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WO2002007755A1 - Modulation d'un influx calcique neuronal induite par la proteine liee au recepteur de lipoproteine via des recepteurs de nmda, et son utilisation - Google Patents

Modulation d'un influx calcique neuronal induite par la proteine liee au recepteur de lipoproteine via des recepteurs de nmda, et son utilisation Download PDF

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Publication number
WO2002007755A1
WO2002007755A1 PCT/US2000/040636 US0040636W WO0207755A1 WO 2002007755 A1 WO2002007755 A1 WO 2002007755A1 US 0040636 W US0040636 W US 0040636W WO 0207755 A1 WO0207755 A1 WO 0207755A1
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lrp
receptor
neuronal cells
calcium influx
agent
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PCT/US2000/040636
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English (en)
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Bradley T. Hyman
Dudley K. Strickland
Brian J. Bacskai
G. William Rebeck
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The General Hospital Corporation
The American National Red Cross
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Priority to CA002416755A priority Critical patent/CA2416755A1/fr
Priority to AU2000277598A priority patent/AU2000277598A1/en
Publication of WO2002007755A1 publication Critical patent/WO2002007755A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/451—Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/46—8-Azabicyclo [3.2.1] octane; Derivatives thereof, e.g. atropine, cocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'

Definitions

  • the present invention relates to methods of treating neurological disorders with agents that bind to low-density lipoprotein receptor-related protein (LRP) receptors.
  • LRP low-density lipoprotein receptor-related protein
  • the present invention also relates to methods of modulating calcium influx and inhibiting cell death in neuronal cells by treating the neuronal cells with agents that bind to LRP.
  • the present invention also relates to methods of identifying agents that modulate calcium influx in neuronal cells by binding to LRP.
  • the low-density lipoprotein receptor is one of the best studied examples of an endocytic receptor, delivering cholesterol containing lipoproteins and other ligands to acidic compartments within cells for further metabolism.
  • a family of homologous receptors plays similar roles in various tissues, including the very low-density lipoprotein receptor (VLDL-r), the apolipoprotein E receptor 2 (APOER2), the low-density lipoprotein receptor related protein (LRP), and megalin (or GP330).
  • LRP is a widely expressed endocytic receptor which is strongly expressed in brain on neurons and reactive astrocytes (Rebeck, G. W., et al, Neuron 11:575-
  • LRP is a >600 kDa (4454 amino acid) protein cleaved in the trans- Golgi network to form a heterodimer with a single transmembrane spanning domain, a ⁇ 515 kDa extracellular region containing 4 ligand binding repeat regions, multiple EGF and growth factor repeats, and a smaller intracellular domain containing two NPXY sequences which direct endocytosis of the receptor to clathrin coated pits (Herz, J., et al., EMBO J. 7:4119-27 (1988); Strickland,
  • LRP has more than 15 identified ligands, which fall into several broad categories such as proteases, protease inhibitors, such as activated alpha-2-macroglobulin (a2M*), protease/protease inhibitor complexes, protein- lipid complexes, and other proteins and molecules such as lactoferrin.
  • proteases such as activated alpha-2-macroglobulin (a2M*)
  • protease/protease inhibitor complexes such as activated alpha-2-macroglobulin (a2M*)
  • a2M* activated alpha-2-macroglobulin
  • protease/protease inhibitor complexes such as activated alpha-2-macroglobulin (a2M*)
  • a2M* activated alpha-2-macroglobulin
  • protease/protease inhibitor complexes such as activated alpha-2-macroglobulin (a2M*)
  • RAP 39 kDa receptor associated protein
  • LRP binds and imports these ligands into intracellular vesicles, acidified compartments where the ligand is released, and the receptor is recycled to the surface.
  • LRP Strickland, D.K., et al, J. Biol. Chem. 265:17401-4 (1990)
  • APOER2 APOER2
  • the present invention is directed to a method of treating a subject in need of treatment of a neurological disorder, the method comprising: administering to the subject a pharmaceutically effective amount of an agent that binds to low- density lipoprotein receptor-related protein (LRP) receptor of LRP on neuronal cells, and modulates calcium influx in said neuronal cells.
  • LRP low- density lipoprotein receptor-related protein
  • the present invention is directed to a method of inhibiting cell death in neuronal cells, the method comprising: providing neuronal cells with an agent that binds to low-density lipoprotein receptor-related protein (LRP) receptor on the neuronal cells, and modulates calcium influx in the neuronal cells.
  • LRP low-density lipoprotein receptor-related protein
  • the present invention also is directed to a method of modulating calcium influx in neuronal cells, the method comprising: treating the neuronal cells with an agent that binds to low-density lipoprotein receptor-related protein (LRP) receptor on the neuronal cells, and modulates calcium influx in the neuronal cells.
  • LRP low-density lipoprotein receptor-related protein
  • the present invention is directed to a method of identifying an agent that modulates calcium influx in neuronal cells by binding to low-density lipoprotein receptor-related protein (LRP) receptors on the neuronal cells, the method comprising: (a) treating neuronal cells with an agent and assaying for calcium influx; (b) treating neuronal cells with a known modulator of LRP- mediated calcium influx and the agent in (a) and assaying for calcium influx; and (c) comparing the levels of calcium influx in (a) and (b) to determine if the agent in (a) modulates calcium influx by interacting with an LRP receptor.
  • LRP low-density lipoprotein receptor-related protein
  • Figure 1 Shows that a2M* increases [Ca 2+ ], specifically in neurons.
  • Primary cultures of mouse cortex were loaded for 30 min with 1 ⁇ M indo-1/AM and imaged using a Biorad Multiphoton confocal microscope.
