US20020160933A1

US20020160933A1 - Methods and compositions for producing a neurosalutary effect in a subject

Info

Publication number: US20020160933A1
Application number: US09/949,200
Authority: US
Inventors: Larry Benowitz
Original assignee: Childrens Medical Center Corp
Current assignee: Childrens Medical Center Corp
Priority date: 2000-09-07
Filing date: 2001-09-07
Publication date: 2002-10-31
Also published as: US20070231376A1; CA2419456A1; WO2002020056A3; AU8711801A; AU2001287118B2; US7172871B1; WO2002020056A2; JP2004523470A; EP1315514A2

Abstract

Methods and compositions for producing a neurosalutary effect in a subject are provided. These methods generally involve administering to a subject a therapeutically effective amount of a compound that modulates the activity of N-kinase, or analog thereof. Pharmaceutical and packaged formulations including the compounds of the invention, e.g., compounds that modulate the activity of N-kinase, are also provided.

Description

RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No. 09/656,915, filed on Sep. 7, 2000, the entire contents of which are incorporated herein by this reference.[0001]

BACKGROUND OF THE INVENTION

Disorders of the peripheral and central nervous system are widespread, and for many of these conditions effective therapeutic interventions are lacking.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for producing a neurosalutary effect in a subject, e.g., a subject suffering from a neurological condition. Such an effect includes promoting neuronal survival, axonal outgrowth, neuronal regeneration or normalized neurological function in a subject. The present invention is based, at least in part, on the isolation of a highly purified form of the N-kinase polypeptide from normal mammalian neuronal tissue, molecular characterization of its chemical structure (including amino acid sequence), demonstration of its sensitivity to purine regulation and the discovery that this kinase plays an active role in the axonal outgrowth of CNS neurons, including mammalian CNS neurons, such as retinal ganglion neurons. The identification of N-kinase as a critical intracellular mediator of axonal outgrowth, and the chemical characterization of its structure, now provides for the ability to produce a neurosalutary effect in a subject by modulating N-kinase activity. Furthermore, this purification and characterization of N-kinase now allows for its use in screening assays to identify additional compounds capable of producing a neurosalutary effect in a subject.

Accordingly, in one aspect, the present invention provides a method which includes administering to a subject a therapeutically effective amount of a compound that modulates the activity of N-kinase, thereby producing a neurosalutary effect in the subject.

In one aspect, the compound that modulates the activity of N-kinase is administered to a subject in accordance with the present invention such that the compound is brought into contact with neurons of the central nervous system of the subject. For example, the compound may be administered into the cerebrospinal fluid of the subject into the intrathecal space by introducing the compound into a cerebral ventricle, the lumbar area, or the cisterna magna. In such circumstances, the compound that modulates the activity of N-kinase can be administered locally to cortical neurons or retinal ganglion cells to produce a neurosalutary effect.

In certain embodiments, the compound that modulates the activity of N-kinase may be administered to a subject using a pharmaceutically acceptable formulation. The pharmaceutically acceptable formulation may allow for sustained delivery, providing effective amounts of the compound that modulates the activity of N-kinase to a subject for at least one week, or in other embodiments, at least one month, after the pharmaceutically acceptable formulation is initially administered to the subject. Approaches for achieving sustained delivery of a compound of the invention include the use of a slow release polymeric capsule, a bioerodible matrix, or an infusion pump that disperses the compounds of the invention. The infusion pump may be implanted subcutaneously, intracranially, or in other locations as would be medically desirable. In certain embodiments, the compounds of the invention would be dispensed by the infusion pump via a catheter either into the cerebrospinal fluid, or to a site where local delivery was desired, such as a site of neuronal injury or a site of neurodegenerative changes.

In one embodiment of the invention, the compound that modulates the activity of N-kinase is a small molecule, the N-kinase polypeptide or fragment thereof, an anti-N-kinase antibody, an antisense N-kinase nucleic acid molecule, a ribozyme, or the N-kinase gene or fragment thereof.

In yet another aspect, the invention features a method which includes administering a therapeutically effective amount of a compound that modulates the activity of N-kinase to a subject suffering from a neurological disorder, thereby treating the subject suffering from a neurological disorder. In one embodiment, the method further includes making a first assessment of a nervous system function, e.g., a sensory function, cholinergic innervation, or a vestibulomotor function, prior to administering the compound that modulates the activity of N-kinase to the subject and making a second assessment of the nervous system function after administering the compound to the subject.

In a further aspect, the invention features a method for identifying a compound capable of producing a neurosalutary effect in a subject by contacting N-kinase, or a biologically active fragment thereof, with a test compound and determining the ability of the test compound to modulate the activity of N-kinase, thereby identifying a compound capable of producing a neurosalutary effect in a subject. In a preferred embodiment, the ability of the test compound to modulate the activity of N-kinase is determined by assessing the ability of the test compound to modulate N-kinase dependent phosphorylation of a substrate, e.g., a histone HF-1 protein.

In one embodiment, the N-kinase used in the methods of the invention is a human N-kinase, such as a recombinantly produced human N-kinase. In another embodiment, the N-kinase used in the methods of the invention is a bovine N-kinase, such as an N-kinase which is purified from a bovine source, e.g., neonatal bovine brain tissue.

In another embodiment, the screening method of the invention further includes determining the ability of the test compound to modulate axonal outgrowth of a central nervous system neuron.

In another aspect, the invention features a method for identifying a compound capable of producing a neurosalutary effect in a subject. The method includes contacting N-kinase, or a biologically active fragment thereof, with a test compound, an N-kinase substrate (e.g., a histone HF-1 protein), radioactive ATP (e.g., [γ- ³²P] ATP), and Mn⁺²; and determining the ability of the test compound to modulate N-kinase dependent phosphorylation of the substrate, thereby identifying a compound capable of producing a neurosalutary effect in a subject. In a preferred embodiment, the method of the invention further includes determining the ability of the test compound to modulate axonal outgrowth of a central nervous system neuron.

In another aspect, the invention features a compound capable of producing a neurosalutary effect in a subject identified by any of the foregoing methods.

In yet another aspect, the invention features an isolated N-kinase polypeptide of the type that: (a) is present in neonatal brain tissue (e.g, neonatal human, bovine, rat, or mouse brain tissue); (b) is inhibited by 6-thioguanine; (c) is activated by Mn ⁺²but not by Mg⁺²or Ca⁺²; (d) has a molecular weight of approximately 49 kDa; and (e) is eluted from a Cibacron Blue column at a NaCl concentration of 1.5-1.75 M.

In a further aspect, the invention features an antibody, e.g., an intracellular antibody, which is specifically reactive with an epitope of the N-kinase polypeptide. In a preferred embodiment, the antibody is reactive with an epitope which includes the ATP binding domain of the N-kinase.

In another aspect, the invention features a fragment of the N-kinase polypeptide, for example, a fragment that includes at least 15, 20, 25, 30, 40, 50, 100, 150, or 200 contiguous amino acids of the N-kinase polypeptide. In a preferred embodiment, the fragment of the N-kinase polypeptide is able to elicit an immune response.

In a further aspect, the invention features an isolated nucleic acid molecule that encodes the polypeptide of SEQ ID NO:1.

