JP2010285413A - Therapeutic use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for treating neurodegenerative diseases such as multiple sclerosis by administering an EphA4 antagonist or an EphA4 ligand antagonist to a subject in need. <P>SOLUTION: The antagonist may be a soluble EphA4 or EphA4-binding ephrin or their functional variants such as an EphA4-Fc or an ephrin A5-Fc. The antagonist may be a nucleic acid, polypeptide, peptide or organic molecule that binds to EphA4 or an EphA4-ligand or a nuclei acid encoding EphA4 or the EphA4-ligand and downregulates its activity. Combination therapies are also disclosed. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present description relates to therapeutic neuroprotective and neural regeneration molecules. In particular, the present specification relates to therapeutic molecules that improve the clinical progression of neurodegenerative diseases such as multiple sclerosis (MS) and variations of MS.

Bibliographic details of references in the subject specification are also listed at the end of the specification.
The nervous system, particularly the central nervous system, has a limited ability to regenerate after disease or injury, often with devastating consequences. The nervous system is an area of intense research and seems to be somewhat optimistic in the art now that mammalian models of spinal cord injury can repair at least some forms of acute spinal cord injury to some extent.
A number of different molecules, cells and mechanisms are involved in the neural regeneration process. These were reviewed by Horner and Gage, Nature. 407 (6807): 963-970, 2000. Of particular relevance here are the Eph and Ephrin receptor-ligand teams. Especially in humans, mice and chickens, four strains of Eph and ephrin have been identified. EphA receptors (whose members are currently 10) bind to group A ephrin ligands (6 members) which are GPI-linked membrane molecules, and EphB receptors (6 members) are ephrin B ligands that are transmembrane molecules Join (3 members). Binding is promiscuous, with most EphA receptors binding to most ephrin A ligands and most EphB receptors binding to most ephrin B ligands. Two exceptions are EphA4, which also binds to ephrin B2 and ephrin B3, and EphB2, which binds to ephrin A5.

  The intracellular domain of the transmembrane Eph receptor contains a kinase domain that is linked to the membrane with a proximate membrane domain. The carboxy side of the kinase domain includes a SAM domain and a carboxy terminal PDZ binding domain. The extracellular protein contains two fibronectin type III repeats, a cysteine rich region and an amino terminal ephrin binding domain. Ephrin B ligand has an extracellular Eph binding domain, an intracellular protein containing a short carboxy tail and a carboxy terminal PDZ binding domain. The EphB-Ephrin B complex has been crystallized and its structure can be used for identification based on the computational structure of the modulator of interaction. The high affinity binding site between the receptor and the ligand contains a 15 amino acid long loop, and the G-H loop in the Eph binding domain is inserted into the gap of the Eph receptor. The low affinity interaction site appears to facilitate inter alia binding between the two Eph-ephrin complexes. Receptor interactions and clustering result in activation of signaling pathways mediated by phosphorylation of residues in the cytoplasmic domain and promote receptor kinase activity. Bidirectional signaling occurs and cell-based ephrin binding to cell-bound Eph receptors can induce signaling at either ephrin or Eph receptors. Antagonists of Eph / ephrin interactions are generally designed to block high or low affinity interactions between members and thus receptor or ligand activation.

Eph was first studied in connection with embryonic development and found that Eph is an important axon guide molecule for several nervous systems. In these systems, the complementary expression gradient of Eph / ephrin molecules bound to specific cells effectively attracts or rejects axons that grow in the context of their proper target. Currently, Eph / ephrin interactions such as the soluble form of action are thought to be involved in various ways in the development, maintenance and repair of several different systems such as the nervous system, cell system and immune system (Wilkinson Nat Rev Neurosci. 2 (3): 155-164, 2001, Pasquale, Nat Rev Mol Cell Biol. 6 (6): 462-75, 2005 and Horner and Gage, 2000 (see above)). Many studies have shown downregulation of Eph and ephrin in neural tissue and reactive astrocytes after acute spinal cord injury or hemisection of the spinal cord. Re-expression of these receptors in adults was postulated to be important in regulating nerve regeneration after acute injury (Goldshmit et al., 2004 (supra), Goldshmit et al., Brain Res Brain Res Rev 52: 327-45, 2006). Blocking EphA4 on astrocytes with soluble ligands reduces any direct inhibitory effect of EphA4 on axonal regrowth, as shown in vitro (Goldshmit et al., 2004 (above)) .
Eph and ephrin polypeptides can be used to promote or block Eph receptor activation depending on their stoichiometry. Eph receptor activation requires ephrin-Fc ligand clustering or ephrin ligand membrane attachment; if not clustered, they block Eph receptor activation and downstream cellular responses (Goldshmit et al ., 2004 (above); Davis et al., Science 266: 816-9, 1994; Ohta et al.,. Mech Dev 64: 127-35, 1997; Stein et al., Genes Dev 12: 667-78, 1998).
Several studies have shown that soluble Eph-Fc ligands are effective in vivo, particularly in the context of tumor growth inhibition (Brantley et al., Oncogene 21: 7011-26, 2002; Cheng et al., Neoplasia 5: 445-56, 2003, Dobrzanski et al., Cancer Res 64: 910-9, 2004).