  • the traces represent a time-course of intracellular calcium concentration in a field of cells in a single, representative experiment. Each trace is the average of 6 cells within the field, ⁇ std deviation.
  • the cells that did respond resembled neurons morphologically and also responded to NMDA application.
  • Non-responders had the generally flat appearance of glia and/or fibroblasts and did not respond to NMDA addition.
  • FIG. 2 Shows that all cells in the mixed cultures expressed LRP. Immunohistochemistry using the anti-LRP antibody R777 revealed that all cells in the cultures expressed LRP (top left panel).
  • the top right panel is a dual label with an antibody to MAP2, which was expressed exclusively in neurons (Izant, J.G. and Mclntosh, J.R., Proc. Nat. Acad. Sci. U.S.A. 77:4741-5 (1980)).
  • the scale bar is 25 ⁇ m.
  • FIG. 3 Shows that the calcium increase requires extracellular calcium.
  • Figure 4 Shows that calcium entry occurs through NMDAR channels.
  • the cells were pretreated with 5 ⁇ M MK-801 for 5 minutes, and the
  • NMDA antagonist remained in the bath throughout the procedure.
  • a2M* 35 nM was added to the bath, resulting in a small but insignificant increase in [Ca 2+ ]j in this field of cells.
  • NMDA 100 ⁇ M was added, and no change in [Ca 2+ ]j was observed.
  • Figure 5 Shows that a2M* increases calcium in neurons in which LRP receptors are blocked by RAP.
  • FIG. 6 Shows that an antibody to the ligand binding domain of LRP increases [Ca 2+ ] ! , but an antibody to an intracellular domain of LRP does not.
  • This figure illustrates two experiments utilizing rabbit polyclonal antibodies directed against LRP.
  • R777 which recognizes the ligand binding domain of LRP, was added.
  • Addition of R777 increased [Ca 2+ ] j in a neuron specific manner.
  • addition of R704 which recognizes an intracellular domain of LRP, was unable to elicit an increase in [Ca 2+ ];.
  • subsequent addition of a2M* was able to generate a calcium response in these cells.
  • the LRP receptor is a widely expressed endocytic receptor which is strongly expressed on neuronal cells, including neurons and glial cells of the central and peripheral nervous systems (Rebeck, G. W., et al., Neuron 11:575-80 (1993)).
  • a novel neuron-specific signaling role for LRP is reported herein. Namely, LRP ligand binding and receptor dimerization led to calcium influx in neuronal cells via NMDAR channels. A robust, spatially and temporally discrete calcium signal was observed in neurons treated with ligand competent a2M*, which was blocked by RAP. Non-neuronal cells, which also expressed LRP, in the same cultures did not elicit a calcium response.
  • the calcium signal was dependent on extracellular calcium and was blocked by the NMDA receptor antagonist MK-801 (Tolax, M., et al, J. Neurosci. 19:7100-10 (1999)). Calcium influx in neurons also occurred after treatment with R777, an antibody directed against the extracellular domain of LRP, and this response was also blocked with MK-801. Calcium entiy did not occur after treatment with Fab fragments of R777, suggesting that receptor dimerization may be critical. These results demonstrate a novel signaling role for the multifunctional receptor LRP in neurons.
  • an LRP receptor is a protein that is recognized in the art as such, and forms a heterodimer with a single transmembrane spanning domain, contains an extracellular region containing 4 ligand binding repeat regions, contains multiple EGF and growth factor repeats, and contains a smaller intracellular domain containing two NPXY sequences (where N symbolizes the amino acid asparagine, P symbolizes proline, X is any amino acid and Y symbolizes tyrosine) which direct endocytosis of the receptor to clathrin coated pits (Herz, J., etal, EMBOJ. 7:4119-27 (1988); Strickland, D.K., etal, J. Biol. Chem.
  • the present invention is directed to a method of treating a subject in need of treatment of a neurological disorder, the method comprising: administering to the subject a pharmaceutically effective amount of an agent that binds to low- density lipoprotein receptor-related protein (LRP) receptor on neuronal cells, and modulates calcium influx in said neuronal cells.
  • LRP low- density lipoprotein receptor-related protein
  • the term receptor is meant to include a molecule that binds to a ligand and causes a cellular or physiological response.
  • the receptor can be cytosolic, membrane-bound, membrane-spanning, or it can be an extracellular molecule.
  • the receptor can be in the form of a monomer or a multimer (i.e., dimer, trimer, or higher multimer) .
  • the ter multimer encompasses a homomultirner or a heteromultimer.
  • homomultimer is used to mean a multimer molecule where all of the individual proteins or other molecules that constitute the multimer are identical.
  • a heteromultimer is used herein to mean a multimer molecule where any of the individual proteins or other molecules that constitute the multimer are not identical.
  • a monomeric receptor causes the physiological or cellular response solely, while the multimeric receptor may require two or more proteins or other molecules, acting in concert with one another, to cause a physiological or cellular response.
  • molecules that can act as receptors include, but are not limited to, proteins, polysaccharides, glycoproteins, proteoglycans, nucleic acids, lipids, and lipoproteins.
  • Examples of cellular responses that receptors initiate or propagate include, but are not limited to ion influx or efflux, initiation of second messenger pathways, synthesis of DNA, translation of mRNA, entry of the cells into the cell cycle, arrest of the cell in the cell cycle, endocytosis, release of molecules from the cell, exocytosis, and apoptosis.
  • the cellular response on which the current invention focuses is modulating calcium influx in the affected neuronal cell.