Pharmaceutical compositions, and packaged formulations, comprising a composition of the invention (e.g., a compound that modulates the activity of N-kinase) and a pharmaceutically acceptable carrier are also provided by the invention.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. [0020] 1A-C are SDS-PAGE gels depicting the purification of the N kinase polypeptide. The N-kinase band at each stage of the purification is indicated by an asterisk. FIG. 1A depicts a prominent 49 kDa band which binds strongly to a Cibacron Blue column and requires 1.5-1.75 M NaCl to be eluted. FIG. 1B depicts the protein fractions obtained from the separation on a C4 hydrophobic-interaction column. Fractions 24-26 contain the N-kinase polypeptide. FIG. 1C depicts the final stage of purification which was accomplished by SDS-PAGE. The band indicated by the asterisk coincided in its migration position with N-kinase activity, as visualized in a parallel gel assayed with the in-gel kinase method.
FIG. 2 depicts the amino acid (SEQ ID NO:1) sequence of the human N-kinase. Direct matches between the purified protein and the published sequence are shown in blue. K65 (bold type) lies in the ATP-binding region of the kinase domain.[0021]

DETAILED DESCRIPTION

The present invention provides methods and compositions for producing a neurosalutary effect in a subject, e.g., a subject suffering from a neurological disorder. The invention is based, at least in part, on the isolation of a highly purified form of the N-kinase polypeptide from a mammalian source and the discovery that this kinase plays an active role in the axonal outgrowth of CNS neurons, including mammalian CNS neurons, such as retinal ganglion neurons, or cortical pyramidal cells. [0022]
Accordingly, the present invention is directed to methods which include administering to a subject a therapeutically effective amount of a compound that modulates the activity of N-kinase, thereby producing a neurosalutary effect in the subject. [0023]
As used herein, the term “N-kinase” includes all forms of N-kinase including but not limited to human N-kinase, bovine N-kinase, murine N-kinase, rat N-kinase, and porcine N-kinase. The amino acid and nucleotide sequences of the human N-kinase are described in Zhou T -H. et al. (2000) [0024] J. Biol. Chem. 275(4):2513-2519 and in GenBank Accession Number AF083420, the contents of which are incorporated herein by reference. The amino acid sequence of the human N-kinase is shown in FIG. 2 and in SEQ ID NO:1. In a preferred embodiment, the term “N-kinase” includes the isoform of N-kinase that is inhibited by 6-thioguanine, that is activated by Mn⁺²but not by Mg⁺²or Ca⁺², and that has a molecular weight of approximately 49 kDa.
As used herein, the language “a compound that modulates the activity of N-kinase” includes any compound which has the ability to modulate, e.g., stimulate or inhibit, the activity of N-kinase as determined by, for example, the assays described herein. Such compounds are able to modulate one or more of the following: (a) the ability of N-kinase to phosphorylate a substrate, e.g., a histone HF-1 protein; (b) the ability of N-kinase to interact with, e.g., bind to, a non-N-kinase molecule, such as a downstream molecule in the axonal outgrowth signaling pathway; (c) the ability of N-kinase to bind ATP or Mn[0025] ⁺²; or (d) the ability of N-kinase to modulate the axonal outgrowth of central nervous system neurons.
In a preferred embodiment, the compound that modulates the activity of N-kinase acts downstream of AF-1 or other growth factors in the axonal outgrowth signaling pathway. The ability of the compound to act downstream of AF-1 or other growth factors in the axonal outgrowth signaling pathway may be determined using one of the assays described herein. For example, once a compound has been determined to be capable of stimulating the activity of N-kinase (e.g., stimulating the N-kinase dependent phosphorylation of a substrate), N-kinase may be contacted both with this compound and with 6-thioguanine. The inability of 6-thioguanine to inhibit the stimulatory effect of the compound would indicate that the compound is acting downstream of 6-thioguanine in the axonal outgrowth signaling pathway, whereas the ability of 6-thioguanine to inhibit the stimulatory effect of the compound would indicate that the compound is acting upstream (or at the same point) in the signaling pathway. Alternatively, once a compound has been determined to be capable of inhibiting the activity of N-kinase (e.g., inhibiting the N-kinase dependent phosphorylation of a substrate), N-kinase may be contacted both with this compound and with inosine. The inability of inosine to counteract the inhibitory effect of the compound would indicate that the compound is acting downstream of inosine in the axonal outgrowth signaling pathway, whereas the ability of inosine to counteract the inhibitory effect of the compound would indicate that the compound is acting upstream (or at the same point) in the signaling pathway. [0026]
In certain embodiments of the invention, the compound that modulates the activity of N-kinase can be any compound with the proviso that it is not a purine base (e.g., guanine, inosine, adenosine, and xanthine), such as a purine base linked to sugars, such as ribose, deoxyribose, and analogs and derivatives thereof. In certain other embodiments of the invention, the compound that modulates the activity of N-kinase can be any compound with the proviso that it is not a purine base analog or derivative thereof. [0027]
Examples of compounds that modulate the activity of N-kinase include small molecules, the N-kinase polypeptide or fragments thereof, an anti-N-kinase antibody, an antisense N-kinase nucleic acid molecule, a ribozyme, or the N-kinase gene or fragments thereof. [0028]
As used herein, the term “small molecule” includes, but is not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. [0029]
As used herein, a “neurosalutary effect” means a response or result favorable to the health or function of a neuron, of a part of the nervous system, or of the nervous system generally. Examples of such effects include improvements in the ability of a neuron or portion of the nervous system to resist insult, to regenerate, to maintain desirable function, to grow or to survive. The phrase “producing a neurosalutary effect” includes producing or effecting such a response or improvement in function or resilience within a component of the nervous system. For example, examples of producing a neurosalutary effect would include stimulating axonal outgrowth after injury to a neuron; rendering a neuron resistant to apoptosis; rendering a neuron resistant to a toxic compound such as β-amyloid, ammonia, or other neurotoxins; reversing age-related neuronal atrophy or loss of function; or reversing age-related loss of cholinergic innervation. [0030]
As used herein, the language “modulating the axonal outgrowth of central nervous system neurons” is intended to include the capacity to stimulate or inhibit axonal outgrowth of central nervous system neurons to various levels, e.g., to levels which allow for the treatment of a targeted neurological disorder. [0031]
As used herein, the term “outgrowth” (i.e., axonal outgrowth) refers to the process by which axons grow out of a CNS neuron. The outgrowth can result in a totally new axon or the repair of a partially damaged axon. Outgrowth is typically evidenced by extension of an axonal process of at least 5 cell diameters in length. Moreover, axonal outgrowth can be evidenced by GAP-43 expression (which can be detected by, for example, immunostaining). [0032]
As used herein, the term “CNS neurons” is intended to include the neurons of the brain and the spinal cord which are unresponsive to nerve growth factor (NGF). The term is not intended to include support or protection cells such as astrocytes, oligodentrocytes, microglia, ependyma and the like, nor is it intended to include peripheral nervous system (e.g., somatic, autonomic, sympathetic or parasympathetic nervous system) neurons. Preferred CNS neurons are mammalian neurons, more preferably human neurons. [0033]
The term “administering” to a subject includes dispensing, delivering or applying an active compound in a pharmaceutical formulation to a subject by any suitable route for delivery of the active compound to the desired location in the subject, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route. [0034]
As used herein, the language “contacting” is intended to include both in vivo or in vitro methods of bringing a compound that modulates the activity of N-kinase into proximity with a CNS neuron, such that the compound that modulates the activity of N-kinase can modulate the outgrowth of axonal processes from the CNS neuron. [0035]
As used herein, the term “subject” is intended to include animals. In particular embodiments, the subject is a mammal, a human or nonhuman primate, a dog, a cat, a horse, a cow or a rodent. [0036]
As used herein, the term “effective amount” includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, such as sufficient to produce a neurosalutary effect in a subject. An effective amount of an active compound as defined herein may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the active compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects of the active compound are outweighed by the therapeutically beneficial effects. [0037]
A therapeutically effective amount or dosage of an active compound may range from about 0.001 to 30 mg/kg body weight, with other ranges of the invention including about 0.01 to 25 mg/kg body weight, about 0.1 to 20 mg/kg body weight, about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, and 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an active compound can include a single treatment or a series of treatments. In one example, a subject is treated with an active compound in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, alternatively between 2 to 8 weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of an active compound used for treatment may increase or decrease over the course of a particular treatment. [0038]