Despite considerable work done on the role of Eph receptors in the nervous system, some regulation of Eph / ephrin expression in CNS disease, especially MS and its model conditions, experimental autoimmune encephalomyelitis (EAE) Or do not know if there is a role.
MS is an inflammatory demyelinating neurodegenerative disease of the CNS. MS is the most common cause of neurological disability in young adults, leading to many years of permanent and severe neurological disability.
For many years, MS was primarily regarded as an inflammatory demyelinating disease characterized by axonal demyelination (with relative axonal protection) in local lesions within the CNS. Axon pathology has also been noted (Barnes et al., Brain 114: 1271-80, 1991), but it has only recently become apparent that there is considerable axonal damage and loss in MS lesions. (Trapp et al., N Engl J Med 338: 278-85, 1998; Trapp et al., Curr Opin Neurol 12: 295-302, 1999). It is now widely considered that axonal loss is responsible for the irreversible permanent disability observed in the secondary or chronic progression stages of MS (Bjartmar et al., J Neurol Sci 206: 165 -71, 2003). Histological analysis (Trapp et al., 1998 (supra); Ferguson et al., Brain 120: 393-9, 1997; Kornek et al., Am J Pathol 157: 267-76, 2000) and human MS patients Axonal loss is a disease as shown in the brain imaging (De Stefano et al., Brain 121 (Pt 8): 1469-77, 1998; Gonen et al., Neurology 54: 15-9, 2000). It is also clear that it occurs in the early relapse-remission phase of and is related to the degree of inflammation within the lesion. Furthermore, axonal loss is in the absence of myelin loss, in normal white matter (Bjartmar et al., Neurology 57: 1248-52, 2001), and in gray matter cortical lesions (Kidd et al., Brain 122 17-26, 1999; Peterson et al., Ann Neurol 50: 389-400, 2001). These and related findings, together with an analysis of the EAE model correlating the degree of axonal loss with clinical scores (Wujek et al., J Neuropathol Exp Neurol 61: 23-32, 2002), A hypothesis was derived that initiation was related to the tolerance limit for axonal integrity loss (Bjartmar et al, 2003 (above)). In the relapse-remission phase of MS, remyelination of demyelinating lesions occurs, but there is little evidence to suggest that any damaged axon can be regenerated. In this respect, the EAE's MOG model is a useful tool because it causes considerable axonal loss (> 40% in many areas) (Lo et al., J Neurophysiol 90: 3566-71, 2003; Wu et al Neuroimage 37: 1138-47, 2007).

In MS and EAE, one of the pathological features is the occurrence of glial scars in demyelinating plaques formed by astrocyte activation (Raine et al., Lab Invest 31: 369-80, 1974). Smith et al., Brain Res 264: 241-53, 1983). After CNS injury, astrocytes become activated and become hypertrophic and proliferative, increasing the expression of glial fibrillary acidic protein (GFAP). Astrocytes also give rise to various soluble factors and extracellular matrix molecules. EphA4 is also expressed on astrocytes in human MS tissue along with EphA7, A6, A3 and A1. However, it is not known whether this expression is simply the result of injury or evidence of regeneration or both (Sobel, Brain Pathol. 15 (1): 35-45, 2005).
Thus, the association of astrocyte gliosis with MS and EAE has long been accepted. However, it is unclear whether this gliosis plays a role in the clinical progression and pathology of MS and AEA. In fact, several studies suggest that gliosis has beneficial aspects in these conditions. For example, EAE in GFAP null mice has resulted in more severe clinical results (Liedtke et al., Am J Pathol 152: 251-9, 1998).
Thus, as a result of investigations on the molecular mechanisms and cellular events that characterize neurodegenerative diseases, complex molecular / cellular pathways that can be up-regulated or down-regulated and involved in causing or repairing neuronal damage Increased understanding. However, these studies have so far left little conclusions about future directions for treatment. There is a need for new treatments for neurodegenerative diseases. Neuroprotective compositions are sought for a wide variety of applications.