  • modulation of calcium entry into the cytoplasm of the affected cell includes such responses as increasing or decreasing the quantity of calcium ions that normally enter the cell from the extracellular environment, in conjunction with another stimulus.
  • modulation of calcium entry into the cytoplasm of the affected cell also includes such responses as increasing or decreasing the quantity of calcium ions that normally enter the cell from the extracellular environment, in the absence of another stimulus.
  • modulation of calcium influx is meant to encompass increasing or decreasing the quantity of release or uptake of calcium ions from or to intracellular stores, such as mitochondria, in conjunction with another stimulus.
  • modulation of calcium influx is also meant to encompass increasing or decreasing the quantity of release or uptake of calcium ions from or to intracellular stores, such as mitochondria, in the absence of another stimulus.
  • the channel through which calcium influx in the neuronal cells is mediated is not LRP. More preferably, the channel through which calcium influx in the neuronal cells is mediated is through a class of receptors that binds to the ligand N-methyl-D-aspartate (NMDA), dubbed NMDA receptors.
  • NMDA receptors are ligand-gated ion channels that are a subclass of the larger family of glutamate receptors.
  • a ligand-gated channel is a receptor that binds a ligand and subsequently opens to allow the flow of ions, such as Na + , K + or Ca 2+ , into or out of the cell.
  • LTP long-term potentiation
  • LTD long-term depression
  • LTD is the phenomenon where a postsynaptic neuron has a prolonged decrease response to a presynaptic stimulus.
  • dimerization is the process that is well-recognized in the art where two separate proteins form an association.
  • the dimer formed may be a heterodimer or a homodimer.
  • the association forming the dimer can be temporary or permanent.
  • the association of the two proteins can serve to enhance or diminish the normal function or signaling capacity of each of the two proteins.
  • the association of the two proteins can lead to a completely different function, e.g., second messenger propagation, than the normal function of either of the two proteins.
  • agent ligand or compound is intended to mean a protein, nucleic acid, carbohydrate, lipid or a small molecule.
  • the types of agents or compounds which can be envisioned are limited only by their ability to bind to LRP and mediate calcium influx, particularly through the NMDA receptor channels.
  • LRP has at least four documented binding sites. Each of these four binding sites has its own ligand binding specificity domains, such that the various ligands that LRP binds do not bind to the same binding domain.
  • lactoferrin is a ligand of LRP, there are binding sites on LRP that are unresponsive to lactoferrin.
  • the agent binds to a site on LRP that is unresponsive to lactoferrin.
  • the agent can bind to a site on the LRP that does not bind to amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • the agent that binds to LRP and modulates calcium does not interfere with the amount or rate of binding of APP to LRP.
  • the agent binds to a site on LRP that is unresponsive to lactoferrin, and the agent also does not bind to the APP binding site.
  • agents of the present invention include agents selected from, but are not limited to, protein-lipid complexes, proteases, protease inhibitors, protease-inhibitor complexes, intracellular proteins, small molecules, LRP receptor antibodies, and LRP-interacting proteins.
  • proteins of the aforementioned classes include, but are not limited to, protein-lipid complexes involved in lipid and/or cholesterol metabolism such as apolipoprotein, proteases such as plasminogen, protease inhibitors such as activated alpha-2-macroglobulin, proteins such as beta-amyloid precursor protein, small molecules such as aminoglycosides (e.g., gentamicin), LRP receptor antibodies, and receptor associated protein (RAP) or compounds based on the structure of RAP.
  • preferable agents of the present invention include, but are not limited to, activated alpha-2- macroglobulin, apolipoprotein E, and apolipoprotein E4.
  • a preferred agent of the current invention is activated alpha-2-macroglobulin.
  • the current invention can be useful in treating a subject in need of treatment of a neurological disorder where aberrant calcium influx in neuronal cells is either causal or symptomatic.
  • a neurological disorder as used in the current context, should be obvious to one skilled in the art, but is meant to include any abnormal physical or mental behavior or experience where neuronal cells are involved in the etiology of the disorder, or are affected by the disorder.
  • neurological disorders encompass disorders affecting the central and peripheral nervous systems, and include such afflictions as memory loss, stroke, dementia, personality disorders, gradual, permanent or episodic loss of muscle control.
  • Examples of neurological disorders for which the current invention can be used include, but are not limited to : Alzheimer' s Disease, Parkinson' s Disease, Huntington's Disease, amyotrophic lateral sclerosis, epilepsy, and stroke. More preferably, the current invention can be used to treat Alzheimer's Disease.
  • the term subj ect can be used to mean an animal, preferably a mammal including a human or non-human. The term patient is used to indicate a subject in need of treatment of a neurological disorder.
  • the treatment envisioned by the current invention can be used for patients with a pre-existing neurological condition, or for patients pre-disposed to a neurological disorder. Additionally, the method of the current invention can be used to correct cellular or physiological abnormalities involved with a neurological disorder in patients, and/or to alleviate symptoms of a neurological disorder in patients, or as a preventative measure in patients.
  • the present invention is further directed to a method of inhibiting cell death in neuronal cells, the method comprising: providing neuronal cells with an agent that binds to low-density lipoprotein receptor-related protein (LRP) receptor and modulates calcium influx in the neuronal cells.
  • LRP low-density lipoprotein receptor-related protein
  • the invention can be practiced in vitro or in vivo.
  • cell death includes a process or event that causes the cell to cease or diminish normal metabolism in vivo or in vitro.
  • cell death includes, but are not limited to, programmed cell death (i.e., apoptosis), gradual death of the cells as occurs in diseased states (i.e., necrosis), and more immediate cell death such as acute toxicity.
