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WO1998003191A1 - Inhibiteurs de la calpaine permettant le traitement de l'ischemie cerebrale, des lesions de la moelle epiniere ou des commotions - Google Patents

Inhibiteurs de la calpaine permettant le traitement de l'ischemie cerebrale, des lesions de la moelle epiniere ou des commotions Download PDF

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WO1998003191A1
WO1998003191A1 PCT/US1997/012672 US9712672W WO9803191A1 WO 1998003191 A1 WO1998003191 A1 WO 1998003191A1 US 9712672 W US9712672 W US 9712672W WO 9803191 A1 WO9803191 A1 WO 9803191A1
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calpain
inhibitor
injury
hours
administered
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Ronald L. Hayes
Guy L. Clifton
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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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • 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

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 .
  • 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 . ,
  • 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 neurofilaments
  • MAP2 microtubule associated protein 2
  • 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 i ⁇ 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) .
  • 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
  • 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) .
  • calpain inhibitors such as for example calpain inhibitor I and calpain inhibitor II
  • calpain inhibitors I and II for use in treating traumatic brain injury, cerebral ischemia, spinal injury and stroke, for at least the following several reasons .
  • 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 i ⁇ highly selective (Wang and Yuen, 1994) .
  • Povlishock 1993
  • traumatic brain injury 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.
  • a method for treating cerebral ischemia, spinal cord injury or stroke 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 cerebral ischemia, spinal cord injury, or stroke 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.
  • the Ca 2 " -activated neutral protease calpain is just one of many cellular proteins involved in Ca 2+ signaling 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 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.
  • 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 asse ⁇ sments of contusion, mass legion and/or infarction.
  • Brain/tis ⁇ ue assessments may be made non-invasively, by computerized tomograph, and/or by magnetic resonance imaging or spectroscopy .
  • 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 .
  • 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 "event” i.e., traumatic brain injury, cerebral ischaemia, spinal cord injury or stroke
  • 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.
  • 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 i ⁇ 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 wall ⁇ of spastic " arteries and that this proteolytic response plays an important role in the response of the injured vessel.
  • Calpain substrates found in vascular smooth muscle and endothelial cells include structural and regulatory proteins that participate in the maintenance of cerebrovascular function.
  • PKC protein kinase C
  • Stimulation of PKC in normal vessels elicits a strong and long-lasting constriction.
  • kinase inhibitors capable of blocking PKC activity can attenuate the vaso ⁇ pastic response.
  • calpain substrates in vascular smooth muscle that participate in the control of vascular tone include myosin light-chain kinase (MLCK) , calponin, and caldesmon.
  • MLCK myosin light-chain kinase
  • calponin calponin
  • caldesmon calpain light-chain kinase
  • 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- a ⁇ ociated protein ⁇ that inhibit actomyo ⁇ in 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.
  • 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 vasospam ⁇ .
  • 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.
  • administration of 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 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 minute ⁇ 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/tis ⁇ ue indice ⁇ , and/or to provide for reduction of cyto ⁇ keletal protein lo ⁇ , 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. 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.
  • 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 compres ⁇ ed 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 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.
  • 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.
  • the cooling process continues until the patient is ⁇ ufficiently cooled to a holding temperature below normal body temperature ⁇ uitable to provide an improvement in neurological outcome.