“Neurological disorder” is intended to include a disease, disorder, or condition which directly or indirectly affects the normal functioning or anatomy of a subject's nervous system. Elements of the nervous system subject to disorders which may be effectively treated with the compounds and methods of the invention include the central, peripheral, somatic, autonomic, sympathetic and parasympathetic components of the nervous system, neurosensory tissues within the eye, ear, nose, mouth or other organs, as well as glial tissues associated with neuronal cells and structures. Neurological disorders may be caused by an injury to a neuron, such as a mechanical injury or an injury due to a toxic compound, by the abnormal growth or development of a neuron, or by the misregulation (such as downregulation or upregulation) of an activity of a neuron. Neurological disorders can detrimentally affect nervous system functions such as the sensory function (the ability to sense changes within the body and the outside environment); the integrative function (the ability to interpret the changes); and the motor function (the ability to respond to the interpretation by initiating an action such as a muscular contraction or glandular secretion). Examples of neurological disorders include traumatic or toxic injuries to peripheral or cranial nerves, spinal cord or to the brain, cranial nerves, traumatic brain injury, stroke, ischemia, cerebral aneurism, and spinal cord injury. Other neurological disorders include cognitive and neurodegenerative disorders such as Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease), diabetic neuropathy, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease. Autonomic function disorders include hypertension and sleep disorders. Also to be treated with compounds and methods of the invention are neuropsychiatric disorders such as depression, schizophrenia, schizoaffective disorder, Korsakoff's psychosis, mania, anxiety disorders, or phobic disorders, learning or memory disorders (such as amnesia and age-related memory loss), attention deficit disorder, autism, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, bipolar affective disorder, psychogenic pain syndromes, and eating disorders. Other examples of neurological disorders include injuries to the nervous system due to an infectious disease (such as meningitis, high fevers of various etiologies, HIV, syphilis, or post-polio syndrome) and injuries to the nervous system due to electricity (including contact with electricity or lightning, and complications from electro-convulsive psychiatric therapy). The developing brain is a target for neurotoxicity in the developing central nervous system through many stages of pregnancy as well as during infancy and early childhood, and the methods of the invention may be utilized in preventing or treating neurological deficits in embryos or fetuses in utero, in premature infants, or in children with need of such treatment, including those with neurological birth defects. Further neurological disorders include, for example, those listed in Harrison's Principles of Internal Medicine (Braunwald et al., McGraw-Hill, 2001) and in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders DSM-IV (American Psychiatric Press, 2000) both incorporated herein by reference in their entirety. [0039]
The term “stroke” is art recognized and is intended to include sudden diminution or loss of consciousness, sensation, and voluntary motion caused by rupture or obstruction (for example, by a blood clot) of an artery of the brain. [0040]
“Traumatic brain injury” is art recognized and is intended to include the condition in which, a traumatic blow to the head causes damage to the brain or connecting spinal cord, often without penetrating the skull. Usually, the initial trauma can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure, and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow. [0041]
In another aspect, the invention features an isolated N-kinase polypeptide of the type that: (a) is present in neonatal brain tissue (e.g., neonatal human, bovine, rat, or mouse brain tissue); (b) is inhibited by 6-thioguanine; (c) is activated by Mn[0042] ⁺²but not by Mg⁺²or Ca⁺²; (d) has a molecular weight of approximately 49 kDa; and (e) is eluted from a Cibacron Blue column at a NaCl concentration of 1.5-1.75 M. As used herein, an “isolated” N-kinase polypeptide is substantially free (i.e., greater than 95% free) of cellular material or other contaminating proteins from the cell or tissue source from which the N-kinase protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of N-kinase in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of N-kinase protein having less than about 20% (by dry weight) of non-N-kinase protein (i.e., contaminating protein), more preferably less than about 10% of non-N-kinase protein, still more preferably less than about 5% of non-N-kinase protein, and most preferably less than about 3% non-N-kinase protein. When the N-kinase protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
In yet another aspect, the invention features an isolated nucleic acid molecule encoding an N-kinase polypeptide, e.g., the N-kinase polypeptide of SEQ ID NO:1. [0043]
Various aspects of the invention are described in further detail in the following subsections: [0044]
Methods for Identifying a Compound Capable of Producing a Neurosalutary Effect in a Subject [0045]
In one aspect, the invention features a method for identifying a compound capable of producing a neurosalutary effect in a subject by contacting an N-kinase polypeptide, or a biologically active fragment thereof, with a test compound and determining the ability of the test compound to modulate the activity of N-kinase, thereby identifying a compound capable of producing a neurosalutary effect in a subject. [0046]
The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) [0047] Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) [0048] Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten (1992) [0049] Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc Natl. Acad. Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses an N-kinase protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate N-kinase activity is determined. Determining the ability of the test compound to modulate N-kinase activity can be accomplished by monitoring, for example, the production of one or more specific metabolites in a cell which expresses N-kinase (see, e.g., Saada et al. (2000) [0050] Biochem Biophys. Res. Commun. 269: 382-386). The cell, for example, can be of mammalian origin, e.g., a neuronal cell.
Determining the ability of the test compound to modulate N-kinase activity can further be accomplished by, for example, determining the ability of N-kinase to phosphorylate a substrate. The ability of N-kinase to phosphorylate a substrate (e.g., a histone HF-1 protein) can be determined by, for example, an in vitro kinase assay. Briefly, the N-kinase can be incubated with the substrate and radioactive ATP, e.g., [γ-[0051] ³²P] ATP, in a buffer containing MnCl₂, e.g., 5 mM MnCl₂. Following the incubation, the substrate can be immunoprecipitated or precipitated with TCA or collected on a filter (if no other kinases are present) and separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to a membrane, e.g., a PVDF membrane, and autoradiographed. The appearance of detectable bands on the autoradiograph indicates that the substrate has been phosphorylated. Alternatively, the in-gel assays described in Example 1 may be used to determine the ability of N-kinase to phosphorylate a substrate. Phosphoaminoacid analysis of the phosphorylated substrate can also be performed in order to determine which residues on the protein are phosphorylated. Briefly, the radiophosphorylated protein band can be excised from the SDS gel and subjected to partial acid hydrolysis. The products can then be separated by one-dimensional electrophoresis and analyzed on, for example, a phosphoimager and compared to ninhydrin-stained phosphoaminoacid standards.
The ability of the test compound to modulate N-kinase binding to a non-N-kinase molecule, such as a downstream molecule in the axonal outgrowth signaling pathway, can also be determined. Determining the ability of the test compound to modulate N-kinase binding to a non-N-kinase molecule can be accomplished, for example, by coupling the non-N-kinase molecule with a radioisotope or enzymatic label such that binding of the non-N-kinase molecule to N-kinase can be determined by detecting the labeled non-N-kinase molecule in a complex. [0052]
It is also within the scope of this invention to determine the ability of a test compound to interact with, e.g., bind to, N-kinase or biologically active portions thereof. Preferred biologically active portions of the N-kinase proteins to be used in assays of the present invention include fragments which participate in interactions with non-N-kinase molecules, e.g., fragments with high surface probability scores. Determining the ability of the test compound to bind N-kinase can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to N-kinase can be determined by detecting the labeled N-kinase compound in a complex. For example, test compounds can be labeled with [0053] ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
Determining the ability of a test compound to interact with N-kinase may also be accomplished without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a compound with N-kinase without the labeling of either the compound or the N-kinase. McConnell, H. M. et al (1992) [0054] Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and N-kinase.
Determining the ability of the test compound to bind to N-kinase or a biologically active portion thereof, can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) [0055] Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
In an alternative embodiment, determining the ability of the test compound to modulate the activity of N-kinase can be accomplished by determining the ability of the N-kinase protein to further modulate the activity of a downstream effector of an N-kinase target molecule. For example, the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described. [0056]
In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize any of the reactants, e.g., N-kinase or a non-N-kinase molecule, to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to N-kinase, or interaction of N-kinase with a non-N-kinase molecule in the presence and absence of a test compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/N-kinase fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed non-N-kinase molecule or N-kinase, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of N-kinase binding or activity determined using standard techniques. [0057]
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either N-kinase or a non-N-kinase molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated N-kinase or non-N-kinase molecule can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with N-kinase or a non-N-kinase molecule but which do not interfere with the binding of N-kinase to its target non-N-kinase molecule can be derivatized to the wells of the plate, and unbound non-N-kinase molecule or N-kinase trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with N-kinase or a non-N-kinase molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with N-kinase or a non-N-kinase target molecule. [0058]
In another embodiment, modulators of N-kinase expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of N-kinase mRNA or protein in the cell is determined. The level of expression of N-kinase mRNA or protein in the presence of the candidate compound is compared to the level of expression of N-kinase mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of N-kinase expression based on this comparison. For example, when expression of N-kinase mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of N-kinase mRNA or protein expression. Alternatively, when expression of N-kinase mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of N-kinase mRNA or protein expression. The level of N-kinase mRNA or protein expression in the cells can be determined by methods described herein for detecting N-kinase mRNA or protein. [0059]
In yet another aspect of the invention, the N-kinase proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) [0060] Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with N-kinase (“N-kinase-binding proteins” or “N-kinase-bp”) and are involved in N-kinase activity. Such N-kinase-binding proteins are also likely to be involved in the propagation of signals by the N-kinase proteins or N-kinase targets as, for example, downstream elements of an N-kinase-mediated signaling pathway. Alternatively, such N-kinase-binding proteins are likely to be N-kinase inhibitors.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for an N-kinase protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming an N-kinase-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the N-kinase protein. [0061]