  • the inhibition of cell death for which the current invention provides can be a complete or partial inhibition of cell death.
  • the inhibition of cell death for which the current invention provides can be a complete or partial reversal of the process of cell death.
  • the present invention inhibits cell death by modulating calcium influx in neuronal cells. As the current invention contemplates, modulation of calcium influx has been previously described herein.
  • the present invention provides for inhibiting cell death by modulating calcium influx in neuronal cells.
  • the channel through which calcium influx in the neuronal cells is mediated is not LRP. More preferably, the channel through which calcium influx in the neuronal cells is mediated is through the NMDA class of receptors.
  • Agents of the current invention useful for inhibiting neuronal cell death include any agent that binds to LRP and subsequently modulates calcium influx in neuronal cells.
  • the agents of the current invention useful for inhibiting cell death include, but are not limited to, receptor associated protein (RAP), small molecules or peptides that mimic RAP, LRP receptor antibodies, and proteins that interact with LRP. Such agents have been previously described herein.
  • RAP receptor associated protein
  • the present invention also is directed to a method of modulating calcium influx in neuronal cells, the method comprising: treating the neuronal cells with an agent that binds to low-density lipoprotein receptor-related protein (LRP) receptor on neuronal cells and modulates calcium influx in the neuronal cells.
  • LRP low-density lipoprotein receptor-related protein
  • the channel through which calcium influx in the neuronal cells is mediated is not LRP . More preferably, the channel through which calcium influx in the neuronal cells is mediated is through the NMDA class of receptors.
  • modulation of calcium influx has previously been described. The invention can be practiced in vitro or in vivo.
  • agents that interact with LRP on neuronal cells and modulate calcium influx include, but are not limited to protein-lipid complexes, proteases, protease inhibitors, protease-inhibitor complexes, proteins, small molecules, LRP receptor antibodies, and proteins that interact with LRP.
  • agents have been previously described herein.
  • the types of agents that can be envisioned include agonists and antagonists of LRP and are limited only by their ability to bind to LRP and modulate calcium influx, particularly through the NMDA receptor channels.
  • an agonist is a protein, nucleic acid, carbohydrate, lipid or a small molecule that binds to LRP and mimics the calcium influx that activated alpha-2-macroglobulin elicits under similar, or identical, conditions.
  • the cellular response that the agonist mimics does not have to be identical in magnitude, duration or character.
  • an antagonist is a protein, nucleic acid, carbohydrate, lipid or a small molecule that binds to LRP and attenuates, or reverses the calcium influx that activated alpha-2-macroglobulin elicits under similar, or identical, conditions.
  • the cellular response that the antagonist prevents does not have to be a total prevention or reversal of the response that the ligand elicits.
  • Agents of the present invention that increase calcium influx in neuronal cells are agonists or stimulators of LRP.
  • the term stimulator is meant to include any agent that produces an increase in calcium movement into the cell.
  • a stimulator of LRP-mediated calcium movement is any agent that binds to LRP and causes an increase in calcium movement into or out of the cell.
  • a preferred agent of the current invention that is an agonist of LRP and modulates calcium influx in neuronal cells is activated alpha-2-macroglobulin.
  • preferable agonists of LRP that are used to modulate calcium influx in neuronal cells also include antibodies that increase LRP-mediated calcium influx in neurons through the NMDA receptor channels, such as R777.
  • the antibody R777 is an antibody that binds to LRP and can be used to block the binding of activated alpha-2-macroglobulin and other ligands or agents that bind to LRP.
  • the R777 antibody was obtained from the fusion of spleen cells of mice, which had been immunized with activated a2M, with the myeloma cell line P3 -X63 -Ag8.653.
  • the production and binding specificity of R777 towards LRP has been previously described in Strickland, D.K., et al, Biochemistry 27: 1458-1466 (1988), and Strickland, D.K., et al., J. Biol. Chem. 265:17401-17404 (1990).
  • Agents of the present invention that decrease calcium influx in neuronal cells are antagonists or inhibitors of LRP.
  • the term inhibitor is meant to include any agent that produces a complete or partial blocking of calcium movement into the cell.
  • an inhibitor of LRP-mediated calcium movement is any agent that binds to LRP and produces a complete or partial blocking of calcium movement into the cell.
  • the agents of the current invention that are antagonists of LRP modulate calcium influx in neuronal cells are receptor associated protein (RAP), antibodies that inhibit LRP-mediated calcium influx in neurons through the NMDA receptor channels, and small molecules or peptides that mimic RAP.
  • RAP receptor associated protein
  • the antibodies used in the invention can be, but are not limited to, chimeric, humanized, and human and nonhuman monoclonal and polyclonal antibodies.
  • Antibodies may be used as an isolated whole antibody, or can be used as a source for generating antibody fragments which contain the antigen binding site of the antibody. Examples of such antibody fragments include, but are not limited to the F v , the F(ab), the F(ab) 2 , fragment and single chain antibodies.
  • Various methods known in the art can be used to generate such whole antibodies or antibody fragments without undue experimentation. For example, apolypeptide of interest or an antigenic fragment thereof can be administered to an animal to induce the production of sera containing polyclonal antibodies.
  • Monoclonal antibodies can be prepared using a wide of techniques known in the art including the use of hybridoma and recombinant technology. See, e.g., Harlow et al,
  • the present invention is directed to a method of identifying an agent that modulates calcium influx in neuronal cells by binding to low-density lipoprotein receptor-related protein (LRP) receptor on the neuronal cells, the method comprising: (a) treating neuronal cells with an agent and assaying for calcium influx; (b) treating neuronal cells with a known modulator of LRP- mediated calcium influx and the agent in (a) and assaying for calcium influx; and (c) comparing the levels of calcium influx in (a) and (b) to determine if the agent in (a) modulates calcium influx by interacting with an LRP receptor.
