayer beneath morphologically mature neuron ⁇ a ⁇ judged by process formation and the ability to sequester microtubule as ⁇ ociated protein 2 (MAP2) and tau.
  • MAP2 microtubule as ⁇ ociated protein 2
  • depolarization injury was performed by replacing normal media containing 5.3 mM KCl and 1.8 mM CaCl with medium containing 60 mM KCl and 1.8, 2.8, or 5.8 mM CaCl 2 .
  • the selection for dose ranges of extracellular calcium was chosen to provide reliable difference ⁇ 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 CaCl 2 . 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
  • 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 ⁇ M (calpain inhibitor 1) or 5, 25, 37.5, 75, 100 ⁇ M (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 ba ⁇ ed on preliminary ⁇ tudie ⁇ and the manufacturer ' ⁇ recommendation.
  • Samples were prepared using a modification of the method of Taft et al . (1992). Twenty-four hours after depolarization, media were removed, cultures were rin ⁇ ed two time ⁇ with sterile PBS, and 100 ⁇ l of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 1% Nonidet P-40TM (NP-40) , 0.5% SDS, and 0.02% sodium azide. To restrict protease activity and cytoskeletal protein degradation during sample proces ⁇ ing, 2 mM EGTA, 1 mM EDTA, 100 ⁇ g/ml PMSF, 1 ⁇ g/ml aprotinin, and 0.1 mM leupeptin were added to culture wells. Lysates from 3 identical culture wells were pooled for each sample.
  • Antibodies binding NF proteins were NR4, recognizing phosphorylated and non-phosphorylated NF-L, NN18 recognizing phosphorylated and non-phosphorylated NF-M, and N52 recognizing phosphorylated and non-phosphorylated NF-H (Shaw et al . ,
  • GFAP glial fibrillary acidic protein
  • Antibody 38 a polyclonal antibody that recognizes a calpain 1 mediated BDP of ⁇ - ⁇ pectrin (Robert ⁇ -Lewi ⁇ et al . , 1994) was obtained from Cephalon
  • NF-L immunoreactivity decreased 18% (17.63 ⁇ 1.41%) , NF-M immunoreactivity 12% (11.86 ⁇ 1. 1%) and NF-H immunoreactivity 18% (17.80 ⁇ 1.47%) of control values .
  • Glial fibrillary acidic protein (GFAP) levels were unchanged after depolarization with 60 mM KCl and 2.8 mM CaCl- or 5.8 mM CaCl 2 .
  • Coomassie blue ⁇ taining confirmed that the ⁇ ame protein amount wa ⁇ loaded in each lane .
  • Tables 1 and 2 summarize the result ⁇ of ⁇ tudies systematically examining the effects of administration of varying dose ⁇ of calpain inhibitor 1 and 2 following depolarization at 2.8 mM and 5.8 mM extracellular calcium.
  • calpain inhibitor 1 Two concentrations, 25 ⁇ M and 50 ⁇ M of calpain inhibitor 1 protected against NF loss after depolarization in the presence of 2.8 mM CaCl 2 .
  • 50 ⁇ M 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 loss to 27% (26.50 ⁇ 2.95%) .
  • 25 ⁇ M calpain inhibitor 1 revealed smaller but still significant protective effects against losses of all three NF proteins. NF-L loss was reduced to 46% (45.76+2.83%), NF-M loss to 25% (25.36 ⁇ 1.40) and NF-H loss to 37% (36.90 ⁇ 1.191%) .
  • Calpain inhibitor 2 was ineffective against NF protein loss after depolarization with 60 mM KCl and 2.8 mM CaCl 2 .
  • NF-L loss was reduced to 38% (38.46 ⁇ 1.51%)
  • NF-M loss decreased to 37% (36.76 ⁇ 2.05%)
  • NF-H loss decreased to 34%
  • NF-L protein loss decreased to 18% (18.10 ⁇ 2.41%)
  • NF-M loss to 18% (17.85+2.00%)
  • NF-H loss to 14% (13.50 ⁇ 2.37%)
  • 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 CaCl ? and 5.8 mM CaCl ? .