In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating compound can be identified using a cell-based or a cell free assay, and the ability of the compound to modulate the activity of an N-kinase protein can be confirmed in vivo, e.g., in an animal such as an animal model for a condition characterized by aberrant, e.g., insufficient axonal outgrowth of central nervous system neurons. Examples of such animal models are described in, for example, Benowitz et al. (1999) [0062] PNAS 96(23):13486-90. Epilepsy animal models are also known in the art.
This invention further pertains to novel compounds identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use a compound identified as described herein in an appropriate animal model. For example, a compound identified as described herein (e.g., an N-kinase modulating compound, an antisense N-kinase nucleic acid molecule, an N-kinase-specific antibody, or an N-kinase-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such a compound. Alternatively, a compound identified as described herein can be used in an animal model to determine the mechanism of action of such a compound. Furthermore, this invention pertains to uses of novel compounds identified by the above-described screening assays for treatments as described herein. [0063]
In one embodiment, the N-kinase used in the methods of the invention is a human N-kinase, such as a recombinantly produced N-kinase. N-kinase, e.g., human N-kinase, may be introduced into a recombinant expression vector using standard techniques and expressed in prokaryotic or eukaryotic cells. For example, N-kinase, e.g., human N-kinase, can be expressed in bacterial cells such as [0064] E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, a recombinant expression vector containing N-kinase, e.g., human N-kinase, can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
In another embodiment, the N-kinase used in the methods of the invention is a bovine N-kinase, such as an N-kinase which is purified from a bovine source, e.g., neonatal bovine brain tissue, as described herein in, for instance, Example 1. [0065]
In another embodiment, the method of the invention further includes determining the ability of the test compound to modulate axonal outgrowth of a central nervous system neuron. Determining the ability of the test compound to modulate axonal outgrowth of a central nervous system neuron can be accomplished by, for example, using dissociated cultures of purified rat retinal ganglion cells. Dissociated cultures of purified rat retinal ganglion cells can, for example, be prepared by immunopanning as described in Barres et al., Neuron, 1: 791-803,1988, the contents of which are incorporated herein by reference. In brief, retinas from Sparague-Dawley rats can be dissociated using papain activated with cysteine. Macrophages are removed by incubation with an anti-rat macrophage antibody (Accurate) followed by immunopanning with an anti-rabbit IgG antibody. Ganglion cells are isolated by immunopanning with an anti-Thy-1 antibody, then dislodged with trypsin for use in low-density cultures. Rat retinal ganglion cells are maintained at 37° C. in a CO[0066] ₂incubator using the same medium described above except for the presence of 30 mM bicarbonate.
Samples are plated in quadruplicate in randomized positions of a 24-well plate, contacted with the test compound, and the code is concealed to ensure that growth is evaluated in a blinded fashion. Each experiment may contain 4 wells of a negative control (media plus supplements only) and 4 wells of a positive control (e.g., a standardized AF-1 sample of known activity). Growth and survival are assessed after 6 days for all ganglion cells in 25 consecutive fields of each well using phase contrast microscopy at 400× magnification (c. 150 ganglion cells counted per well). Extension of a process 5 cell diameters in length is used as the criterion for growth, since it clearly distinguishes stimulated cells from negative controls (Schwalb et al., 1995). After the completion of counting, the code is broken, the data tabulated, and means and standard errors are calculated for the 4 replicate wells of each sample using Cricket Graph (CA Associates, Islandia, N.Y.). Data are normalized by subtracting the growth in the negative controls (usually 4-5%) and dividing by the net growth in the positive controls. [0067]
Goldfish retinal ganglion cells (Benowitz et al. (1998) [0068] J. Biol. Chem. 273(45):29626-34) as well as mixtures of rat and goldfish ganglion cells may also be used.
The ability of a test compound to produce a neurosalutary effect in a subject may further be assessed using any of a variety of known procedures and assays. For example, the ability of a test compound to re-establish neural connectivity and/or function after an injury, such as a CNS injury, may be determined histologically (either by slicing neuronal tissue and looking at neuronal branching, or by showing cytoplasmic transport of dyes). The ability of compounds of the invention to re-establish neural connectivity and/or function after an injury, such as a CNS injury, may also be assessed by monitoring the ability of the test compound to fully or partially restore the electroretinogram after damage to the neural retina or optic nerve; or to fully or partially restore a pupillary response to light in the damaged eye. [0069]
Other tests that may be used to determine the ability of an N-kinase modulator to produce a neurosalutary effect in a subject include standard tests of neurological function in human subjects or in animal models of spinal injury (such as standard reflex testing, urologic tests, urodynamic testing, tests for deep and superficial pain appreciation, proprioceptive placing of the hind limbs, ambulation, and evoked potential testing). In addition, nerve impulse conduction can be measured in a subject, such as by measuring conduct action potentials, as an indication of the production of a neurosalutary effect. [0070]
Animal models suitable for use in the assays of the present invention include the rat model of partial transection (described in Weidner et al. (2001) [0071] Proc. Natl. Acad. Sci. USA 98:3513-3518). This animal model tests how well a compound can enhance the survival and sprouting of the intact remaining fragment of an almost fully-transected cord. Accordingly, after administration of the N-kinase modulator these animals may be evaluated for recovery of a certain function, such as how well the rats may manipulate food pellets with their forearms (to which the relevant cord had been cut 97%).
Another animal model suitable for use in the assays of the present invention includes the rat model of stroke (described in Kawamata et al. (1997) [0072] Proc. Natl. Acad. Sci. USA 94(15):8179-8184). This paper describes in detail various tests that may be used to assess sensorimotor function in the limbs as well as vestibulomotor function after an injury. Administration to these animals of an N-kinase modulator of the invention can be used to assess whether a given compound, route of administration, or dosage provides a neurosalutary effect, such as increasing the level of function, or increasing the rate of regaining function or the degree of retention of function in the test animals.
Standard neurological evaluations used to assess progress in human patients after a stroke may also be used to evaluate the ability of an N-kinase modulator to produce a neurosalutary effect in a subject. Such standard neurological evaluations are routine in the medical arts, and are described in, for example, “Guide to Clinical Neurobiology” Edited by Mohr and Gautier (Churchill Livingstone Inc. 1995). [0073]
For assessing function of the peripheral nervous system, standard tests include electromyography, nerve conduction velocity measurements, evoked potentials assessment and upper/lower extremity somato-sensory evoked potentials. Electromyography tests record the electrical activity in muscles, and is used to assess the function of the nerves and muscles. The electrode is inserted into a muscle to record its electrical activity. It records activity during the insertion, while the muscle is at rest, and while the muscle contracts. The nerve conduction velocity test evaluates the health of the peripheral nerve by recording how fast an electrical impulse travels through it. A peripheral nerve transmits information between the spinal cord and the muscles. A number of nervous system diseases may reduce the speed of this impulse. Electrodes placed on the skin detect and record the electrical signal after the impulse travels along the nerve. A second stimulating electrode is sends a small electrical charge along the nerve; the time between the stimulation and response will be recorded to determine how quickly and thoroughly the impulse is sent. [0074]
Standard tests for function of the cranial nerves, as known to those skilled in the neurological medical art, include facial nerve conduction studies; orbicularis oculi reflex studies (blink reflex studies); trigeminal-facial nerve reflex evaluation as used in focal facial nerve lesions, Bell's palsy, trigeminal neuralgia and atypical facial pain; evoked potentials assessment; visual, brainstem and auditory evoked potential measurements; thermo-diagnostic small fiber testing; and computer-assisted qualitative sensory testing. [0075]
Compounds Capable of Producing a Neurosalutary Effect in a Subject [0076]
In another aspect, the invention features a compound capable of producing a neurosalutary effect in a subject identified by any of the foregoing methods. [0077]
In one embodiment, the compound capable of producing a neurosalutary effect in a subject is an antisense N-kinase nucleic acid molecule. An “antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to the entire N-kinase coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding an N-kinase. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding N-kinase. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions). [0078]
Given the coding strand sequence encoding N-kinase, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of N-kinase mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of N-kinase mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of N-kinase mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection). [0079]
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an N-kinase protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site, e.g., in the brain. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. [0080]
In yet another embodiment, the antisense N-kinase nucleic acid molecule may be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) [0081] Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
In still another embodiment, the compound that modulates axonal outgrowth of a central nervous system neuron is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) [0082] Nature 334:585-591)) can be used to catalytically cleave N-kinase mRNA transcripts to thereby inhibit translation of N-kinase mRNA. A ribozyme having specificity for an N-kinase-encoding nucleic acid can be designed based upon the nucleotide sequence of an N-kinase cDNA. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an N-kinase-encoding mRNA. See, e.g., Cech et al. U.S. Pat. Nos. 4,987,071; and 5,116,742. Alternatively, N-kinase mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.
Alternatively, N-kinase gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the N-kinase (e.g., the N-kinase promoter and/or enhancers) to form triple helical structures that prevent transcription of the N-kinase gene in target cells. See generally, Helene, C. (1991) [0083] Anticancer Drug Des. 6(6): 569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.