  • LRP low-density lipoprotein receptor-related protein
  • the channel through which calcium influx in the neuronal cells is mediated is not LRP . More preferably, the channel through which calcium influx in the neuronal cells is mediated is through the NMDA class of receptors.
  • the known modulator of LRP- mediated calcium influx is an NMDA receptor channel antagonist.
  • NMDA receptor channel antagonists include, but are not limited to, MK-801, D(-)-2- Amino- 5- phosphonopentanoic acid, D(-)- 2- Amino- 4- phosphonobutyric acid, ketamine, ifenprodil or phencyclidine. More preferably, the NMDA receptor channel antagonist is MK-801.
  • an NMDA receptor channel antagonist is a protein, nucleic acid, carbohydrate, lipid or a small molecule that binds to an NMDA receptor and blocks, attenuates, or reverses the calcium influx that N-methyl-D-aspartate elicits under similar, or identical, conditions.
  • the method of identifying an agent that modulates calcium influx in neuronal cells is performed on a single population of cells, and (b) is performed on the identical population after the agent in (a) is removed.
  • the method of identifying an agent that modulates calcium influx in cells is performed on two nearly identical populations of cells, under the same culture conditions, where (a) is performed on one population and (b) is performed on another population, and (c) is a comparison of the levels of calcium influx between the two populations of cells.
  • assaying for calcium influx can be accomplished by using any means that can detect differences in intracellular or extracellular calcium levels.
  • means include, but are not limited to, the use of fluorescent dyes in conjunction with microscopy (calcium imaging) (Grynkiewicz, G., etal, J. Biol. Chem. 260:3440- 50 (1985)), enzyme-linked immunosorbent assays (ELIS A), radioactively-labeled isotopes, and detecting local or systemic changes in membrane potential or voltage.
  • agents that can be tested can be proteins, nucleic acids, carbohydrates, lipids or small molecules.
  • agents or compounds which can be envisioned are limited only by their ability to bind to LRP and modulate LRP-mediated calcium influx in neurons through the NMDA receptor channels.
  • the agents of the present invention may be identified and/or prepared according to any of the methods and techniques known to those skilled in the art. These agents, particularly peptide agents and antibody agents, may occur or be produced as monomer, dimers, trimers, tetrameres or multimers. Such multimers can be prepared using enzymatic or chemical treatment of the native receptor molecules or be prepared using recombinant techniques. Preferably, the agents of the present invention are selected and screened at random or rationally selected or designed using protein modeling techniques.
  • candidate agents are selected at random and assayed for their ability to bind to LRP and cause calcium influx in neurons. Any of the suitable methods and techniques known to those skilled in the art may be employed to assay candidate agents.
  • the agent is selected based on the configuration of the LRP binding site found on an LRP ligand, e.g. a2M*, or a ligand binding site found on the LRP.
  • LRP ligand e.g. a2M*
  • a ligand binding site found on the LRP Any of the suitable methods and techniques kno wn to those skilled in the art may be employed for rational selection or design.
  • one skilled in the art can readily adapt currently available procedures to generate antibodies, peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence of LRP. Illustrative examples of such available procedures are described, for example, in Hurby et al. , "Application of
  • Synthetic Peptides Antisense Peptides," in Synthetic Peptides, A User's Guide, W.H. Freeman, NY, pp. 289-307 (1992); Kaspczak et ⁇ /., Biochemistry 28:9230 (1989); and Harlow, Antibodies, Cold Spring Harbor Press, NY (1990).
  • agents of the present invention can alternatively be identified using modification of methods known in the art.
  • suitable peptide agents may be identified using the filter binding assay described by Mischak et al. (Mischak et /., J. Gen. Virol. 69:2653-2656 (1988) and Mischak etal, Virology 163:19-25 (1988)), wherein the peptide is applied to a suitable membrane, such as nitrocellulose, and the membrane is saturated with a detergent mixture in order to block any non-specific binding. The treated membrane is then incubated with labeled LRP (labeled with 125 I-iodine), to check the specific binding. After washing and diying of the membrane, specific binding can be visualized by autoradiography .
  • labeled LRP labeled with 125 I-iodine
  • a pharmaceutically effective amount is intended an amount effective to elicit a cellular response that is clinically significant, without excessive levels of side effects.
  • a pharmaceutical composition of the invention comprising an agent of the invention useful for treatment of a neurological disorder and a pharmaceutically acceptable carrier or excipient.
  • Direct techniques usually involve placement of a drug delivery catheter into the host's ventricular system to bypass the blood-brain barrier.
  • Indirect techniques which are generally preferred, involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs. Latentiation is generally achieved through blocking of the hydroxyl, carboxyl, and primary amine groups present on the drug to render the drug more lipid-soluble and amenable to transportation across the blood-brain barrier.
  • the delivery of hydrophilic drugs can be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier.
  • the blood-brain barrier is a single layer of brain capillary endothelial cells that are bound together by tight junctions.
  • the BBB excludes entry of many blood-borne molecules.
  • the agent can be modified for improved penetration of the blood-brain barrier using methods known in the art.
  • a compound with increase permeability of the BBB can be administered to the subject.
  • RMP-7 a synthetic peptidergic bradykinin agonist was reported to increase the permeability of the blood-brain barrier by opening the tight junctions between the endothelial cells of brain capillaries (Elliott, P.J. etal, Exptl. Neurol. 141:214-224 (1996)).