  • Both calpain inhibitors, applied immediately after depolarization with 60 mM KCl and 2.8 mM CaCl ? or 5.8 mM CaCl ? protected against losses of fine neurites. For example, 37.5 ⁇ M 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.
  • NF protein loss following a six min potassium depolarization was accompanied by appearance of calpain 1 mediated BDP of -spectrin.
  • Losses of the high-, medium-, and low molecular weight NF proteins and appearance of spectrin BDP in vi tro were calcium dependent and not due to cell death.
  • calpain inhibitors 1 and 2 provided strong protection against depolarization induced loss of the NF triplet proteins.
  • 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 cathepsins, trypsin and/or chymotrypsin are primarily responsible for losses of these cytoskeletal proteins in the in vi tro system.
  • calpain inhibitors 1 and 2 can reduce depolarization-induced loss of the NF triplet proteins in vi tro . Since NFs, together with microtubule polypeptides and spectrin, are components of three major cytoskeletal systems of neurons, these data support the hypothesis that these inhibitors are potential agents for protecting cytoskeletal integrity following experimental brain injury in vivo and in vi tro .
  • calpain inhibitors 1 and 2 provided marked protection against losses of all three NF proteins even after experimental neuronal injury in vi tro, supporting the therapeutic potential of these agents. Both calpain inhibitors 1 and 2 strongly protected against losses of all three NF proteins after depolarization injury with 60 mM KCl and 5.8 mM CaCl,.
  • 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 ? .
  • GFAP glial fibrillary acidic protein
  • calpain 1 mediated BDP of ⁇ -spectrin were undetectable in control cultures, they were readily apparent after depolarization in the presence of 5.8 mM CaCl ? .
  • Calpain inhibitors 1 and 2 were applied immediately after depolarization injury of septo-hippocampal neurons in vi tro with 5.8 M CaCl ? at final concentrations of 5, 25, 50, 100 and 150 ⁇ M for calpain inhibitor 1 and of 5, 25, 37.5, 75 and 100 ⁇ M 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 analysis.
  • the present Example demonstrates that calpain inhibitors 1 and 2 can reduce loss of tau protein after brief potassium depolarization of CNS cells in vi tro .
  • 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 .
  • 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 observed in immunofluorescence were evident in apical, basal, and arborized dendrites within (overlaying subarachnoid hemorrhage) and beyond areas of cortical contusion.
  • This Example employed a controlled cortical impact device at a magnitude sufficient to produce cortical contusions similar to those seen after severe human injury
  • Treatment groups included naive, sham-injured and injured rats at 3 hours post-TBI.
  • Core body temperature was monitored continuously by a rectal thermistor probe and maintained at 37-38°C.
  • 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 analyses of time/displacement parameters of the impactor.
  • LVDT linear velocity differential transformer
  • anesthetic gases were discontinued in order to minimize the anesthetic effects on the acute neurological assessments.
  • TBI animals were immediately assessed for recovery of reflexes (Dixon et al . , 1991). Animals which recovered
  • 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 was then removed and incubated in 30% sucrose overnight for adequate cryoptection.
  • the brain was then grossly sectioned, frozen, and mounted in a Hacker-Bright cryostat. Coronal sections of 30-40 ⁇ m thickness were cut at -15°C and immediately placed into wells containing PBS (136 mM NaCI, 81 mM KCl and 1.6 mM Na ? HP0 4 and 14 mM KH ? P0 4 , pH 7.4) .
  • 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, N52, and AP20) in blocker solution (Tween- PBS:10 mM NaP0 gown [pH 7.5], 0.9% NaCI, Tween-20 ® , 1% antifoam A (Sigma A-5758) and 5% non-fat dry milk (Carnation, Inc. were incubated for 3 hours at 25°C. The tissue slices were then washed with blocker solutions 3 x 10 minutes. Secondary antibody (anti-mouse IgG) linked to a specific fluorophore was applied for two hours.