  • this holding temperature will be les ⁇ 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 i ⁇ 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 mo ⁇ t preferably at lea ⁇ t 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/4hr ⁇ , 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.
  • 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 los ⁇ es 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 inhibitor ⁇ 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 inhibitor ⁇ 1 and 2 were influenced by different levels of extracellular calcium.
  • Qualitative immunohistochemical evaluations and Western blot data confirmed protection of NF los ⁇ 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.
  • calpain inhibitors 1 and 2 which inhibit calpain ⁇ , cathepsins, and to a le ⁇ er extent chymotrypsin, strongly protected against los ⁇ es of all three NF protein ⁇ 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 2+ /Mg 2+ -free Hanks balanced salt solution (HBSS) . After rinsing, cells were dis ⁇ ociated 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 5 cells/well to 16 mm poly-L-ly ⁇ ine coated pla ⁇ tic 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 time ⁇ a week.
  • DMEM Dulbecco's modified Eagle's medium
  • 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.
  • MAP2 microtubule associated protein 2
  • depolarization injury was performed by replacing normal media containing 5.3 mM KCl and 1.8 M CaCl 2 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.
  • 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 .
  • 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 based on preliminary studies and the manufacturer's recommendation.
  • Sample ⁇ 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 ⁇ 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.
  • 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 electrophoresi ⁇ chamber u ⁇ ing a 4% acrylamide ⁇ tacking gel over a 7.5% acrylamide resolving gel. Gel ⁇ were run at constant voltage (200 V) for approximately 1 hour. 20 ⁇ g of sample protein was resolved in each lane.
  • 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-20TM at 4°C. Monoclonal antibodies specific for individual NF proteins were used for immunolabeling.
  • Antibodies binding NF proteins were NR4 , recognizing phosphorylated and non-pho ⁇ phorylated 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 -spectrin (Roberts-Lewis et al . , 1994) was obtained from Cephalon
  • Control and depolarized cultures were stained 24 hours after depolarization insult with fluorescein diacetate (FDA 11.5 ⁇ g/ml), which labels live cell ⁇ and with propidium iodine (PI 3.2 ⁇ g/ml) which stains dead cells. Both compounds were dissolved in phosphate buffer and left in contact with the cells for 10 minute ⁇ . No fixation wa ⁇ done before treatment. Cells were then observed with a fluorescence microscope. Cell loss was calculated for five wells in each ⁇ tudy as a percentage of total cell number.
  • FDA 11.5 ⁇ g/ml fluorescein diacetate
  • PI 3.2 ⁇ g/ml propidium iodine
  • 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 .
  • Glial fibrillary acidic protein (GFAP) levels were unchanged after depolarization with 60 M KCl and 2.8 mM CaCl 2 or 5.8 M CaCl, . Coomassie blue staining confirmed that the same protein amount was loaded in each lane.
  • Tables 1 and 2 summarize the results of studies 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.
  • NF-L los ⁇ was reduced to 46% (45.76+2.83%), NF-M los ⁇ to 25% (25.36 ⁇ 1.40) and NF-H loss to 37% (36.90+1.191%).
  • Calpain inhibitor 2 was ineffective against NF protein los ⁇ after depolarization with 60 mM KCl and 2.8 mM CaCl 2 .
  • calpain inhibitor 1 All concentrations of calpain inhibitor 1 protected against loss of NF proteins after depolarization with 5.8 mM CaCl 2 . 50 ⁇ M of calpain inhibitor 1 revealed the highest protection against los ⁇ of all three NF protein ⁇ . NF-L lo ⁇ 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%
  • 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 2 and 5.8 M CaCl, .
  • Both calpain inhibitors, applied immediately after depolarization with 60 mM KCl and 2.8 mM CaCl 2 or 5.8 mM CaCl protected against losses of fine neurites.
  • 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.
  • calpain inhibitors 1 and 2 provided strong protection against depolarization induced loss of the NF triplet protein ⁇ .
  • 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 cathep ⁇ in ⁇ , trypsin and/or chymotrypsin are primarily responsible for losses of these cytoskeletal protein ⁇ in the in vi tro sy ⁇ tem.
  • calpain inhibitors 1 and 2 can reduce depolarization- induced loss of the NF triplet proteins in vi tro . Since NF ⁇ , together with microtubule polypeptides and spectrin, are components of three major cytoskeletal sy ⁇ tem ⁇ of neurons, these data support the hypothesi ⁇ that these inhibitors are potential agents for protecting cytoskeletal integrity following experimental brain injury in vivo and in vi tro .