  • the invention further contemplates the use of prodrugs which are converted in vivo to the therapeutic compounds of the invention (Silverman, R.B., "The Organic Chemistry of Drug Design and Drug Action ' Academic Press, Ch.
  • Such prodrugs can be used to alter the biodistribution (e.g., to allow compounds which would not typically cross the blood-brain barrier to cross the blood-brain barrier) or the pharmacokinetics of the therapeutic compound.
  • an anionic group e.g., a sulfate or sulfonate
  • the ester is cleaved, enzymatically or non-enzymatically, to reveal the anionic group.
  • Such an ester can be cyclic, e.g., a cyclic sulfate or sultone, or two or more anionic moieties can be esterified through a linking group.
  • An anionic group can be esterified with moieties (e.g., acyloxymethyl esters) which are cleaved to reveal an intermediate compound which subsequently decomposes to yield the active compound.
  • an anionic moiety can be esterified to a group which is actively transported in vivo, or which is selectively taken up by target organs. The ester can be selected to allow specific targeting of the therapeutic moieties to particular organs, as described below for carrier moieties.
  • the therapeutic compounds or agents of the invention can be formulated to cross the blood-brain-barrier, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs thus providing targeted drug delivery (Ranade, J., Clin. Pharmacol. 29:685 (1989)).
  • exemplary targeting moieties include folate or biotin (U.S. Pat. No.5,416,016), mannosides (Umezawa et al, Biochem. Biophys. Res. Comm.
  • the pharmaceutical composition can be administered orally, nasally, parenterally, intrasystemically, intraperitoneally, topically (as by drops or transdermal patch), bucally, or as an oral or nasal spray.
  • pharmaceutically acceptable carrier is intended, but not limited to, a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • a pharmaceutical composition of the present invention for parenteral injection can comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions of the present invention can also contain adjuvants such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the absorption from subcutaneous or intramuscular injection In some cases, in order to prolong the effect of the drugs, it is desirable to slow the absorption from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules.
  • the active compounds are mixed with at least one item pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrohdone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, acetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay
  • the dosage form can also comprise buffering agents.
  • Solid compositions of a similar type can also be employed as fillers in soft and hardfilled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms can contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye.
  • Compositions for topical administration can be prepared as a dry powder which can be pressurized or non-pressurized.
  • the active ingredients in finely divided form can be used in admixture with a larger- sized pharmaceutically acceptable inert carrier comprising particles having a size, for example, of up to 100 ⁇ m in diameter.
  • suitable inert carriers include sugars such as lactose.
  • at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 ⁇ m.
  • the composition can be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant.
  • a compressed gas such as nitrogen or a liquefied gas propellant.
  • the liquefied propellant medium and indeed the total composition is preferably such that the active ingredients do not dissolve therein to any substantial extent.
  • the pressurized composition can also contain a surface active agent.
  • the surface active agent can be a liquid or solid non-ionic surface active agent or can be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.
  • compositions of the present invention can also be administered in the form of liposomes.
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to the compounds of the invention, stabilizers, preservatives, excipients, and the like.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art (see, for example, Prescott, Ed., Meth. Cell Biol.
  • agents of the invention can be determined empirically and can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodrug form.
  • the agents can be administered to a subject, in need of treatment of a neurological disorder, as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the agents or composition of the present invention will be decided by the attending physician within the scope of sound medical judgement.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well kno wn in the medical arts .
  • satisfactoiy results are obtained by oral administration of the compounds at dosages on the order of from 0.05 to 10 mg/kg/day, preferably 0.1 to 7.5 mg/kg/day, more preferably 0.1 to 2 mg/kg/day, administered once or, in divided doses, 2 to 4 times per day.
  • dosages on the order of from 0.01 to 5 mg/kg/day, preferably 0.05 to 1.0 mg/kg/day and more preferably 0.1 to 1.0 mg/kg/day can be used.
  • Suitable daily dosages for patients are thus on the order of from 2.5 to
  • 500 mg p.o. preferably 5 to 250 mg p.o., more preferably 5 to 100 mg p.o., or on the order of from 0.5 to 250 mg i.v., preferably 2.5 to 125 mg i.v. and more preferably 2.5 to 50 mg i.v.
  • Dosaging can also be arranged in a patient specific manner to provide a predetermined concentration of the agents in the blood, as determined by techniques accepted and routine in the art (HPLC is preferred).
  • HPLC is preferred.
  • patient dosaging can be adjusted to achieve regular on-going blood levels, as measured by HPLC, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.
  • Example 1 serves only to illustrate the invention, and is not to be construed as in any way to limit the invention.
  • mice Primary cultures of mouse cortex were prepared from embryonic day 15-17 CD 1 mice. The cortices were isolated and triturated in Ca + free PBS and plated onto 35 mm poly-lysine coated culture dishes at a density of
  • indo-1/AM Calbiochem, La Jolla, CA
  • pluronic F- 127 Molecular Probes, Eugene, Oregon
  • DMSO DMSO and then added to the culture dishes at a final concentration of 1 ⁇ M indo- 1/AM and 0.02% pluronic F-127 for 30 min (Grynkiewicz, G., et al, J. Biol. Chem. 260:3440-50 (1985)).
  • HBSS Hanks Balanced Salt Solution
  • the dishes were placed on the stage of an upright microscope (Olympus
  • Time course experiments were performed by acquiring an image pair (512x512 pixels, 8 bits per pixel) at a relatively slow rate (generally, 0.2 Hz) and saving the images to disk.