  • Primary antibodies Sigma NR4, N52, and AP20
  • blocker solution Tween- PBS:10 mM NaP0 gown [pH 7.5], 0.9% NaCI, Tween-20 ® , 1% antifoam A (Sigma A-5758) and 5% non-fat dry
  • Fluorescein (Vector labs) was used to detect NF68, as well as MAP2 , and rhodamine (Boehringer-Mannheim) was used to detect NF200.
  • the tissue slices were then washed 3 times in PBS solution, mounted on subbed slides (Fisher 12-550-15) , and allowed to dry. Dried sections were then coverslipped with Elvanol ® (Dupont) and stored in the dark prior to classical immunofluorescence and/or confocal microscopy (Molecular Dynamics) examination. Control sections without primary antibodies did not stain.
  • 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 proces ⁇ es 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 assessment by classical immunofluorescence. All tissue section ⁇ were serially scanned at 600X.
  • NR4 anti-NF68
  • N52 anti-NF200
  • AP-20 anti-MAP2 detects MAP2 only in neuronal somato-dendritic regions at a site independent of phosphorylation state and does not detect lower molecular weight proteolytic fragments.
  • 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 corcoup 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 revealed pallor and dark shrunken neurons only in the superficial ipsilateral and contralateral cortical layers (1-3), with the most remarkable pathology occurring ipsilateral 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 .
  • 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 processes was evident throughout the cortical layers in the ipsilateral 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 cortices.
  • NF200 immunoreactivity loss 3 hours post-TBI was not apparent in white matter tracts including the corpus callosum and internal capsule.
  • TBI produced a loss of MAP2 immunoreactivity in cortical neuronal processes using anti-MAP2 (AP-20) , as compared to sham-injured animals. Losses 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 .
  • 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 animals. 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 hour ⁇ post-TBI that was not apparent in sham-injured animals.
  • MAP2 immunofluorescence alterations i.e., fragmented apical dendrites
  • MAP2 immunofluorescence alterations were observed rostral to cortical impact (at +0.2 Bregma) as compare to sham injured animals following TBI.
  • changes in MAP2 immunofluorescence could be detected in areas (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.
  • This Example is a systematic immunohistochemical examination of derangements in cytoskeletal proteins following TBI in rats.
  • the data presented here indicate that preferential dendritic rather than axonal damage occurs within three hours post TBI .
  • 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.
  • confocal microscopy revealed varying degrees of NF200 disassembly within injured apical dendrites.
  • 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 analyses of fragmented apical dendrites imaged with N52 contained varying degrees of NF200 disassembly. Possible explanations for observed differences in immunolabeling patterns post-TBI include the binding characteristics of the selected antibodies, the role of the tertiary structure of the individual subunits, and potential post-tran ⁇ lational 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 ⁇ -helical domain common to all NF proteins. These low MW fragments have been reported after calpain mediated proteolysis in vivo (Schlaepfer).
  • the appearance of fragmented apical dendrites observed with classical and confocal microscopy after TBI could represent the inability of the N52 antibody to detect the low MW ⁇ -helical containing NF fragments and/or physically broken dendrites.
  • 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) .
  • 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.
  • dephosphorylation of NF200 and MAP2 has been shown to increase susceptibility to calpain proteolysis in vi tro (Pant, 1988; Johnson and Foley, 1993) .
  • MAP2 is a cytoskeleton protein restricted to so ato-dendritic neuronal domains.
  • 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 loss was also similar 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 consistent with the binding characteristics of the antibodies.
  • 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.
  • NF68 with NR4 provides complementary data to NF200 immunoreactivity.
  • a unique morphological feature detected with NR4 (anti-NF68) immunofluorescence was the presence of regular spaced vacuoles within apical dendrites.
  • the increased immunoreactivity found adjacent to the plasmalemma could have been produced by immunopositive degraded or disassembled NF subunits.
  • the rostrocaudal extent of morphopathological changes 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 sites of contusion occurred in cortical layers 1 and 3-5.