  Historically, mouse models of experimental autoimmune encephalomyelitis (EAE) have reproduced specific features of histopathology and neuropathology of multiple sclerosis (MS) and are putative modulators in MS It has been found to provide a useful indicator of the therapeutic effect.

Surprisingly, the clinical course of experimental autoimmune encephalomyelitis (EAE) was less severe in EphA4 knockout mice and reduced axonal damage in EAE EphA4 knockout mice compared to EAE controls Turned out to be.
As shown in FIGS. 1 and 2, EphA4-deficient mice showed significantly less limb weakness and axonal damage than control EAE mice. Therefore, by antagonizing EphA4 or EphA4 binding ligand (hereinafter “EphA4 ligand”), the clinical course of multiple sclerosis (MS) and neurodegenerative diseases (NDD) such as multiple sclerosis variants, and so on Propose to attenuate it if not.
Unless otherwise specified, each embodiment of this specification shall be modified where applicable and applied to any other embodiment.
In one embodiment, the present invention contemplates a method of treating a neurodegenerative disease (NDD) such as MS in a subject comprising administering to said subject an EphA4 antagonist or an EphA4 ligand antagonist. In certain embodiments, the antagonist reduces clinical progression of the disease.
In a related embodiment, the present invention contemplates an EphA4 antagonist or EphA4 ligand antagonist for use in the treatment of a neurodegenerative disease (NDD) such as MS in a subject. In certain embodiments, the antagonist reduces clinical progression of the disease.
In a similar embodiment, the present invention broadly provides for the use of an EphA4 antagonist or EphA4 ligand antagonist in the manufacture of a medicament for the treatment of NDD such as MS in a subject. In certain embodiments, the antagonist reduces clinical progression of the disease. “Manufacturing” encompasses drug selection and design.
In another similar embodiment, the present invention provides a pharmaceutical composition comprising an EphA4 antagonist or an EphA4 ligand antagonist for use in the treatment of NDD such as MS in a subject. In certain embodiments, the antagonist reduces clinical progression of the disease. For example, the antagonist may protect the function or integrity of the subject's neural tissue or reduce the rate, occurrence, or amount of nerve damage that occurs in the subject.

In yet another embodiment, the present invention is a method of treating NDD, such as MS, in a subject comprising the following steps:
i) screening a biological sample from said subject for axonal damage;
ii) administering an EphA4 antagonist or EphA4 ligand antagonist under conditions sufficient to protect the function or integrity of the subject's neural tissue or to reduce the rate, occurrence or amount of nerve damage that occurs in the subject A method comprising the steps is contemplated.
In the above embodiments, the subject may be at risk for NDD or may exhibit clinical signs of NDD.
In some embodiments, the NDD is MS or a variation of MS. References herein to “MS” include MS and variations of MS.
In some embodiments, the antagonist is administered for a time and under conditions sufficient to reduce the clinical progression of the disease. In some embodiments, the subject exhibits or may exhibit less nerve damage or less progressive neurological dysfunction. In some embodiments, the neurological dysfunction is limb weakness. In some embodiments, an effective amount of an antagonist is provided to alleviate the progressive neurological dysfunction.
In some embodiments, the antagonist protects the function or integrity of the subject's neural tissue or reduces the rate, occurrence, or amount of nerve damage that occurs in the subject.
The term “injury” includes nerve (axon) changes that indicate a loss of cellular integrity or loss of function. The term includes cell death, degeneration and fragmentation. In some embodiments, the term extends to reduced nerve conductivity or responsiveness or demyelination. Nerve damage can be assessed by any of the procedures widely accepted and described herein.
In some embodiments, the antagonist protects the function or integrity of the subject's neural tissue or undergoes the rate, occurrence, or amount of axonal damage that occurs in the subject during a chronic progression stage of NDD, such as MS. Decrease. In some embodiments, administration is at or before the occurrence of clinical symptoms or before or at the occurrence of recurrence of clinical symptoms.
In some embodiments, the antagonist is administered adjacent to or adjacent to neural tissue. In some embodiments, it is administered locally to the brain, spinal cord or optic nerve.
In some embodiments, the subject is a mammal such as a human.
In some embodiments, the antagonist is administered to provide about 1 mg / kg to 100 mg / kg systemically every day or every other day or every week, or a smaller amount for local delivery To do.