  • Regions of interest (ROI) within an image were selected, corresponding to the cell bodies of single cells. The average intensity from within each ROI was obtained for each emission wavelength, the appropriate background level was subtracted, and the ratio was calculated.
  • the ratio reflects changes in intracellular calcium ([Ca 2+ ] I ), independently of excitation strength, concentration of indo-1, volume of the cell, or the optical path.
  • the ratios were converted to calcium concentration after calibrating the dye in vitro with a series of calcium buffers (Molecular Probes), and plotted as a function of time.
  • Fab fragments were generated from the polyclonal antibody as follows.
  • R777 Strickland, D.K., et al, Biochemistry 27: 1458-1466 (1988), and Strickland, D.K., et al, J. Biol. Chem. 265:17401-17404 (1990)
  • R777 was dialyzed against 20 mM sodium phosphate, 10 mM EDT A, pH 7.0 , and mixed with 0.5 L Pierce immobilized papain in 20 mM sodium phosphate, 10 mM EDTA, pH 7.0 containing 20 mM cysteine. Digestion was carried out at 37 °C for 12 h with gentle mixing.
  • the digest was applied to Protein A Sepharose, and the nonbinding Fab fragments were collected.
  • the Fab fragments were analyzed by immunoblotting cell extracts (using 5 ⁇ g/ml), which revealed positive reactivity against LRP and no other proteins.
  • a2M* methylamine-activated a2M
  • NMDA 100 ⁇ M
  • Non-neuronal cells generally do not respond to NMDA (Beaman-Hall, CM., et al, J. Neurochem. 77:1993-2005 (1998)), whereas neurons that do express NMDA receptors allow calcium entry in the presence of NMDA (Grant, E.R., et al, J. Biol. Chem. 272:647-56 (1997)).
  • the responding cells were all identified as neurons.
  • the calcium response occurred within several tens of seconds after ligand addition, and, in most cases, the response was sustained for several tens of minutes until the end of the experiment. However, occasionally the calcium response was transient, returning to baseline within several minutes. No consistent difference in these subpopulations in terms of response to NMDA or in morphology was noted.
  • a2M* was examined.
  • Treatment of neurons with native a2M (70 nM) had no effect on intracellular calcium (139 ⁇ 80 nM vs 144 ⁇ 80 nM, n 4 expts, 54 cells, NS, p>0.05).
  • methylamine was added at concentrations up to 100 ⁇ M directly to the cultures with no effect on intracellular calcium.
  • activated a2M appears to be critical for the calcium response.
  • a2M* was added in the presence of 2-5 ⁇ M tetrodotoxin (TTX). At this concentration, the cultured neurons are unable to generate action potentials. However, a2M* was capable of eliciting a calcium response even in the presence of TTX. This result indicates that the observed calcium response elicited by a2M* is not an indirect result of synaptic glutamate release.
  • TTX tetrodotoxin
  • Table 1 The effect of channel blockers on the a2M*-mediated calcium response.
  • the table lists the channel blockers used (at the indicated concentrations), as well as the target of the blockers, and the experimental result. Each experimental test was performed in at least three cultures.
  • a2M* -induced calcium influx was examined to test if this phenomenon was mediated by LRP.
  • Pre-incubation with a specific physiologic inhibitor of LRP, receptor associated protein (RAP, 500 ⁇ M), blocked the response to a2M* (114 ⁇ 17 nM vs 125 ⁇ 9 nM, n 4 expts, 27 cells, NS, p>0.05, Figure 5).
  • RAP blocks ligand-receptor interactions with all members of the LDL receptor family proteins.
  • an anti-LRP antibody was used which specifically interacts with LRP but not other members of the LDL receptor family.
  • the ability of antibody R777 to recognize the ligand binding region of LRP activated the calcium response.
  • a2M* concentrations might provide information about the local microenvironment to neurons, and therefore might alter local dendritic calcium levels in a spatially restricted fashion.
  • a2M* 5 ⁇ M
  • the delivery of a2M* was restricted to a circular area with a radius of about 25 ⁇ m, and this was the only area that responded with a calcium increase.
  • the response was localized, and decays within several tens of seconds. Indeed, a second pressure pulse was able to stimulate the same area again, without affecting the calcium concentration in the cell body.
  • LRP LDL receptor
  • LRP is implicated as the mediator of this response because it is an a2M* receptor on neurons (Rebeck, G.W., et al, Neuron 11:575-80 (1993); Bu, G., et al, J. Biol. Chem. 269:29874-82 (1994)), and the effect is blocked by RAP which is a specific physiologic inhibitor of the LDL family of receptors. That LRP is specifically involved is demonstrated by the observation that an antibody directed against the extracellular domain of LRP can also induce calcium influx in neurons. Of note, both a2M* , which is tetrameric, and the bivalent R777 antibody could potentially lead to dimerization of the receptor.
  • LRP is present on both neurons and astrocytes in culture and in adult brain (Rebeck, G.W., et al, Neuron 11:575-80 (1993); Bu, G., et al, J. Biol. Chem. 269:29874-82 (1994); Bu, G., et al, J. Biol. Chem. 269:18521-8 (1994)), although only neurons have an LRP mediated calcium response.
  • LRP LRP
  • signal receptors 2 classes of a2M methylamine receptors: LRP and a separate "signaling receptor.” Stimulation of the latter leads to a rapid rise in intracellular calcium in macrophages, which is not blocked by RAP or altered by LRP antibodies. By contrast, a2M* binding to LRP does not appear to induce a calcium influx in macrophages, consistent with the lack of response that was observed in non-neuronal cells.
