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Title:  Methods for treating neuropathological states and neurogenic inflammatory states and methods for identifying compounds useful therein
United States Patent: 
7,022,484
Issued: 
April 4, 2006
Inventors: 
High; Karin Westlund (League City, TX); Taglialatela; Giulio (Dickinson, TX)
Assignee: 
Board of Regents, The University of Texas System (Austin, TX)
Appl. No.: 
877220
Filed: 
June 8, 2001


 

George Washington University's Healthcare MBA


Abstract

The present invention provides methods for treating neuropathological states and neurogenic inflammatory states in a subject. The present invention also provides methods for identifying compounds that can be used to treat such states. Preferably, the compounds alter the distribution of NMDA glutamate receptor NR1 subunit in cells, and/or alter the production of TNFα by cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides methods for identifying compounds that alter the distribution of NR1 subunits and/or alter the production of Tumor Necrosis Factor Alpha (TNFα) in a cell. The cell can be ex vivo or in vivo. As used herein, the term "ex vivo" refers to a cell that has been removed from the body of a subject. Ex vivo cells include, for instance, primary cells (e.g., cells that have recently been removed from a subject and are capable of limited growth in tissue culture medium), and cultured cells (e.g., cells that are capable of extended culture in tissue culture medium). As used herein, the term "in vivo" refers to a cell that is within the body of a subject. Preferably, the cell is ex vivo.

Cells useful in the present invention have a glutamate receptor, preferably an NMDA glutamate receptor, on the cell surface. Examples of such cells include, for instance, neurons. Examples of useful neurons that can be used ex vivo include cultured neuroblastoma cells, preferably rat, mouse, or human, more preferably human. An example of a cultured human neuroblastoma cell is SHSY5Y (ATCC CRL-2266). Other examples of useful ex vivo neurons include neurons isolated from the dorsal horn of the spinal cord, dorsal root ganglia and other cell bodies of peripheral nerves, hippocampal or other limbic or cortical neurons. Preferably the neurons are removed from a rat. Examples of in vivo neurons include neurons in the spinal cord, for instance neurons in the dorsal horn and motor horn, the brain, for instance neurons in the basal forebrain and hippocampus, peripheral neurons, for instance dorsal root ganglia.

Examples of other cells useful in the present invention include, for instance, cultured synovial sarcoma cells, preferably rat, mouse, or human, more preferably human. An example of a cultured human synovial sarcoma cell is SW982 (ATCC HTB-93). Examples of in vivo cells include synovial cells lining a knee joint of a subject, preferably a human.

In an aspect of the present invention, the methods include evaluating the effect of different compounds by contacting a cell with a compound, activating a glutamate receptor, preferably an NMDA glutamate receptor, present on the cell, and detecting the distribution of the NR1 subunit in the cell. The distribution of the NR1 subunit in the cell is compared to the distribution in a cell that was not exposed to the compound. Contacting the cell with a compound can occur before, during, or after activating a glutamate receptor present in the cell. Cells ex vivo can be contacted directly with the compound by, for instance, adding the compound to the media in which the cell is bathed. Cells in vivo can be contacted directly with a compound. For instance, cells of the spinal cord can be contacted directly as described in Example 1. Alternatively, a compound can be introduced to the animal systemically as a pharmaceutical composition. Pharmaceutical compositions are detailed herein.

In another aspect of the present invention, the methods include evaluating the effect of different compounds by contacting a cell with a compound, activating a glutamate receptor, preferably an NMDA glutamate receptor, present on the cell, and detecting the production of TNFα by the cell. The amount of TNFα produced by the cell is compared to the amount of TNFα produced by a cell that was not exposed to the compound. Contacting the cell with a compound can occur before, during, or after activating a glutamate receptor present in the cell. Cells ex vivo can be contacted directly with the compound by, for instance, adding the compound to the media in which the cell is bathed. Cells in vivo can be contacted directly with a compound. Alternatively, a compound can be introduced to the animal systemically as a pharmaceutical composition.

The invention is not intended to be limited by the types of compounds that can be screened for activity using the methods described herein. Accordingly, a compound can be, for instance, a polypeptide, an organic molecule, polyketide, or a non-ribosomal peptide. Compounds useful in the methods of the present invention can be produced by natural organisms, or produced using methods known to the art including, for instance, recombinant techniques, or chemical or enzymatic synthesis techniques. Preferably, the compound is not produced by a mammal.