  • calpain inhibitor ⁇ 1 and 2 provided marked protection against losses of all three NF protein ⁇ even after experimental neuronal injury in vi tro, supporting the therapeutic potential of these agents. Both calpain inhibitors 1 and 2 strongly protected against losse ⁇ of all three NF proteins after depolarization injury with 60 mM KCl and 5.8 mM CaCl 2 .
  • rat fetuses Primary septo-hippocampal cells (ratio 1:1) were prepared from 18-day old rat fetuses and distributed at a density of 2.18 x 10 5 cells/well to 16 mm poly-L-lysine coated plastic culture dishes and kept in a humidified C0 2 incubator. After five days in media with serum, cultures were maintained in B18 media (Brewer and Cot an, 1989) . After ten days in culture, astrocyte ⁇ formed a confluent monolayer beneath morphologically mature neurons as judged by proces ⁇ formation and the ability to ⁇ equester microtubule as ⁇ ociated protein 2 and tau.
  • 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.
  • the loss of tau protein was significantly greater after depolarization with 5.8 mM CaCl 2 as compared to 1.8 mM CaCl 2 (p ⁇ 0.001, Student's t-test) .
  • 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 2 .
  • Calpain inhibitors l and 2 were applied immediately after depolarization injury of septo-hippocampal neurons in vi tro with 5.8 mM CaCl 2 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 analysi ⁇ .
  • the present Example demonstrates that calpain inhibitors 1 and 2 can reduce los ⁇ of tau protein after brief pota ⁇ ium depolarization of CNS cell ⁇ 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 .
  • 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 as ⁇ embly state associated with immunofluorescence alterations. Little evidence of axonal involvement was detected at 3 hours post-TBI. These studies sugge ⁇ t that derangements of NF68, NF200 and MAP2 as early as 3 hours after lateral impact injury occur preferentially in dendrites rather than axon ⁇ within and beyond areas of 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 (Dixon et al . , 1991).
  • Cortical impact models utilize a pneumatic piston to deform a specifiable volume of expo ⁇ ed cortex over a range of impact velocities .
  • the magnitude of cortical impact injury in thi ⁇ study (6 m/s) produces over disruption of the overlying vasculature from approximately -1.5 to -4.2 Bregma.
  • thi ⁇ magnitude of impact is associated with significant motor and spatial memory deficits (Ham et al . , 1992).
  • 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 analyse ⁇ of time/di ⁇ placement parameter ⁇ of the impactor.
  • LVDT linear velocity differential transformer
  • the 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). Animal ⁇ which recovered righting re ⁇ ponse in 5 minutes or les ⁇ or omitted from the Example .
  • 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 wa ⁇ then removed and incubated in 30% ⁇ ucrose overnight for adequate cryoptection.
  • the brain wa ⁇ then gro ⁇ ly 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 NaCl, 81 mM KCl and 1.6 mM Na ? HP0 4 and 14 mM KH 2 P0 4 , pH 7.4) .
  • 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 asse ⁇ sment by classical immunofluorescence. All tissue sections were serially scanned at 600X.
  • NR4 anti-NF68
  • NR4 anti-NF68
  • MW low molecular weight
  • N52 anti-NF200
  • AP-20 anti-MAP2 detect ⁇ 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 ip ⁇ ilateral 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 proces ⁇ es wa ⁇ evident throughout the cortical layers in the ip ⁇ ilateral 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 cortice ⁇ .
  • 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 layer ⁇ 3 and 5.
  • NF200 immunoreactivity lo ⁇ s 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) , a ⁇ compared to ⁇ ham-injured animal ⁇ .
  • Lo ⁇ ses 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 animal ⁇ . 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.
  • This Example is a sy ⁇ tematic immunohi ⁇ tochemical 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 analyse ⁇ of fragmented apical dendrite ⁇ imaged with N52 contained varying degrees of NF200 disassembly. Pos ⁇ ible explanations for observed differences in immunolabeling patterns post-TBI include the binding characteri ⁇ tic ⁇ 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 ⁇ -helical domain common to all NF proteins. These low MW fragments have been reported after calpain mediated proteolysis in vivo (Schlaepfer).
  • 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 ha ⁇ been shown to increase su ⁇ ceptibility to calpain proteolysis in vitro (Pant, 1988; Johnson and Foley, 1993).
  • MAP2 is a cytoskeleton protein restricted to somato-dendritic neuronal domain ⁇ .
  • 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 lo ⁇ wa ⁇ also ⁇ imilar 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 con ⁇ i ⁇ tent 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 change ⁇ 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 site ⁇ 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 . 5.