EphA4 antagonists or EphA4 ligand antagonists are well known in the art and are described in further detail in the “Commentary” and “Examples” section.
In some embodiments, the antagonist binds to EphA4 and downregulates its level or activity. In an exemplary embodiment, the antagonist is ephrin that binds to EphA4 and down-regulates its level or activity, including soluble forms or functional variants or analogs thereof. Suitable ephrins are ephrin A ephrins such as ephrin A1, A2, A3, A4, A5, A6, or ephrin B ephrins such as ephrin B2 or B3. In some embodiments, the ephrin is A4, A5, B2 or B3. Ephrin may be monomeric, dimeric, tetrameric or multimeric. In some embodiments, the soluble ephrin is an Fc or His fusion protein. In particular, Fc fusion is contemplated.
In another embodiment, the antagonist binds to an EphA4-binding ephrin molecule and inhibits its level or activity. In an exemplary embodiment, the antagonist is an EphA4 receptor, such as a soluble EphA4 receptor polypeptide or a functional variant or analog thereof. In some embodiments, the EphA4 is a monomer, dimer, tetramer or multimer. In some embodiments, the EphA4 receptor is an EphA4-Fc or His fusion protein. In particular, Fc fusion is contemplated.
In another embodiment, the antagonist is an antisense or inhibitory RNA molecule.
In some embodiments, the antagonist is a protein, polypeptide or peptide, such as, but not limited to, a fixed peptide or peptidomimetic. In other embodiments, the antagonist is (typically small or medium) organic molecule, such as, but not limited to, a molecule derived from a library of pharmaceutically acceptable small organic molecules.
In some embodiments, the antagonist is an antibody or antigen-binding fragment or aptamer of an antibody that binds to EphA4 or an EphA4 ligand.

The present invention targets EphA4 signaling to reduce clinical signs of MS or other NDD, such as nerve damage, and immunomodulators or immunosuppressants or anti-inflammatory agents for use in improving the immune or inflammatory components of the disease Alternatively, a combination therapy that also provides a procedure is provided.
Accordingly, the present invention contemplates a method of treating NDD, such as MS, comprising administering at least one other therapeutic agent or procedure together with an EphA4 antagonist or EphA4 ligand antagonist. In a similar embodiment, the present invention provides at least one other therapeutic agent or procedure for use in the treatment of NDD, such as MS, with an EphA4 antagonist or an EphA4 ligand antagonist. In another related embodiment, the invention provides an EphA4 antagonist or EphA4 ligand antagonist for use in the treatment of NDD, such as MS, administered with at least one other therapeutic agent or procedure. Similarly, the present invention provides at least one therapeutic agent or procedure for use in the treatment of NDD, such as MS, administered with an EphA4 antagonist or an EphA4 ligand antagonist. In some embodiments, the antagonist reduces the clinical progression of the disease. Reference to “both” includes sequential or simultaneous administration.
In another aspect, the invention provides an EphA4 antagonist or EphA4 ligand antagonist in the manufacture of a composition for protecting the function or integrity of a subject's neural tissue or for reducing the rate, occurrence, or amount of a subject's nerve damage. Provide drug use. In some embodiments, the subject has or is at risk of having a condition associated with nerve injury.
In a similar embodiment, a method for protecting the function or integrity of a subject's neural tissue or for reducing the rate, occurrence or amount of nerve damage in a subject, wherein said subject is an EphA4 antagonist or EphA4 ligand antagonist A method comprising the step of administering a drug is provided. In some embodiments, the subject has or is at risk of having a condition associated with nerve injury.
Similarly, an EphA4 antagonist or EphA4 ligand antagonist is provided that is used to protect the function or integrity of a subject's neural tissue or to reduce the rate, occurrence, or amount of nerve damage in a subject. In some embodiments, the subject has or is at risk of having a condition associated with nerve injury.
Similarly, a pharmaceutical composition comprising an EphA4 antagonist or an EphA4 ligand antagonist for use in protecting the function or integrity of a subject's neural tissue or for reducing the rate, occurrence, or amount of nerve damage in a subject is contemplated. In some embodiments, the subject has or is at risk of having a condition associated with nerve injury.