Preferred examples of compounds that can be used in some aspects of the present invention include tyrosine kinase inhibitors. Tyrosine kinase inhibitors are known in the art and include, for instance, Genistein (5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one; 4′,5,7-trihydroxy-isoflavone, Catalog Number G-103 from RBI, Natick, Mass.), Lavendustin A (5-Amino-[N-2,5-dihydroxybenzyl)-N′-2-hydroxybenzyl]salicylic acid, Catalog Number 428150 from Calbiochem, La Jolla, Calif.), and K252a (Catalog Number 420298, from Calbiochem, La Jolla, Calif.). Whether a compound is a tyrosine kinase inhibitor can be detennined using methods known in the art (see, for instance, Akiyama et al., J. Biol. Chem., 262, 5592-5595 (1987) and Ohmichi et al., Biochemistry, 31(16:4034-4039 (1992)). Preferably, a tyrosine kinase inhibitor useful in the present invention decreases phosphorylation of NR1 receptor. Without being limiting, it is expected that a specific tyrosine kinase mediates the translocation of NR1 subunit, and that this specific tyrosine kinase will be a member of one of the known families of tyrosine kinases, for instance, the Src or Jak families. Inhibitors are available that specifically inhibit individual members of the known families of tyrosine kinases. Accordingly, it is expected that specific tyrosine kinase inhibitors will be useful in the methods of the present invention. Alternatively, it is expected that other useful compounds include tyrosine phosphatases and serine/threonine phosphatases. In other aspects of the present invention, preferred examples of compounds that can be used include tyrosine kinases, tyrosine phosphatase inhibitors, or serine/threonine phosphatase inhibitors where increases in NR1 subunit would be advantageous such as for improving memory or slowing the aging process.

The amount of a compound that is administered to alter the distribution of NR1 subunits in a cell, or alter the production of TNFα by a cell, varies depending on the type of compound used. Typically, when a compound is screened for activity in the methods of the present invention, various concentrations of the compound are used.

Activation of a neuron ex vivo can be accomplished by incubating a cell in tissue culture media and adding at least about 5 micromolar of a glutamate receptor agonist, and more preferably at least about 10 micromolar of a glutamate receptor agonist. Typically, translocation of NR1 subunit to the nuclear membrane can be observed about 4-24 hours after exposure. Neurogenic inflammatory states can be experimentally recreated ex vivo by adding at least 5 micromolar, preferably at least about 10 micromolar, of a glutamate receptor agonist to a cell that includes a glutamate receptor, and measuring the resulting production of TNFα by the cells. Alterations in the amount of TNFα produced by a cell can be observed about 4-24 hours after exposure.

Activation of a cell in vivo is typically accomplished by using an animal model that can be used for investigating conditions that result from sensitization of cells or from increased concentrations of glutamate. Without intending to be limiting, such conditions include neuropathological states and neurogenic inflammatory states. Animal models for studying these conditions are known in the art and can be used in the methods of the present invention. Models for the study of pain include those approved by the International Association for the Study of Pain. A preferred animal model for identifying compounds that alter the distribution of NR1 subunits in a cell is the rat arthritis model described in Example 1. The rat arthritis model is a commonly accepted model for the study of pain and arthritis in humans. Other animal models (for instance, using cat, monkey, or rabbit as the animal) are also commonly accepted models for these human conditions (see, e.g., Neugebauer & Schaible, Agents and Actions, 25, 234-236 (1988) and O'Byrne et al., Arthritis and Rheumatism, 33, 1023-1028 (1990)). To study pain in this model, cells in the spinal cord are exposed to a compound for a period of time, and then a knee of the animal is exposed to a stimulus that evokes persistent pain. Methods of evoking persistent pain are known in the art. After a period of time the responsiveness of the animal to an innocuous or noxious stimulus is evaluated using methods known in the art. A compound that causes an animal to have reduced primary allodynia or secondary allodynia, or reduced primary hyperplasia or secondary hyperplasia compared to an animal that has not received the compound indicates that the distribution of NR1 subunits in the spinal cord is altered. This model may also be used to study arthritis. A compound can be injected into the synovial space of a knee joint, and then the knee exposed to a stimulus that evokes arthritis. After a period of time, the responsiveness of the animal to movements in the working range of the joint are evaluated. A compound that causes an animal to have reduced response time when its foot is touched, that is, the animal overreacts, compared to an animal that has not received the compound indicates that the distribution of NR1 subunits in the cells lining the synovial space, and/or the production of TNFα by the cells is altered. Neuropathological states and neurogenic inflammatory states can also be experimentally recreated in vivo by injecting a glutamate receptor agonist into a space containing neurons (for instance, the spinal cord) or other cells (for instance, the synovial space present in a joint) that include a glutamate receptor.