  • immunofluorescence studies detected significant dendritic derangements beyond areas of cortical contusion (+0.2 Bregma) not associated with prominent H&E morphopathology .
  • focal ischemia could have also contributed to possible loss of calcium homeostasis and calpain activation in this Example.
  • the presence of focal ischemia ip ⁇ ilateral to the site of injury could have contributed to the greater loss of NF68, NF200 and MAP2 in the ipsilateral cortex as compared to the cortex contralateral to the site of cortical impact.
  • dendritic processes (apical dendrites) propagate neuronal transmission by low voltage calcium channels and consequently may be more likely to open after TBI.
  • the electrophysiological properties of dendrites can potentially yield immediate focal alterations in intracellular Ca'' homeostasis at sites of ion entry.
  • axons within white matter tracts 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.
  • electrophysiological studies examining long term potentiation (LTP) following TBI also support impaired dendritic functioning as inferred by decreased efficacy of synaptic transmission.
  • cytoskeletal derangements observed with qualitative light immunofluorescence and confocal microscopy occurred focally along dendrites resulting 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 suggesting that cytoskeleton derangements can occur in areas not necessarily 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 loss observed in diffuse process injury (DPI) suggests profound effects on neuronal connectivity and synaptic efficacy that may contribute to neurobehavioral deficits .
  • DPI diffuse process injury
  • 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 levels observed in naive, uninjured rat cortices, respectively.
  • 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 administration into the right external carotid artery for 24 hours, significantly reduced NF200 losses to 17% and 3% of naive levels in the ipsilateral and contralateral cortices, respectively.
  • Calpain inhibitor treatment abolished NF68 loss in the ipsilateral cortex and was accompanied by a reduction of putative calpain mediated NF68 BDPs.
  • Calpain 1-mediated BDPs to brain ⁇ -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 preservation of neuronal structure throughout cortical regions of the traumatically-injured brain.
  • histopathological studies employing hematoxylin and eosin staining also revealed preservation of neuronal somata in areas of contusion.
  • This Example employed a cortical impact model of TBI (Dixon et al . , 1991) that reproduces a number of features of severe TBI in humans. Examination of cytoskeletal proteins was restricted to cortical areas since no alterations in NF protein levels have been observed in hippocampal samples following lateral cortical impact injury (Posmantur et al . , 1994). Using Western blot analyses, the present invention demonstrates that a systemically-administered calpain inhibitor (calpain inhibitor 2) protects against cortical NF protein loss and reduces calpain-mediated spectrin BDP following experimental TBI in vivo . In addition,
  • 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 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 N 2 0/0. 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 compression.
  • Velocity was 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/displacement parameters of the i pactor.
  • Sham-injured animals underwent identical surgical procedures including craniotomy on both hemispheres, but did not receive impact injury.
  • expanded craniotomy was only performed on the (right) ipsilateral cortex, identical to the injured (right) ipsilateral cortex. Naive rats were not exposed to any injury related surgical procedures. Following cortical impact, animals were extubated and immediately assessed for recovery of reflexes (Dixon et al . , 1991).
  • mice 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) .
  • the animals 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.
  • tissue was frozen immediately in liquid N ? .
  • the microdissected tissue was homogenized at 4°C in an ice cold homogenization buffer containing 20 mM PIPES (pH 7.1) 2 mM EGTA, 1 mM EDT2A, 1 mM dithiothreitol, 0.3 mM phenylmethyl- sulfonylflouride (PMSF) and 0.1 mM leupeptin.
  • PIPES pH 7.1
  • PMSF phenylmethyl- sulfonylflouride
  • 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 0.025 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 ⁇ g 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 u ⁇ ing 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 NaCI, 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.
  • N52 NF200
  • NR4 NF68
  • Antibody 38 a polyclonal antibody that recognizes a calpain 1 mediated BDP of ⁇ -spectrin was obtained from Cephalon, Inc.
  • blots were incubated with a secondary antibody linked to horseradish 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-assi ⁇ ted 2-dimen ⁇ ional densitometric scanning (Biorad Model GS-670) . Data acquired in arbitrary densitometric units were transformed to percentages of the densitometric levels observed on scans from naive animals visualized on the same blot.