A pharmaceutical composition is prepared using a pharmaceutically acceptable carrier and / or diluent.
Also provided is a medical kit comprising an EphA4 antagonist or EphA4 ligand antagonist along with instructions for use in the treatment of NDD such as MS.
The present invention is a treatment protocol for treating NDD such as MS in a subject, which in turn is sufficient to reduce nerve damage, a step of screening a biological sample from said subject for axonal damage Further provided is a therapeutic protocol comprising administering an EphA4 antagonist or EphA4 ligand antagonist under the proposed time and conditions. In some embodiments, the subject is re-screened for axonal damage after treatment. In some embodiments, alternatively or in addition, a sample from the subject is tested to determine one or more conditions, such as autoimmunity, inflammation or demyelination. In some embodiments, the antagonist is administered prior to nerve injury to prevent nerve injury.
In another embodiment, the invention comprises screening any one or more Eph or ephrin modulators for the ability to reduce clinical progression of NDD, such as MS. In some embodiments, as demonstrated in the “Examples”, the agent is tested for the ability to protect the function or integrity of the subject's neural tissue or reduce the rate, occurrence, or amount of axon damage to the subject. To do.
In another embodiment, a screen for identifying a therapeutic agent for NDD, such as MS, comprising a screening agent for the ability to modulate the level or activity of a polypeptide having a functional activity of EphA4 or EphA4 ligand I will provide a.
The above summary is in no way an exclusive limitation of all embodiments of the invention, and should not be considered as such.
Some drawings include color representations or components. Color versions of the drawings are available on request from the patentee or from a valid patent office. A fee may be charged if obtained from the JPO.

A graphical representation showing that EphA4 knockout mice show a less severe clinical course of EAE, and the onset and severity of the disease were similar between genotypes at the very early stages of the disease, but the subsequent course was significant In contrast, the maximum clinical grade reached by EphA4 knockout mice was lower than the control (n = 10 each). FIG. 4 is a graphical representation of ELISA data showing that EAE EphA4 knockout mice show a trend for low levels of phospho-neurofilament-H suggesting that EAE axonal damage is lower than wild type mice in EAE. Bone marrow from grade 3 MOG-induced EAE C57BL / 6 mice immunostained for GFAP and EphA4 expression is a photographic representation showing the expression of EphA4 around the EAE lesion site, apparent in the absence of primary EphA4 antibody Indicates that there is no background. A photograph showing the expression of EphA4 around the EAE lesion site where bone marrow derived from C57BL / 6 mice of grade 3 MOG-induced EAE was prepared for GFAP and EphA4 expression (two astrocytes are represented by arrows, lesions Indicates that GFAP-expressing astrocytes derived from these mice also appeared. Fig. 3b is a photographic display similar to Fig. 3b, showing that EphA4 expression can be observed. Figure 3b and 3c merged and photographic representation plus DAPI to show nuclei and EphA4 on all EAE GFAP positive astrocytes as shown by red and green staining of the arrowhead site It expresses. FIG. 4 is a photographic representation showing that ephrinA5-Fc enters the CNS at the injury site after IP injection (mouse was treated with (A) PBS or (B) EphrinA5-Fc for 4 days). Photographic representation showing that monomeric ephrin A5-Fc blocks phosphorylation of EphA4 on astrocytes (under basic conditions or with IFNγ, monomer (blocking) or multimer (activation) It was shown that monomeric ephrin A5-Fc inhibited phosphorylation of EphA4 under both conditions. 1 is a schematic representation of the amino acid sequences of EphA4 and EphA4-binding ephrin Fc and His fusion proteins.