In still another embodiment, the compound that modulates axonal outgrowth of a central nervous system neuron is an anti-N-kinase antibody. A full-length N-kinase protein or, alternatively, antigenic peptide fragments of N-kinase may be used as immunogens to generate anti-N-kinase antibodies. The antigenic peptide of N-kinase comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:1 and encompasses an epitope of N-kinase such that an antibody raised against the peptide forms a specific immune complex with the N-kinase protein. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. [0084]
Preferred epitopes encompassed by the antigenic peptide are regions of N-kinase that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. [0085]
An N-kinase immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed N-kinase protein or a chemically synthesized N-kinase polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic N-kinase preparation induces a polyclonal anti-N-kinase antibody response. [0086]
The term “antibody” as used herein includes immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as an N-kinase. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)[0087] ₂fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind N-kinase molecules. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of N-kinase. A monoclonal antibody composition thus typically displays a single binding affinity for a particular N-kinase protein with which it immunoreacts.
Polyclonal anti-N-kinase antibodies can be prepared as described above by immunizing a suitable subject with an N-kinase immunogen. The anti-N-kinase antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized N-kinase. If desired, the antibody molecules directed against N-kinase can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-N-kinase antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) [0088] Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an N-kinase immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds N-kinase.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-N-kinase monoclonal antibody (see, e.g., G. Galfre et al. (1977) [0089] Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind N-kinase, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-N-kinase antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with N-kinase to thereby isolate immunoglobulin library members that bind N-kinase. Kits for generating and screening phage display libraries are commercially available (e.g, the Pharmacia [0090] Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 2:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.
Additionally, recombinant anti-N-kinase antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) [0091] Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.
In one embodiment, the compound capable of producing a neurosalutary effect in a subject is an intracellular antibody specific for N-kinase. The use of intracellular antibodies to inhibit protein function in a cell is known in the art (see e.g., Carlson, J. R. (1988) [0092] Mol. Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO J. 9:101-108; Werge, T. M. et al. (1990) FEBS Letters 274:193-198; Carlson, J. R. (1993) Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W. A. et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. et al. (1994) Bio/Technology 12:396-399; Chen, S- Y. et al. (1994) Human Gene Therapy 5:595-601; Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen, S- Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. et al. (1994) J. Biol. Chem. 269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys. Res. Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J. 14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad Sci. USA 92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCT Publication No. WO 95/03832 by Duan et al.).
To inhibit protein activity using an intracellular antibody, a recombinant expression vector is prepared which encodes the antibody chains in a form such that, upon introduction of the vector into a cell, the antibody chains are expressed as a functional antibody in an intracellular compartment of the cell. [0093]
In still another embodiment, the compound capable of producing a neurosalutary effect in a subject is a small molecule. As used herein, the term “small molecule” includes, but is not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. [0094]
In still another embodiment, the compound capable of producing a neurosalutary effect in a subject is an N-kinase polypeptide or portion thereof, e.g., a fragment that includes at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 contiguous amino acids of the N-kinase polypeptide. For example, the compound could comprise the constitutively active catalytic domain of the N-kinase. [0095]
In still another embodiment, the compound capable of producing a neurosalutary effect in a subject is an N-kinase nucleic acid molecule or a portion thereof. [0096]
In a preferred embodiment, the compound that modulates the activity of N-kinase is capable of entering a cell and acting intracellularly. For example, membrane translocation domains may be used to guide peptidic modulators of the N-kinase into a cell. Such membrane translocation domains are known in the art and include, but are not limited to, the third helix of the antennapedia homeodomain protein and the HIV-1 protein Tat and are described in, for example, Derossi et al., (1994) [0097] J. Biol. Chem. 269, 10444-10450; Lindgren et al., (2000) Trends Pharmacol. Sci. 21, 99-103; Ho et al., Cancer Research 61, 474-477 (2001); U.S. Pat. Nos. 5,888,762; 6,015,787; 5,846,743; 5,747,641; 5,804,604; and Published PCT applications WO 98/52614, WO 00/29427 and WO 99/29721. The entire contents of each of the foregoing references are incorporated herein by reference.
Further art known techniques for facilitating the transfer of the compound that modulates the activity of N-kinase (i.e., the N-kinase modulator) into a cell include those described in, for example, Lindgren M et al. (2000) [0098] Trends Pharmacol Sci. 21(3):99-103; Gariepy J. et al. (2001) Trends Biotechnol 19(1):21-28; Vyas S P et al. (2001) Crit Rev Ther Drug Carrier Syst 18(1):1-76; Morris M C et al. (2000) Curr Opin Biotechnol 11(5):461-466; and Derossi D et al. (1998) Trends Cell Biol. 8(2):84-87, the contents of all of which are incorporated herein by reference.
Constructs expressing therapeutic genes, antisense oligonucleotides and ribozymes can be delivered into cells by viral vectors, as well as by non-viral delivery systems including those described in, for example, Hope M J et al. (1998) [0099] Mol Membr Biol 15(1):1-14.
Pharmaceutically Acceptable Formulations [0100]
Pharmaceutical compositions and packaged formulations comprising a compound that modulates the activity of N-kinase and a pharmaceutically acceptable carrier are also provided by the invention. [0101]
In a method of the invention, the compound that modulates the activity of N-kinase, can be administered in a pharmaceutically acceptable formulation. Such pharmaceutically acceptable formulation may include the N-kinase modulator as well as a pharmaceutically acceptable carrier(s) and/or excipient(s). As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and anti fungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. For example, the carrier can be suitable for injection into the cerebrospinal fluid. Excipients include pharmaceutically acceptable stabilizers and disintegrants. The present invention pertains to any pharmaceutically acceptable formulations, including synthetic or natural polymers in the form of macromolecular complexes, nanocapsules, microspheres, or beads, and lipid-based formulations including oil-in-water emulsions, micelles, mixed micelles, synthetic membrane vesicles, and resealed erythrocytes. [0102]
In one embodiment, the pharmaceutically acceptable formulations comprise a polymeric matrix. The terms “polymer” or “polymeric” are art-recognized and include a structural framework comprised of repeating monomer units which is capable of delivering an N-kinase modulator such that treatment of a targeted condition, such as a neurological disorder, occurs. The terms also include co-polymers and homopolymers such as synthetic or naturally occurring. Linear polymers, branched polymers, and cross-linked polymers are also meant to be included. [0103]
For example, polymeric materials suitable for forming the pharmaceutically acceptable formulation employed in the present invention, include naturally derived polymers such as albumin, alginate, cellulose derivatives, collagen, fibrin, gelatin, and polysaccharides, as well as synthetic polymers such as polyesters (PLA, PLGA), polyethylene glycol, poloxomers, polyanhydrides, and pluronics. These polymers are biocompatible with the nervous system, including the central nervous system, they are biodegradable within the central nervous system without producing any toxic byproducts of degradation, and they possess the ability to modify the manner and duration of the active compound release by manipulating the polymer's kinetic characteristics. As used herein, the term “biodegradable” means that the polymer will degrade over time by the action of enzymes, by hydrolytic action and/or by other similar mechanisms in the body of the subject. As used herein, the term “biocompatible” means that the polymer is compatible with a living tissue or a living organism by not being toxic or injurious and by not causing an immunological rejection. Polymers can be prepared using methods known in the art. [0104]
The polymeric formulations can be formed by dispersion of the active compound within liquefied polymer, as described in U.S. Pat. No. 4,883,666, the teachings of which are incorporated herein by reference or by such methods as bulk polymerization, interfacial polymerization, solution polymerization and ring polymerization as described in Odian G., Principles of Polymerization and ring opening polymerization, 2nd ed., John Wiley & Sons, New York, 1981, the contents of which are incorporated herein by reference. The properties and characteristics of the formulations are controlled by varying such parameters as the reaction temperature, concentrations of polymer and the active compound, the types of solvent used, and reaction times. [0105]
The active therapeutic compound can be encapsulated in one or more pharmaceutically acceptable polymers, to form a microcapsule, microsphere, or microparticle, terms used herein interchangeably. Microcapsules, microspheres, and microparticles are conventionally free-flowing powders consisting of spherical particles of 2 millimeters or less in diameter, usually 500 microns or less in diameter. Particles less than 1 micron are conventionally referred to as nanocapsules, nanoparticles or nanospheres. For the most part, the difference between a microcapsule and a nanocapsule, a microsphere and a nanosphere, or microparticle and nanoparticle is size; generally there is little, if any, difference between the internal structure of the two. In one aspect of the present invention, the mean average diameter is less than about 45 μm, preferably less than 20 μm, and more preferably between about 0.1 and 10 μm. [0106]
In another embodiment, the pharmaceutically acceptable formulations comprise lipid-based formulations. Any of the known lipid-based drug delivery systems can be used in the practice of the invention. For instance, multivesicular liposomes, multilamellar liposomes and unilamellar liposomes can all be used so long as a sustained release rate of the encapsulated active compound can be established. Methods of making controlled release multivesicular liposome drug delivery systems are described in PCT application Ser. Nos. US96/11642, US94/12957 and US94/04490, the contents of which are incorporated herein by reference. [0107]