The distribution of the NR1 subunits present in a cell exposed to the compound is measured and compared to a cell that has not been exposed to the compound. The distribution of NR1 subunits present in a cell can be measured using methods known in the art for determining the location of a polypeptide in a cell. For instance, the amount of NR1 subunits associated with the cellular membrane, present in the cytoplasm, or associated with the nucleus can be determined. Preferably, the amount of NR1 subunits associated with the nucleus, more preferably associated with the nuclear membrane, most preferably associated with the inner nuclear membrane is determined. The amount of NR1 subunit associated with the nucleus can be increased or decreased, preferably decreased, in a cell contacted with a compound.

Typically, the presence of NR1 is assayed using NR1-specific antibodies and methods known to the art including western immunoblot, immunoprecipitation, and immunocytochemistry. Without intending to be limiting, for example, cells can be fractionated and the different subcellular fractions tested for the presence of NR1 subunits. Alternatively, cells can be fixed and sectioned for analysis by, for instance, immunocytochemical analysis, using light microscopy or electron microscopy.

Alternatively, instead of detecting alterations in the distribution of NR1 subunits in the cell, the total amount of NR1 present in a cell contacted with a compound can be determined and compared to the total amount of NR1 present in a cell not contacted with a compound. The small amount of NR1 subunit in the nucleus normally is at the level of detection while an increase can be measured five hours after induction of knee joint inflammation. Methods for determining the total amount of a polypeptide in a cell or cell fractions are known in the art. In another alternative, instead of detecting alterations in the distribution of NR1 subunits in the cell, the amount of phosphorylated NR1 subunit present in a cell contacted with a compound can be determined and compared to the amount of the activated form of the subunit, phosphorylated NR1 present in a cell not contacted with a compound. Methods for determining whether a polypeptide is phosphorylated are known in the art.

Due to the observed presence of NR1 subunits in the nucleus, it is expected that NR1 subunits alter gene expression. Accordingly, it is expected that changes in gene expression, including alterations in transcription or translation, preferably translation, may also be used to measure alterations in the distribution of NR1 subunits in a cell.

The production of TNFα by a cell can be measured using methods known in the art, including, for instance, Enzyme-linked Immunosorbant Assay (ELISA), and ex vivo biological assays. The TNFα can be TNFα present inside the cell, secreted by the cell, or a combination thereof. Preferably, the TNFα measured is TNFα that has been secreted by the cell. Preferably, the amount of TNFα produced by a cell contacted with a compound is decreased.

The present invention is further directed to methods for treating certain conditions in a subject. The conditions include, for instance, a neuropathological state such as persistent pain, stroke, brain injury, spinal cord injury, epileptogenesis, and viral invasion, and neurogenic inflammation such as arthritis, stroke, ulcerative colitis, inflammatory bowel disease, Crohn's disease, pancreatitis, asthma, spinal cord injury, and viral invasion.

The methods include administering to the subject an effective amount of a compound that decreases or prevents a symptom of a neuropathological state or neurogenic inflammatory state. The compounds useful in this aspect of the invention are described above and can be used alone or in combination. Preferably, the compound is a tyrosine kinase inhibitor, more preferably Genistein, Lavendustin A, or K252a. The subject can be an animal, preferably a rat, a mouse, or a human, most preferably a human.