[Simple invention of table]
Table 1 provides a description of the SEQ ID NO provided herein.
Table 2 provides a subclassification of amino acids.
Table 3 provides typical amino acid substitutions.
Table 4 provides a list of unnatural amino acids contemplated by the present invention.
Table 5 provides a list of abbreviations.

[Detailed Discussion of Embodiment]
(Summary)
Abbreviations used in this specification are defined in Table 5.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The invention can be practiced or tested using any materials and methods similar or equivalent to those described herein. The practitioner is particularly familiar with definitions and terminology in the art and other methods known to those skilled in the art, especially Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, Coligan et al., Current Protocols In Protein Science, John Wiley & Sons, Inc., 1995-1997, especially Chapters 1, 5 and 6 and Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons , New York, 1999.
Throughout this specification, unless the context requires another meaning, variations such as “comprising” or “including” mean including the element or integer or group of elements or integers referred to. To any other element or integer or group of elements or integers.
The sequence identifier number (SEQ ID NO :) represents nucleotide and amino acid sequences. SEQ ID NO: numerically corresponds to the sequence identifiers <400> 1, <400> 2, etc. A summary of the sequence identifier is provided in Table 1. The sequence listing is provided after the claims.

In the present specification, the terms “polypeptide”, “protein” and “peptide” are used interchangeably.
As used herein, the singular form “1” (“a”, “an”) and “the” (“the”) includes plural aspects unless the context clearly dictates otherwise. To do. Thus, for example, “a cell” includes not only a single cell but also two or more cells; “a drug” includes not only one drug but also two or more drugs. Etc.
The terms “genetic material”, “genetic form”, “nucleic acid”, “nucleotide” and “polynucleotide” include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, sense strand and antisense strand. And, as will be readily appreciated by those skilled in the art, may be chemically or biochemically modified, or may include non-natural or derivatized nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more natural nucleotides with analogues (eg morpholine rings), internucleotide modifications, eg uncharged bonds (eg methylphosphonates, phosphotriesters, Phosphoamidates, carbamates, etc.), charged bonds (eg, phosphorothioates, phosphorodithioates, etc.), pendant components (eg, polypeptides), intercalating agents (eg, acridine, psoralen, etc.), chelating agents, alkylating agents, and modifications Examples of the binding include α-anomeric nucleic acid. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to specified sequences via hydrogen bonding and other chemical interactions. Such molecules are well known in the art and include, for example, molecules in which peptide bonds are replaced with phosphate bonds within the main chain of the molecule.
The term “gene” is used in its broadest sense and encompasses cDNAs that correspond to exons of a gene. References herein to “gene” are also to be understood to include: transcriptional and / or translational control sequences and / or coding regions and / or untranslated sequences (ie introns, 5′- and 3 ′ -A classical genomic gene consisting of (untranslated sequences); or the coding region of the gene (ie exon) and the mRNA or cDNA corresponding to the 5'- and 3'-untranslated sequences.
The expression “isolated” means a substance that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated polynucleotide” as used herein is a polynucleotide that is isolated from a sequence that naturally follows it, eg, a DNA fragment (usually adjacent to the fragment). Removed from the array). Alternatively, "isolated peptide" or "isolated polypeptide" as used herein refers to a peptide or polypeptide from its natural cellular environment and with other components of the cell. By in vitro isolation and / or purification from association. Without limitation, an isolated composition, complex, polynucleotide, peptide, or polypeptide may represent a native sequence that has been isolated by purification, or a sequence that has been produced by recombinant or synthetic means. May be expressed.
Citation of any reference herein shall not be construed as an admission that the reference is valid as “prior art” to the present application.
The subject invention is not limited to specific screening procedures for drugs. The specific formulation of the drug and the various medical methodologies can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Reference to “treatment” or “treating” is not only a therapeutic process whose purpose is to at least reduce the severity of clinical signs of a neurodegenerative disease or to delay or attenuate the clinical progression of the disease, as well as prevention. Means are also included. Although not wishing to be bound by theory, the experimental results described herein show that a reduction in EphA4 function may protect the function or integrity of neural tissue or reduce the rate, occurrence or amount of axonal damage Suggests that it contributes to clinical outcomes.