  • Coronal sections of 30-40 ⁇ m thickness were cut at -15°C and immediately placed into wells containing PBS (136 mM NaCI, 81 mM KCl and 1.6 mM NaHP0 4 and 14 mM KH 2 P0 4 , 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 hour ⁇ at 25°C in blocker solution: Tween-PBS: 10 mM NaP0 4 (pH 7.5), 0.9% NaCI, Tween-20 ® , 0.1% antifoam A (Sigma A-5758) and 5% nonfat dry milk (Carnation) . The tissue slices were then washed with blocker solution 3 x 10 minutes. Secondary antibody (anti-mouse IgG, 1:1000) linked to a specific fluorophore was applied for two hours.
  • Fluorescein (Vector labs) was used to detect NF200 and NF68. The tis ⁇ ue slices were then washed 3 times in PBS solution, mounted on subbed ⁇ lides with Elvanol (Dupont) and stored in the dark prior to immunofluorescence examination. Sections without primary antibodies did not stain. 6. Histopathological Assessment
  • 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 cor ⁇ nally at 3 micron 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 microns on a microtome, from +0.2 to -3.8 Bregma, mounted on glass slides and stained with hematoxylin and eosin staining.
  • 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) .
  • 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
  • sham injury groups (receiving vehicle [SV] or calpain inhibitor 2 [SD] ) showed no statistically significant NF68 loss as compared to naive animals.
  • NF68 levels in the contralateral cortex did not differ
  • 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
  • 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 was decreased by 36% (35.82 ⁇ 9.09%) in injured, vehicle treated animals (IV) of naive levels (p ⁇ 0.001).
  • IV vehicle treated animals
  • NF200 immunoreactivity between injured, drug treated animals, sham injured animals and naive animals NF200 immunoreactivity between injured, drug treated animals, sham injured animals and naive animals.
  • sham-injury caused the appearance of spectrin BDPs.
  • Sham injury groups (SD and SV) manifested significant higher amounts of spectrin BDPs as compared to naive animals (p ⁇ O.Ol for SD and SV respectively), and there was no significant difference between sham injured animals treated with calpain inhibitor 2 (SD) or vehicle (SV) .
  • SD calpain inhibitor 2
  • SV vehicle
  • 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 revealed significantly lower spectrin BDPs levels as compared to samples from IV animals (+++p ⁇ 0.001) . The amount of spectrin BDPs from ID animals
  • 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.
  • NF200 immunofluorescence in injured-drug treated animals 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 preservation somal labeling.
  • vehicle treated animals manifested a prominent loss of NF200 immunofluorescence among fine dendritic processes within layer 1.
  • receiving calpain inhibitor 2 showed a preservation of fine dendritic processes within the superficial cortical layers.
  • animals receiving calpain inhibitor 2 showed marked preservation of neuronal processes even in areas of direct impact (-3.4 mm Bregma) and ti ⁇ sue deformation.
  • NF68 immunoreactivity in naive animals 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 axons was observed in the area of contusion (from -1.5 mm Bregma to -3.8 mm Bregma) from cortical layer 1 to layers 5 and 6.
  • 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 somata and neuronal processe ⁇ in cortical pyramidal layers 3 and 5.
  • Alternations in axonal NF68 immunoreactivity i.e. axonal
  • NF68 immunoreactivity in injured, drug treated animals revealed less pronounced axonal alterations including axonal swelling, breaks, and retraction balls, than seen in injured, vehicle treated animals.
  • H&E hematoxylin and eosin
  • Ipsilateral superficial cortical contusion 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 corcoup 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 transver ⁇ e direction, limited to 1-2 mm, in the ipsilateral cortex and contralateral cortex.
  • 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 protein ⁇ of the neuronal cytoskeleton.
  • We ⁇ tern blotting studies showed that calpain inhibitor 2 protected against cortical NF los ⁇ es in both hemispheres, ipsilaterally and contralaterally to the site of impact.