Treatment described herein can be prophylactic (initiated before a subject manifests symptoms of a condition described herein) or, alternatively, can be initiated after the development of a condition described herein. Accordingly, administration of a compound can be performed before, during, or after the occurrence of the conditions described herein. Treatment initiated after the development of a condition may result in decreasing the severity of a symptom of the condition, or completely removing a symptom of the condition. The compound can be administered systemically. When administered systemically, the compound is preferably associated with an agent that will direct the compound to the appropriate cells. For example, the compound can be associated with an antibody to a receptor present on the surface of a cell such that the compound is transported to the interior of the cell, for instance by endocytosis. Preferably, the receptor is endocytosed through clathrin-coated pits to endosomes. The compound can be administered locally. In the treatment of persistent pain that results from cancer or back pain the compound can be administered by, for instance, intraspinal catheter. In the treatment of arthritis the compound can be administered by, for instance, injection of the compound into the synovial space of the affected joint. Preferably, the compound is administered locally.

An aspect of the invention is directed to a method for treating a neuropathological state in a subject. The method includes administering to the subject an effective amount of a compound, preferably a tyrosine kinase inhibitor, more preferably Genistein, Lavendustin A, or K252a.

In some aspects of the invention, the compound used to treat the subject alters the distribution of NR1 subunit in a cell, preferably decreases the amount of NR1 subunit associated with the nucleus of a cell. Alternatively, the compound decreases the total amount of NR1 in the cell, or decreases the amount of phosphorylated NR1 in the cell. Preferably, the compound both decreases the amount of NR1 subunit associated with a cell's nucleus and decreases the total amount of NR1 in the cell. In other aspects of the invention, the compound used to treat the subject alters the production of TNFα by a cell, preferably decreases the production of TNFα by the cell. In some aspects, the compound may both alter the distribution of NR1 in a cell and alter the production of TNFα by the cell.

A neural cell can be present in the spinal cord, for instance in the dorsal horn, in the brain, for instance in the basal forebrain or hippocampus, or in the cell body of a peripheral neuron, for instance a dorsal root ganglia. Other glutamate receptor-containing cells of the present invention can be present, for instance, in the knee joint such as synovial cells. In aspects of the invention that are directed to decreasing a symptom of a neuropathological state, preferably the cell is a sensitized neuron. It is expected that decreasing a symptom of a neuropathological state results in converting a neuron from a sensitized state to a non-sensitized state. In aspects of the invention that are directed to preventing a symptom of a neuropathological state, preferably the cell is a non-sensitized neuron. It is expected that preventing a symptom of a neuropathological state results in preventing the conversion of a cell from a non-sensitized state to a sensitized state.

Also provided by the present invention are methods for treating a neurogenic inflammatory state in a subject. The method includes administering to the subject an effective amount of a compound, preferably a tyrosine kinase inhibitor, more preferably Genistein, Lavendustin A, or K252a.

In the case of a neurogenic inflammatory state of a subject caused by non-neuronal cells producing TNFα in response to a glutamate receptor agonist (for instance, arthritis, ulcerative colitis, inflammatory bowel disease, Crohn's disease, pancreatitis, asthma, stroke, brain injury, and viral invasion), it is expected that decreasing a symptom of a neurogenic inflammatory state will result in decreasing or preventing cells from producing TNFα. Without intending to be limiting, it is expected that, by reducing TNFα production, the capacity of TNFα to stimulate the inflammatory cascade is reduced, thereby decreasing, preferably preventing, a neurogenic inflammatory state.

In the case of a neurogenic inflammatory state located in the nervous system of a subject, such as neurogenic inflammation occurring during a neuropathological state of a subject (for instance, stroke, spinal cord injury, and viral invasion), TNFα expression and/or secretion from the cell is also reduced, thereby decreasing, preferably preventing, neurogenic inflammation occurring during a neuropathological state. For instance, a subject suffering from a severe spinal cord injury may develop a neuropathological state as well as a neurogenic inflammatory state. Under these circumstances, administration of a compound of the present invention is capable of decreasing, preferably preventing, the neuropathological state and the neurogenic inflammatory state.

The present invention provides methods for altering the ability of a subject to retain information, e.g., increasing the memory of a subject. The method includes administering to the subject an effective amount of a compound that increases the ability of a subject to retain information. Whether a subject is able to retain information can be determined using methods known to the art. For instance, when this method is used with non-humans, maze tests can be used, and when this method is used with humans, tests such as the California learning scale or the Wisconsin memory test can be used. Preferably, short-term memory or long-term memory is increased, more preferably, long-term memory is increased.