(Embodiment)
In one embodiment, the present invention contemplates a method of treating a neurodegenerative disease (NDD) such as MS in a subject comprising administering to said subject an EphA4 antagonist or an EphA4 ligand antagonist.
The term “antagonist” is used in a broad context to denote an agent, active agent, modulator, pharmaceutical agent, compound, pharmacologically active agent, drug, molecule, etc. that down regulates the level or activity of a target molecule. In this context, “activity” encompasses one or more biological functions of the target molecule. In some embodiments, the target is an Eph or ephrin polypeptide molecule. As is well known in the art, the activity of these molecules includes, inter alia, intracellular signaling activity, enzyme activity and binding activity that provide its functional ability. Signal transduction activity includes, for example, intracellular transduction of signals to elicit cellular responses mediated by expression, phosphorylation or binding or conformational changes of intracellular factors; enzyme activity includes kinases or Other catalytic activities are included; binding activities include low to moderate or high affinity binding to complementary Ephs, ephrins or other molecules that form complexes or bind to Ephs or ephrins . As is well known in the art, in some embodiments, binding is inhibitory, and in other embodiments, binding results in activation and receptor / ligand signaling. These functions are easily evaluated and quantified. The activity of functional domains such as paranuclear membrane domains, PDZ domains or SAM domains, GPI anchors, fibronectin repeats and cysteine-rich domains can also influence the regulation of Eph and ephrin functional activities. Methods for monitoring the level or activity of Eph / Ephrin or other ligands are well known in the art. For example, EphA4 receptor binding assays, such as Davis et al., 1994 (supra), Murai et al., Mol Cell Neurosci. 24 (4): 1000-1011, 2004 or PubChem Bioassay AID No. 689 ("EphA4 receptor Several antagonists can be identified using the assay described in Colorimetric assay for HTS discovery of chemical inhibitors of EphA4 receptor antagonists ”) . Suitable EphA4 antagonists and EphA4 ligand antagonists can inhibit the binding of EphA4 ligands to EphA4. Similarly, other antagonists can be identified using, for example, the EphA4 receptor phosphorylation assay described in Davis et al., 1994 (supra). Suitable EphA4 antagonists and EphA4 ligand antagonists can inhibit phosphorylation of EphA4 when exposed to EphA4 ligands. Several antagonists can also be identified using functional assays, such as the neural crest migration assay of Murai et al., 2004 (supra). Suitable EphA4 antagonists and EphA4 ligand antagonists can inhibit EphA4 function in vitro or in vivo. Several antagonists can also be identified using computational methods. For example, the crystal structure of the high affinity ephrin-binding channel of the EphA4 receptor can be seen from Qin et al., J Biol Chem. 283 (43): 29473-29484, 2008. This structure can be used for the theoretical design of suitable EphA4 antagonists and EphA4 ligand antagonists.

In some embodiments, co-suppression, antisense or repression, by reducing the level of transcription or translation, for example, by suppressing promoter or enhancer activity, by methylation, or now routine in the art. Gene silencing, such as sex RNA strategy, can be used to reduce the level or activity of the target polypeptide. Thus, the present invention may be used in cases where it is necessary to indirectly regulate the level of EphA4 or EphA4 ligand, eg, gene therapy, or suppression or promotion of gene function, or gene silencing constructs or antisense known to those of skill in the art Or the use of a nucleic acid molecule or a vector comprising a nucleic acid molecule by the use of inhibitory RNA oligonucleotides. Methods for regulating or monitoring gene function such as transcription or translation are well known in the art. For example, Fu et al., Nature Neuroscience 10: 67-76, 2007 discloses a method for regulating or monitoring the expression of EphA4.
The term “subject” as used herein refers to warm-blooded animals, particularly mammals, and more particularly primates, including lower primates, and even more particularly humans that can benefit from the medical uses of the present invention. To do. A subject, whether a human or non-human animal or embryo, may be referred to as an individual, subject, animal or patient. In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject is a subject in need of treatment.
Reference to modulation or reduction of the level or activity of a particular “Eph”, “Eph binding ligand” or “Ephrin” includes all polypeptide forms of these molecules, eg, homologous forms from other species, or References to naturally occurring forms are included. To refer to these molecules as antagonists, homologues and naturally occurring, isolated, synthetic, or recombinant forms, analogs, and parts thereof, eg retain activity Functional fragments or domains and functional variants are included.