  • Protective effect ⁇ against NF degradation were accompanied by reduction of putative calpain mediated BDPs to NF68.
  • the accumulation of calpain mediated BDPs to spectrin was significantly attenuated in ipsi- and contralateral cortices following calpain inhibition treatment.
  • TBI produced significant losses of NF200, while NF68 levels were unchanged. This, however, does not exclude calpain induced proteoly ⁇ i ⁇ of NF200, a ⁇ it ha ⁇ been reported previou ⁇ ly that NF200 has a higher susceptibility to calpain induced proteolysis than NF68 in vivo and in vi tro (Kamakura et al * , 1985) .
  • calpain mediated BDPs of spectrin could be also detected in the contralateral cortex following TBI.
  • Differential ⁇ u ⁇ ceptibility of cyto ⁇ keletal 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 .
  • calpain inhibitor 2 Protein Loss 24 Hours After TBI Calpain inhibitor 2 administration effectively reduced NF68, NF200 protein loss following TBI. As this inhibitor significantly 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.
  • spectrin may be more vulnerable to calpain proteolysis than NFs.
  • ⁇ ham injured animals receiving calpain inhibitor 2 had significant less spectrin BDPs in the contralateral cortex than sham-injured animals, it is not clear why calpain inhibitor 2 had no effect on reduction of spectrin BDPs in the ipsilateral cortex of sham injured animals.
  • Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of post occlusion intra-arterial administration. Stroke 25: 2265-2270.
  • Neurofilament 200 Posmantur et al . , "Neurofilament 68 and Neurofilament 200

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Abstract

Cette invention se rapporte à des procédés de traitement des lésions cérébrales et médullaires et des accidents cérébrovasculaires grâce à l'utilisation d'inhibiteurs de la calpaïne. Dans les réalisations préférées de l'invention, on utilise des inhibiteurs de calpaïne 1 et 2 aux fins de protection contre la perte en protéines des neurofilaments qui résulte à la fois d'une dépolarisation et de la blessure par impact.
PCT/US1997/012668 1996-07-19 1997-07-18 Inhibiteurs de calpaine destines au traitement des lesions traumatiques du cerveau Ceased WO1998003190A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO1999036082A3 (fr) * 1998-01-13 1999-09-23 Julien Jean Pierre Utilisation de taux accrus de proteines de neurofilaments en tant que protection et therapie contre la neurodegenerescence associee au stress oxydatif
JP2007535318A (ja) * 2004-04-15 2007-12-06 バンヤン バイオマーカーズ 癌、器官傷害、および筋肉リハビリテーション／運動過剰訓練についての診断の生物マーカーとしてのタンパク質分解マーカー

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999036082A3 (fr) * 1998-01-13 1999-09-23 Julien Jean Pierre Utilisation de taux accrus de proteines de neurofilaments en tant que protection et therapie contre la neurodegenerescence associee au stress oxydatif
JP2007535318A (ja) * 2004-04-15 2007-12-06 バンヤン バイオマーカーズ 癌、器官傷害、および筋肉リハビリテーション／運動過剰訓練についての診断の生物マーカーとしてのタンパク質分解マーカー
EP1747282A4 (fr) * 2004-04-15 2009-11-11 Univ Florida Marqueurs proteolytiques, biomarqueurs diagnostiques du cancer, et des lesions des organes et des lesions des muscles dues a un surentrainement lors de rehabilitations ou d'exercices
JP4885122B2 (ja) * 2004-04-15 2012-02-29 ユニバーシティ オブ フロリダ リサーチ ファンデーション インコーポレーティッド 癌、器官傷害、および筋肉リハビリテーション／運動過剰訓練についての診断の生物マーカーとしてのタンパク質分解マーカー
US8298835B2 (en) 2004-04-15 2012-10-30 University Of Florida Research Foundation, Inc. Proteolytic markers as diagnostic biomarkers for cancer, organ injury and muscle rehabilitation/exercise overtraining

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