The compounds useful in this aspect of the invention are described above. Preferably, the compound is a tyrosine kinase, a tyrosine phosphatase inhibitor, a serine/threonine phosphatase inhibitor, or combinations thereof. Typically, the compound alters the distribution of NR1 subunit in a neuron or cells targeted by neurons, preferably by increases the amount of NR1 subunit associated with the nucleus of a neuron or cell. Alternatively, the compound increases the total amount of NR1 in the neuron or target cell, or increases the amount of phosphorylated NR1 in the neuron or target cell. Preferably, the compound both increases the amount of NR1 subunit associated with the nucleus of a neuron and increases the total amount of NR1 in the neuron. The subject can be an animal, preferably a rat, a mouse, or a human, most preferably a human. The neuron is typically present in the brain, preferably in the hippocampus.

The compound(s) useful in the methods disclosed herein is optionally and preferably present in a pharmaceutically acceptable carrier. The compounds useful in the present invention, preferably a tyrosine kinase inhibitor, may be formulated in pharmaceutical preparations in a variety of forms adapted to the chosen route of administration. Formulations include those suitable for parental administration (for instance intramuscular, intraperitoneal, intraspinal catheter, intrasynovial, or intravenous), oral, transdermal, or nasal administration. Dosages of the compositions of the invention are typically from about 0.1 mg/kg up to about 50 mg/kg intravenous or 50-250 mg/kg oral dose. Preferably, an intraspinal dose is about 10 microliters of a solution containing from about 01 micromolar to about 1 micromolar of the solution.

The formulations may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. All methods of preparing a pharmaceutical composition include the step of bringing the active compound (e.g., a tyrosine kinase inhibitor) into association with a carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

Typically, the compositions of the invention will be administered from about 1 to about 4 times per day. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound. The amount of compound in such therapeutically useful compositions is such that the dosage level will be effective to prevent or suppress the neuropathological state or the neurogenic inflammatory state.

Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the composition, or dispersions of sterile powders that include the composition, which are preferably isotonic with the blood or synovial fluid of the recipient. Isotonic agents that can be included in the liquid preparation include sugars, buffers, and sodium chloride. Solutions of the composition can be prepared in water, and optionally mixed with a nontoxic surfactant. Dispersions of the composition can be prepared in water, ethanol, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, glycerol esters, and mixtures thereof. The ultimate dosage form is sterile, fluid, and stable under the conditions of manufacture and storage. The necessary fluidity can be achieved, for example, by using liposomes, by employing the appropriate particle size in the case of dispersions, or by using surfactants. Sterilization of a liquid preparation can be achieved by any convenient method that preserves the bioactivity of the composition, preferably by filter sterilization. Preferred methods for preparing powders include vacuum drying and freeze drying of the sterile injectable solutions. Subsequent microbial contamination can be prevented using various antimicrobial agents, for example, antibacterial, antiviral and antifungal agents including parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Absorption of the composition by the animal over a prolonged period can be achieved by including, for example, aluminum monostearate or gelatin.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as tablets, troches, capsules, lozenges, wafers, or cachets, each containing a predetermined amount of the active compound as a powder or granules, as liposomes containing the active compound, or as a solution or suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, an emulsion or a draught.

The tablets, troches, pills, capsules, and the like may also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch, or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, fructose, lactose or aspartame; and a natural or artificial flavoring agent. When the unit dosage form is a capsule, it may further contain a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir may contain one or more of a sweetening agent, a preservative such as methyl or propylparaben, an agent to retard crystallization of the sugar, an agent to increase the solubility of any other ingredient, such as a polyhydric alcohol, for example glycerol or sorbitol, a dye, and flavoring agent. The material used in preparing any unit dosage form is substantially nontoxic in the amounts employed. The compound may be incorporated into sustained-release preparations and devices.
 


Claim 1 of 28 Claims

1. A method for altering NR1 subunit distribution in a test cell by decreasing the amount of NR1 subunit associated with a nucleus of the test cell, the method comprising:

contacting a test cell with a tyrosine kinase inhibitor,

activating an NMDA glutamate receptor present on the test cell and on a control cell not contacted with a tyrosine kinase inhibitor; and

detecting the distribution of NR1 subunit associated with the nucleus in the test cell and the control cell, wherein a decrease in the amount of NR1 subunit associated with the nucleus of the test cell relative to the control cell indicates the alteration of NR1 subunit distribution.
 

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