  • proteolytic fragments of apolipoprotein E or a tandem dimer repeat peptide derived from apolipoprotein E elicited calcium responses in both hippocampal cultures and chick sympathetic neurons, with the calcium increases being blocked by RAP and by the NMDA receptor antagonist MK-801 (Tolar, M., et al, J. Neurosci. 79:7100-10 (1999)).
  • MK-801 NMDA receptor antagonist
  • LRP Long Term Evolution
  • a2M*, apolipoprotein E, and other LRP ligands have been shown to promote neurite outgrowth via LRP (Holtzman, D.M., etal, Proc. Natl. Acad. Sci. U.S.A. 92:9480-4 (1995); Ishii, M., etal, Brain Res. 737:269-74 (1996); Mori, T., etal, Brain Res. 567:355-7 (1991); Postuma, R.B., etal, FEBSLett. 428:13-6 (1998)).
  • LRP intracellular domain of LRP can bind both disabled and FE65, adapter proteins implicated in signal transduction (Tro msdorff, M., et al, J. Biol. Chem. 273:33556-60 (1998)).
  • LRP has also been reported to interact with a heterotrimeric G protein (Goretzki, L. and Mueller, B. M., Biochem. J. 356:381- 6 (1998)).
  • LRP-expressing cell lines with the LRP ligands lactoferrin or urokinase-type plasminogen activator caused a significant elevation in cAMP and stimulated PKA activity in a dose-dependent manner (Goretzki, L. and Mueller, B. M., Biochem. J. 336:381-6 (1998)).
  • Reelin is a ligand for APOER2 and VLDL-r, supporting the idea that these receptors directly mediate reelin signal transduction (Trommsdorff, M., et al, Cell 97:689-701 (1999); D'Arcangelo, G., et al, Neuron 24:471-9 (1999)). It is interesting to note that both VLDL-r and APOER2 are also strongly expressed on mature neurons (Christie, R.H., etal, J. Neuropathol. Exp.
  • a ⁇ the major constituent of senile plaques, is a peptide fragment of the amyloid precursor protein, itself a protease inhibitor and ligand for LRP (Kounnas, M.Z., et al, Cell 82:331 -40 (1995)).
  • Apolipoprotein E and a2M* bind A ⁇ and the complexes can be cleared by LRP (Qiu, Z., et al, J. Neurochem. 73:1393-8 (1999); Jordan, J., et al, J. Neurosci. 18:195-204 (1998); Narita, M., et al, J.

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Abstract

L'invention concerne des méthodes permettant de traiter des troubles neurologiques au moyen d'agents qui se lient à des récepteurs de la protéine liée au récepteur de lipoprotéine (LRP) faible densité. L'invention concerne également des méthodes permettant de moduler un influx calcique et d'inhiber la mort cellulaire des cellules neuronales par traitement desdites cellules neuronales au moyen d'agents qui se lient à LRP. L'invention concerne enfin des méthodes permettant d'identifier les agents qui modulent l'influx calcique dans les cellules neuronales par liaison avec LRP.
PCT/US2000/040636 2000-07-24 2000-08-15 Modulation d'un influx calcique neuronal induite par la proteine liee au recepteur de lipoproteine via des recepteurs de nmda, et son utilisation WO2002007755A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
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WO2003039575A2 (fr) * 2001-11-09 2003-05-15 Neuronova Ab Role fonctionnel et utilisation therapeutique potentielle de proteines s, gas6 et reelin en relation avec des cellules souches neuronales adultes
US7179462B2 (en) 2000-06-02 2007-02-20 University Of Connecticut Health Center α (2) macroglobulin receptor as a heat shock protein receptor and uses thereof
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WO2007098417A2 (fr) * 2006-02-21 2007-08-30 Oklahoma Medical Research Foundation TRAITEMENT DE LA MALADIE D'ALZHEIMER AVEC DES INHIBITEURS DE LA FIXATION DE L'ApoE AU RÉCEPTEUR DE L'ApoE
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7179462B2 (en) 2000-06-02 2007-02-20 University Of Connecticut Health Center α (2) macroglobulin receptor as a heat shock protein receptor and uses thereof
US7186515B1 (en) 2000-06-02 2007-03-06 University Of Connecticut Health Center Alpha(2) macroglobulin receptor as a heat shock protein receptor and uses thereof
US7449557B2 (en) 2000-06-02 2008-11-11 University Of Connecticut Health Center Complexes of alpha (2) macroglobulin and antigenic molecules for immunotherapy
WO2003039575A2 (fr) * 2001-11-09 2003-05-15 Neuronova Ab Role fonctionnel et utilisation therapeutique potentielle de proteines s, gas6 et reelin en relation avec des cellules souches neuronales adultes
WO2003039575A3 (fr) * 2001-11-09 2003-10-16 Neuronova Ab Role fonctionnel et utilisation therapeutique potentielle de proteines s, gas6 et reelin en relation avec des cellules souches neuronales adultes
WO2007098417A2 (fr) * 2006-02-21 2007-08-30 Oklahoma Medical Research Foundation TRAITEMENT DE LA MALADIE D'ALZHEIMER AVEC DES INHIBITEURS DE LA FIXATION DE L'ApoE AU RÉCEPTEUR DE L'ApoE
WO2007098417A3 (fr) * 2006-02-21 2007-10-18 Oklahoma Med Res Found TRAITEMENT DE LA MALADIE D'ALZHEIMER AVEC DES INHIBITEURS DE LA FIXATION DE L'ApoE AU RÉCEPTEUR DE L'ApoE

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