  • 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 los ⁇ of NF68, NF200 and MAP2 in the ipsilateral cortex as compared to the cortex contralateral to the site of cortical impact.
  • LTP long term potentiation
  • cytoskeletal derangements may not solely be a function of the contusion, but also may reflect more global, neuronal responses to injury.
  • DPI diffuse proces ⁇ 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 level ⁇ ob ⁇ erved in naive, uninjured rat cortice ⁇ , re ⁇ pectively .
  • NF68 protein levels decreased by 35% of naive levels only in the ipsilateral cortex.
  • Calpain inhibitor 2 administered ten minutes after injury via continuous
  • Calpain inhibitor treatment abolished NF68 los ⁇ in the ipsilateral cortex and wa ⁇ 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 .
  • calpain inhibitor 2 a systemically-administered calpain inhibitor (calpain inhibitor 2) protects against cortical NF protein los ⁇ and reduces calpain-mediated spectrin BDP following experimental TBI in vivo .
  • calpain inhibitor 2 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 .
  • Velocity wa ⁇ 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/di ⁇ placement parameter ⁇ of the impactor.
  • 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) ip ⁇ ilateral cortex, identical to the injured (right) ip ⁇ ilateral cortex.
  • Naive rat ⁇ were not expo ⁇ ed to any injury related surgical procedures. Following cortical impact, animals were extubated and immediately assessed for recovery of reflexes (Dixon et al . , 1991) .
  • ICA 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 2 .
  • the microdis ⁇ ected 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.
  • PIPES pH 7.1
  • PMSF phenyl ethyl- sulfonylflouride
  • the amount of protein in samples was determined using BCA ® reagents (Pierce) with albumin standards. Protein- balanced samples were prepared for polyacrylamide gel electrophoresi ⁇ 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 .
  • 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.
  • N52 NF200
  • NR4 NF68
  • Antibody 38 a polyclonal antibody that recognizes a calpain 1 mediated BDP of ⁇ -spectrin was obtained from Cephalon, Inc.
  • Coronal section ⁇ of 30-40 ⁇ m thickne ⁇ 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 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 hours at 25°C in blocker solution: Tween-PBS : 10 mM NaP0 4 (pH 7.5), 0.9% NaCl, Tween-20 ® , 0.1% antifoam A (Sigma A-5758) and 5% non- fat dry milk (Carnation) . The tissue slice ⁇ were then wa ⁇ hed with blocker ⁇ olution 3 x 10 minute ⁇ . Secondary antibody (anti-mouse IgG, 1:1000) linked to a specific fluorophore was applied for two hour ⁇ .
  • 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 section ⁇ and optimal hematoxylin and eosin (H&E) staining. Brain section ⁇ 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 slide ⁇ 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 wa ⁇ decreased by 36% (35.82+9.09%) in injured, vehicle treated animals (IV) of naive level ⁇ (p ⁇ 0.001).
  • NF200 immunoreactivity in the contralateral cortex wa ⁇ decreased by 36% (35.82+9.09%) in injured, vehicle treated animals (IV) of naive level ⁇ (p ⁇ 0.001).
  • IV vehicle treated 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.
  • NF200 immunoreactivity was observed from -1.5 mm Bregma through -3.8 mm Bregma rostrocaudally, corresponding to regions showing morphopathology characteristic of injured neurons.
  • Los ⁇ of NF200 immunolabeling wa ⁇ also detected in a well defined area in the contralateral cortex, 1-2 mm wide.
  • NF200 immunofluorescence in injured-drug treated animal ⁇ 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 pre ⁇ ervation somal labeling.
  • 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.
  • 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 ⁇ omata 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 ⁇ uch a ⁇ 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 ⁇ een in injured, vehicle treated animal ⁇ .
  • H&E hematoxylin and eosin
  • Ip ⁇ ilateral superficial cortical contu ⁇ ion 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 transverse direction, limited to 1-2 mm, in the ipsilateral cortex and contralateral cortex.
  • TBI produced significant losses of NF200, while NF68 levels were unchanged. This, however, does not exclude calpain induced proteolysis of NF200, a ⁇ 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.
  • 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 .
  • sham injured animals receiving calpain inhibitor 2 had significant less ⁇ pectrin BDPs in the contralateral cortex than sham-injured animals, it is not clear why calpain inhibitor 2 had no effect on reduction of spectrin BDP ⁇ 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.
  • MAP2 Microtubule Associated Protein 2
  • Rat Brain Abstr. 24th Annu . Meet . Soci . Neurosci . , 460.2, 1994.