The composition of the synthetic membrane vesicle is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. [0108]
Examples of lipids useful in synthetic membrane vesicle production include phosphatidylglycerols, phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, sphingolipids, cerebrosides, and gangliosides, with preferable embodiments including egg phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol. [0109]
In preparing lipid-based vesicles containing an active compound such variables as the efficiency of active compound encapsulation, lability of the active compound, homogeneity and size of the resulting population of vesicles, active compound-to-lipid ratio, permeability, instability of the preparation, and pharmaceutical acceptability of the formulation should be considered. [0110]
Prior to introduction, the formulations can be sterilized, by any of the umerous available techniques of the art, such as with gamma radiation or electron beam sterilization. [0111]
Administration of the Pharmaceutically Acceptable Formulation [0112]
The pharmaceutically acceptable formulations of the invention are administered such that the active compound comes into contact with a subject's nervous system to thereby produce a neurosalutary effect. Both local and systemic administration of the formulations are contemplated by the invention. Desirable features of local administration include achieving effective local concentrations of the active compound as well as avoiding adverse side effects from systemic administration of the active compound. In one embodiment, the active compound is administered by introduction into the cerebrospinal fluid of the subject. In certain aspects of the invention, the active compound is introduced into a cerebral ventricle, the lumbar area, or the cisterna magna. In another aspect, the active compound is introduced locally, such as into the site of nerve or cord injury, into a site of pain or neural degeneration, or intraocularly to contact neuroretinal cells. [0113]
The pharmaceutically acceptable formulations can be suspended in aqueous vehicles and introduced through conventional hypodermic needles or using infusion pumps. [0114]
In one embodiment, the active compound formulation described herein is administered to the subject in the period from the time of, for example, an injury to the CNS up to about 100 hours after the injury has occurred, for example within 24, 12, or 6 hours from the time of injury. [0115]
In another embodiment of the invention, the active compound formulation is administered into a subject intrathecally. As used herein, the term “intrathecal administration” is intended to include delivering an active compound formulation directly into the cerebrospinal fluid of a subject, by techniques including lateral cerebroventricular injection through a burrhole or cisternal or lumbar puncture or the like (described in Lazorthes et al. Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1: 169-179, the contents of which are incorporated herein by reference). The term “lumbar region” is intended to include the area between the third and fourth lumbar (lower back) vertebrae. The term “cisterna magna” is intended to include the area where the skull ends and the spinal cord begins at the back of the head. The term “cerebral ventricle” is intended to include the cavities in the brain that are continuous with the central canal of the spinal cord. Administration of an active compound to any of the above mentioned sites can be achieved by direct injection of the active compound formulation or by the use of infusion pumps. Implantable or external pumps and catheter may be used. [0116]
For injection, the active compound formulation of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the active compound formulation may be formulated in solid form and re-dissolved or suspended immediately prior to use. Lyophilized forms are also included. The injection can be, for example, in the form of a bolus injection or continuous infusion (such as using infusion pumps) of the active compound formulation. [0117]
In one embodiment of the invention, the active compound formulation is administered by lateral cerebroventricular injection into the brain of a subject, preferably within 100 hours of when an injury (resulting in a condition characterized by aberrant, insufficient or inadequate axonal outgrowth of central nervous system neurons) occurs (such as within 6, 12, or 24 hours of the time of the injury). The injection can be made, for example, through a burr hole made in the subject's skull. In another embodiment, the formulation is administered through a surgically inserted shunt into the cerebral ventricle of a subject, preferably within 100 hours of when an injury occurs (such as within 6, 12 or 24 hours of the time of the injury). For example, the injection can be made into the lateral ventricles, which are larger, even though injection into the third and fourth smaller ventricles can also be made. In yet another embodiment, the active compound formulation is administered by injection into the cisterna magna, or lumbar area of a subject, preferably within 100 hours of when an injury occurs (such as within 6, 12, or 24 hours of the time of the injury). [0118]
An additional means of administration to intracranial tissue involves application of compounds of the invention to the olfactory epithelium, with subsequent transmission to the olfactory bulb and transport to more proximal portions of the brain. Such administration can be by nebulized or aerosolized prerparations. [0119]
In another embodiment of the invention, the active compound formulation is administered to a subject at the site of injury, preferably within 100 hours of when an injury occurs (such as within 6, 12, or 24 hours of the time of the injury). [0120]
Duration and Levels of Administration [0121]
In a preferred embodiment of the method of the invention, the active compound is administered to a subject for an extended period of time to produce a neurosalutary effect, such as effect modulation of axonal outgrowth. Sustained contact with the active compound can be achieved by, for example, repeated administration of the active compound over a period of time, such as one week, several weeks, one month or longer. More preferably, the pharmaceutically acceptable formulation used to administer the active compound provides sustained delivery, such as “slow release” of the active compound to a subject. For example, the formulation may deliver the active compound for at least one, two, three, or four weeks after the pharmaceutically acceptable formulation is administered to the subject. Preferably, a subject to be treated in accordance with the present invention is treated with the active compound for at least 30 days (either by repeated administration or by use of a sustained delivery system, or both). [0122]
As used herein, the term “sustained delivery” is intended to include continual delivery of the active compound in vivo over a period of time following administration, preferably at least several days, a week, several weeks, one month or longer. Sustained delivery of the active compound can be demonstrated by, for example, the continued therapeutic effect of the active compound over time (such as sustained delivery of the active compound can be demonstrated by continued production of a neurosalutary effect in a subject). Alternatively, sustained delivery of the active compound may be demonstrated by detecting the presence of the active compound in vivo over time. [0123]
Preferred approaches for sustained delivery include use of a polymeric capsule, a minipump to deliver the formulation, a bioerodible implant, or implanted transgenic autologous cells (as described in U.S. Pat. No. 6,214,622). Implantable infusion pump systems (such as Infusaid; see such as Zierski, J. et al. (1988) [0124] Acta Neurochem. Suppl. 43:94-99; Kanoff, R. B. (1994) J. Am. Osteopath. Assoc. 94:487-493) and osmotic pumps (sold by Alza Corporation) are available in the art. Another mode of administration is via an implantable, externally programmable infusion pump. Suitable infusion pump systems and reservoir systems are also described in U.S. Pat. No. 5,368,562 by Blomquist and U.S. Pat. No. 4,731,058 by Doan, developed by Pharmacia Deltec Inc.
It is to be noted that dosage values may vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the active compound and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention. [0125]
The invention, in another embodiment, provides a pharmaceutical composition consisting essentially of an N-kinase modulator and a pharmaceutically acceptable carrier, as well as methods of use thereof to modulate axonal outgrowth by contacting CNS neurons with the composition. By the term “consisting essentially of” is meant that the pharmaceutical composition does not contain any other modulators of neuronal growth such as, for example, nerve growth factor (NGF). In one embodiment, the pharmaceutical composition of the invention can be provided as a packaged formulation. The packaged formulation may include a pharmaceutical composition of the invention in a container and printed instructions for administration of the composition for producing a neurosalutary effect in a subject having a neurological disorder. Use of an N-kinase modulator derived factor in the manufacture of a medicament for modulating the axonal outgrowth of neurons is also encompassed by the invention. [0126]
In vitro Treatment of CNS Neurons [0127]
CNS neurons can further be contacted with a compound that modulates the activity of N-kinase, in vitro to modulate axonal outgrowth in vitro. Accordingly, CNS neuron cells can be isolated from a subject and grown in vitro, using techniques well known in the art, and then treated in accordance with the present invention to modulate axonal outgrowth. Briefly, a CNS neuron cell culture can be obtained by allowing neuron cells to migrate out of fragments of neural tissue adhering to a suitable substrate (e.g., a culture dish) or by disaggregating the tissue, e.g., mechanically or enzymatically, to produce a suspension of CNS neuron cells. For example, the enzymes trypsin, collagenase, elastase, hyaluronidase, DNase, pronase, dispase, or various combinations thereof can be used. Trypsin and pronase give the most complete disaggregation but may damage the cells. Collagenase and dispase give a less complete dissagregation but are less harmful. Methods for isolating tissue (e.g., neural tissue) and the disaggregation of tissue to obtain cells (e.g., CNS neuron cells) are described in Freshney R. I., Culture of Animal Cells, A Manual of Basic Technique, Third Edition, 1994, the contents of which are incorporated herein by reference. [0128]
Such cells can be subsequently contacted with a compound that modulates the activity of N-kinase in amounts and for a duration of time as described above. Once modulation of axonal outgrowth has been achieved in the CNS neuron cells, these cells can be re-administered to the subject, e.g., by implantation. It is preferred that the cells are not allowed to differentiate extensively in vitro, as cells that integrate most successfully in a subject are primitive cells. [0129]
The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing are hereby incorporated by reference. [0130]