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Abstract

La présente invention concerne des procédés se rapportant au traitement des lésions de l'encéphale et de la moelle épinière et au traitement des commotions par utilisation d'inhibiteurs de la calpaïne. Selon des réalisation préférées, on utilise des inhibiteurs des calpaïnes 1 et 2 en protection contre les pertes de protéines des neurofilaments imputables à une dépolarisation et au traumatisme.
PCT/US1997/012672 1996-07-19 1997-07-18 Inhibiteurs de la calpaine permettant le traitement de l'ischemie cerebrale, des lesions de la moelle epiniere ou des commotions Ceased WO1998003191A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074381A3 (fr) * 2000-03-31 2002-01-31 Parker Hughes Inst Inhibiteurs de calpaines pour le traitement du cancer
DE102004011655A1 (de) 2004-03-10 2005-09-29 Daimlerchrysler Ag Fahrberechtigungssystem, zugehöriges Authentikationselement sowie Energieversorgungsverfahren

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5444042A (en) * 1990-12-28 1995-08-22 Cortex Pharmaceuticals Method of treatment of neurodegeneration with calpain inhibitors
US5506243A (en) * 1993-04-28 1996-04-09 Mitsubishi Kasei Corporation Sulfonamide derivatives

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5444042A (en) * 1990-12-28 1995-08-22 Cortex Pharmaceuticals Method of treatment of neurodegeneration with calpain inhibitors
US5506243A (en) * 1993-04-28 1996-04-09 Mitsubishi Kasei Corporation Sulfonamide derivatives

Non-Patent Citations (4)

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Title
BRAIN RESEARCH, Vol. 609, 1993, RAMI A. et al., "Protective Effects of Calpain Inhibitors Against Neuronal Damage Caused by Cytotoxic Hypoxia in Vitro and Ischemia in Vivo", pages 67-70. *
NEUROLOGICAL RESEARCH, August 1995, Vol. 17, BARTUS R.T. et al., "Calpain as a Novel Target for Treating Acute Neurodegenerative Disorders", pages 249-257. *
STROKE, Vol. 25, No. 3, March 1994, HONG SEUNG-CHYUL et al., "Neuroprotection with a Calpain Inhibitor in a Model of Focal Cerebral Ischemia", pages 663-669. *
TIPS, November 1994, Vol. 15, WANG K.W. et al., "Calpain Inhibition: An Overview of Its Therapeutic Potential", pages 412-419. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074381A3 (fr) * 2000-03-31 2002-01-31 Parker Hughes Inst Inhibiteurs de calpaines pour le traitement du cancer
DE102004011655A1 (de) 2004-03-10 2005-09-29 Daimlerchrysler Ag Fahrberechtigungssystem, zugehöriges Authentikationselement sowie Energieversorgungsverfahren

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