EXAMPLES

Example I

Isolation and Characterization of the N-kinase Polypeptide [0131]
Neocortical gray matter from bovine brain was homogenized in buffer containing protease and phosphatase inhibitors, and particulate material was centrifuged down. The soluble fraction from approximately 1 kg of tissue was used as the starting material for isolation of the kinase. [0132]
During the purification process, kinase activity was monitored using an in-gel kinase method. In this method, a histone HF-1 substrate protein is polymerized into a 10% polyacrylamide gel before the samples to be analyzed are electrophoresed in the gel. Following completion of the electrophoresis, proteins are partially renatured with guanidium isothiocyanate and incubated in the presence [[0133] ³²P]-ATP plus Mn²⁺, with or without 6-TG present. (Activation by Mn²⁺, but not by Mg²⁺ or Ca²⁺, is a distinctive property of N-kinase). The purification of the kinase was monitored by looking for a 6-TG-inhibitable radioactive band corresponding to the site where the kinase phosphorylates the HF-1 substrate in the gel.
In the first step of the purification process the starting material was subjected to cation-exchange chromatography (using a Fast-S column, Pharmacia). A 6-TG inhibitable kinase activity bound strongly to this column and eluted with 0.3 M NaCl. The cation-exchange column fraction containing N-kinase was subsequently separated on a Cibacron Blue column (Pharmacia), which allows for the separation of adenine nucleotide-binding proteins. A 6-TG inhibitable, 47-50 kDa polypeptide bound strongly and required a NaCl concentration of 1.5 M NaCl for elution (see FIG. 1A). [0134]
The eluted fraction containing the N-kinase polypeptide was, then, subjected to reversed-phase chromatography with a C4 hydrophobic interaction column (Pharmacia). Briefly, the Cibacron Blue column fraction containing the N-kinase was applied to the C4 column and eluted with a gradient of increasing acetonitrile-isopropanol concentration. Evaluation of the kinase activity of the column fractions by in-gel kinase assays showed that the 6-TG-inhibitable, HF-1-phosphorylating activity eluted in fractions 24-26 (FIG. 1B). To achieve a higher level of purification, these fractions were pooled, re-applied to the same column, and the separation was repeated with a gradient of increasing acetonitrile-isopropanol concentration. The N-kinase polypeptide, again, eluted at fractions 24-26. [0135]
The column fractions containing the highest concentrations of N-kinase were lyophilized and applied to a 10% polyacrylamide SDS gel. A small portion of the sample was run on a parallel gel to carry out in-gel kinase assays. A band at 49 kDa was clearly visible after staining the gel with Coomassie blue; this coincided in its migration position with the 6-TG-inhibitable kinase activity. This band was cut out and it was verified that it contained HF-1-phosphorylating activity. [0136]
The gel band containing the N-kinase polypeptide was then subjected to partial proteolytic digestion, the proteolytic fragments were analyzed by mass spectroscopy and the masses of the fragments compared to those of various known peptides using the process described in, for example, Eng J. K. et al. (1994) [0137] J. Am. Soc. Mass. Spectrom. 5:976-989; Chittum H. S. et al. (1998) Biochemistry 37:10866-870; and LeRoy G. et al. (1998) Science 282:1900-04, the contents of which are incorporated herein by reference.
This analysis revealed that N-kinase is an isoform of MST-3, i.e., either MST-3 itself, MST-3b, or an as yet undefined isoform. These proteins are members of the STE family of serine-threonine kinases that are found throughout the animal kingdom. STE family members are generally components of modular signaling cassettes that are involved in various aspects of cellular differentiation. FIG. 2 depicts the amino acid sequence of the N-kinase. Direct matches between the purified protein and the published sequence are shown in blue. K65 (bold type) lies in the ATP-binding region of the kinase domain. [0138]
Equivalents [0139]
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. [0140]
1 1 1 272 PRT Homo sapiens 1 Met Asp Ser Arg Ala Trp Gly Ala Asn Lys Arg Arg Ala Thr His Gly 1 5 10 15 Gly Ser Thr Asn Lys Ala Asp Thr Lys Lys Gly Lys Gly Ser Gly Val 20 25 30 Lys Gly Asp Asn Arg Thr Lys Val Val Ala Lys Asp Ala Asp Asp Thr 35 40 45 Val Ser Cys Asp Ser Tyr Val Thr Lys Tyr Tyr Gly Ser Tyr Lys Asp 50 55 60 Thr Lys Trp Met Tyr Gly Gly Gly Ser Ala Asp Gly Asp Thr Ala Thr 65 70 75 80 Arg Lys Gly Asp Tyr His Ser Lys Lys His Arg Asp Lys Ala Ala Asn 85 90 95 Val Ser His Gly Val Lys Ala Asp Gly Val Ala Gly Thr Asp Thr Lys 100 105 110 Arg Asn Thr Val Gly Thr Trp Met Ala Val Lys Ser Ala Tyr Asp Ser 115 120 125 Lys Ala Asp Trp Ser Gly Thr Ala Ala Arg Gly His Ser His Met Lys 130 135 140 Val Lys Asn Asn Thr Gly Asn Tyr Ser Lys Lys Val Ala Cys Asn Lys 145 150 155 160 Ser Arg Thr Ala Lys Lys His Lys Arg Asn Ala Lys Lys Thr Ser Tyr 165 170 175 Thr Asp Arg Tyr Lys Arg Trp Lys Ala Ser His Asp Asp Ser Ser Ser 180 185 190 Asp Ser Asp Ala Thr Asp Gly Ala Ser Gly Gly Ser Asp Ser Gly Asp 195 200 205 Trp Thr Arg Lys Asp Lys Asn Asn Gly Ala Ser Asp Asp Arg Asn Lys 210 215 220 Met Lys Asp Lys Arg Ser Cys Ser Thr Ser Ala Lys Lys Ser Ala Cys 225 230 235 240 Gly Gly Asn Gly Ser Arg Gly Ala Tyr Ala Ala Cys Gly Ser Asp Thr 245 250 255 Met Val Ala Val Arg Arg Tyr Ser Ser Gly Gly Gly Thr Ser Ser His 260 265 270

Claims

We claim:

1. A method comprising administering to a subject a therapeutically effective amount of a compound that modulates the activity of N-kinase, thereby producing a neurosalutary effect in said subject.

2. The method of claim 1, wherein the neurosalutary effect is produced in said subject by modulating neuronal survival.

3. The method of claim 1, wherein the neurosalutary effect is produced in said subject by modulating neuronal regeneration.

4. The method of claim 1, wherein the neurosalutary effect is produced in said subject by modulating neuronal axonal outgrowth.

5. The method of claim 1, wherein the neurosalutary effect is produced in said subject by modulating axonal outgrowth of central nervous system neurons.

6. The method of claim 5, wherein the central nervous system neurons are retinal ganglion cells.

7. The method of claim 1, wherein the compound that modulates the activity of N-kinase is administered by introduction into a region of neuronal injury.

8. The method of claim 1, wherein the compound that modulates the activity of N-kinase is introduced into the cerebrospinal fluid of the subject.

9. The method of claim 1, wherein the compound that modulates the activity of N-kinase is introduced to the subject intrathecally.

10. The method of claim 1, wherein the compound that modulates the activity of N-kinase is introduced into a region selected from the group consisting of a cerebral ventricle, the lumbar area, and the cisterna magna of the subject.

11. The method of claim 1, wherein the compound that modulates the activity of N-kinase is administered to the subject in a pharmaceutically acceptable formulation.

12. The method of claim 11, wherein the pharmaceutically acceptable formulation is a dispersion system.

13. The method of claim 11, wherein the pharmaceutically acceptable formulation comprises a lipid-based formulation.

14. The method of claim 13, wherein the pharmaceutically acceptable formulation comprises a liposome formulation.

15. The method of claim 13, wherein the pharmaceutically acceptable formulation comprises a multivesicular liposome formulation.

16. The method of claim 11, wherein the pharmaceutically acceptable formulation comprises a polymeric matrix.

17. The method of claim 11, wherein the pharmaceutically acceptable formulation is contained within a minipump.

18. The method of claim 11, wherein the pharmaceutically acceptable formulation provides sustained delivery of the compound that modulates the activity of N-kinase, to a subject for at least one week after the pharmaceutically acceptable formulation is administered to the subject.

19. The method of claim 13, wherein the pharmaceutically acceptable formulation provides sustained delivery of the compound that modulates the activity of N-kinase, to a subject for at least one month after the pharmaceutically acceptable formulation is administered to the subject.

20. The method of claim 1, wherein the subject is a mammal.

21. The method of claim 20, wherein the mammal is a human.

22. The method of claim 1, wherein said subject is suffering from a neurological disorder.

23. The method of claim 22, wherein said neurological disorder is a spinal cord injury.

24. The method of claim 23, wherein the spinal cord injury is characterized by monoplegia, diplegia, paraplegia, hemiplegia and quadriplegia.

25. The method of claim 22, wherein said neurological disorder is epilepsy.

26. The method of claim 22, wherein said neurological disorder is stroke.

27. The method of claim 22, wherein said neurological disorder is Alzheimer's disease.

28. A method comprising administering a therapeutically effective amount of a compound that modulates the activity of N-kinase to a subject suffering from a neurological disorder, thereby treating said subject suffering from a neurological disorder.

29. The method of claim 28, further comprising making a first assessment of a nervous system function prior to administering the compound that modulates the activity of N-kinase to the subject and making a second assessment of the nervous system function after administering the compound that modulates the activity of N-kinase to the subject.

30. The method of claim 29, wherein the nervous system function is a sensory function, cholinergic innervation, or a vestibulomotor function.

31. A method for identifying a compound capable of producing a neurosalutary effect in a subject, comprising contacting N-kinase, or a biologically active fragment thereof, with a test compound and determining the ability of the test compound to modulate the activity of N-kinase, thereby identifying a compound capable of producing a neurosalutary effect in a subject.

32. The method of claim 31, wherein the N-kinase is human N-kinase.

33. The method of claim 32, wherein the human N-kinase is a recombinantly produced N-kinase.

34. The method of claim 31, wherein the N-kinase is bovine N-kinase.

35. The method of claim 34, wherein the bovine N-kinase is purified from a bovine source.

36. The method of claim 31, further comprising determining the ability of the test compound to modulate axonal outgrowth of a central nervous system neuron.

37. The method of claim 31, wherein the test compound inhibits the activity of N-kinase.

38. The method of claim 31, wherein the test compound stimulates the activity of N-kinase.

39. The method of claim 31, wherein the ability of the test compound to modulate the activity of N-kinase is determined by assessing the ability of the test compound to modulate N-kinase dependent phosphorylation of a substrate.

40. A method for identifying a compound capable of producing a neurosalutary effect in a subject, comprising

contacting N-kinase or a biologically active fragment thereof, with a test compound, an N-kinase substrate, radioactive ATP, and Mn⁺²; and

determining the ability of the test compound to modulate N-kinase dependent phosphorylation of the substrate, thereby identifying a compound capable of producing a neurosalutary effect in a subject.

41. The method of claim 40, wherein the N-kinase substrate is a histone HF-1 protein.

42. The method of claim 40, wherein the radioactive ATP is [γ-³²P] ATP.

43. The method of claim 40, wherein the N-kinase is human N-kinase.

44. The method of claim 43, wherein the human N-kinase is a recombinantly produced N-kinase.

45. The method of claim 40, wherein the N-kinase is bovine N-kinase.

46. The method of claim 45, wherein the bovine N-kinase is purified from a bovine source.

47. The method of claim 40, further comprising determining the ability of the test compound to modulate axonal outgrowth of a central nervous system neuron.

48. A compound capable of producing a neurosalutary effect in a subject identified by the method of claim 40.

49. An isolated N-kinase polypeptide of the type that:

(a) is present in neonatal brain tissue;

(b) is inhibited in the presence of 6-thioguanine;

(c) is activated in the presence of Mn⁺²but not by Mg⁺²or Ca⁺²;

(d) has a molecular weight of approximately 49 kDa; and

(e) is eluted from a Cibacron Blue column at a NaCl concentration of 1.5-1.75 M.

50. An antibody which is specifically reactive with an epitope of the N-kinase polypeptide of claim 49.

51. The antibody of claim 50, wherein the antibody is an intracellular antibody.

52. The antibody of claim 50, wherein the epitope comprises an ATP binding domain.

53. A fragment of the N-kinase polypeptide of claim 49, wherein the fragment comprises at least 15 contiguous amino acids.

54. The fragment of claim 53, wherein the fragment comprises at least 30 contiguous amino acids.

55. The fragment of claim 53, wherein the fragment comprises at least 50 contiguous amino acids.

56. The fragment of claim 53, wherein the fragment comprises at least 100 contiguous amino acids.

57. A fragment of the N-kinase polypeptide of claim 49, wherein the fragment is able to elicit an immune response.

58. An isolated nucleic acid molecule that encodes the polypeptide of SEQ ID NO:1.