In some embodiments, the subject is at risk for NDD or exhibits one or more clinical symptoms of NDD. The subject may be at risk or possibility of developing a genetically, clinically or physiologically demonstrable NDD, for example. For example, early clinical symptoms of MS include optic neuritis in a 15 to 50 year old subject.
Neurodegenerative diseases (NDD) include those of the CNS and / or PNS tissues and include the following: peripheral neuropathy, motor neuropathy, amyotrophic lateral sclerosis (ALS, Lou Gehrig) Disease), facial paralysis, Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's chorea, Down's syndrome, hypoxia-ischemic encephalopathy, incidental Lewy bodies disease, amyloid angiopathy , Traumatic spinal softening, and Meniere's disease. Peripheral neuropathy is a neurodegenerative disease that affects peripheral nerves and often manifests as one or a combination of motor, sensory, sensorimotor, or autonomous dysfunction. Peripheral neuropathy may be acquired, for example, may result from systemic disease, or may be induced by toxic factors, such as neurotoxins, such as antitumor drugs, or industrial or environmental pollutants. Peripheral sensory neuropathy is characterized by degeneration of peripheral sensory nerves, which may be idiopathic, for example, may occur as a result of diabetes (diabetic neuropathy), cancer cell division-inhibiting drug therapy Can occur as a result of alcoholism, acquired immune deficiency syndrome (AIDS), or genetic predisposition. Genetically acquired peripheral neuropathies include, for example, Refsum's disease, Krabbe's disease, Metachromatic leukodystrophy, Fabry's disease, Dejerine-Sottas Sottas syndrome, Abetalipoproteinemia, and Charcot-Marie-Tooth (CMT) disease (Proneal Muscular Atrophy) or hereditary motor and sensory neuropathy (HMSN) ), Also known as). Peripheral neuropathy usually acts on both sensory and motor nerves to cause mixed sensory and motor neuropathy, but pure sensory neuropathy and pure motor neuropathy are also known. In some embodiments, symptoms of NDD develop for months or years, in this case referred to as chronic or progressive.

In some embodiments, the neurodegenerative disease is associated with one or more of demyelination, inflammation and autoimmunity. Examples of diseases associated with demyelination, inflammation and autoimmunity are multiple sclerosis and its variants or Guillain-Barre syndrome (GBS) and its variants.
In some embodiments, the neurodegenerative disease is multiple sclerosis or a variant of MS. Examples of MS variants include Marburg's variant, Schilder's disease, Balo concentric sclerosis and Devic's disease. In an exemplary embodiment, the disease (condition) is multiple sclerosis.
In some embodiments, the antagonist is administered for a time and under conditions sufficient to reduce the clinical progression of the disease. In some embodiments, the subject exhibits or may exhibit less nerve damage or less progressive neurological dysfunction. In some embodiments, the neurological dysfunction is limb weakness. In some embodiments, an amount of antagonist is provided that is effective to reduce progressive neurological dysfunction.
In some embodiments, the antagonist protects the function or integrity of the subject's neural tissue or reduces the rate, occurrence, or amount of nerve damage that occurs in the subject.
In some embodiments, the antagonist is provided in an “effective amount” sufficient to alleviate or delay the progression of clinical symptoms of NDD, such as MS.
An “effective amount” in the context of treating NDD is active for a subject found to be effective in a single dose or as part of a continuous or delayed release system in showing therapeutic effects in some subjects Means the amount of administration. The effective amount will vary depending on the health and physical condition of the subject, the formulation of the composition used, the assessment of the medical situation, and other relevant factors. The amount is expected to fall within a relatively broad range that can be determined through routine trials.
A pharmaceutical composition comprising the subject antagonist is considered to exhibit therapeutic activity when administered in an amount that depends on the individual circumstances. Variations in the amount will depend, for example, on the human or animal and on the drug selected. An antagonist is typically an agent that is bioavailable at a concentration sufficient to produce an effect. A wide range of doses are applicable. Considering the subject, for example, about 0.1 mg to 0.9 mg per kg body weight every day or every other day or every week or every month (i.e. 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, Including about 0.6 mg, 0.7 mg, 0.8 mg, and 0.9 mg), about 15 mg to 35 mg, about 1 mg to 30 mg, or 5 to 50 mg, or 10 mg to 100 mg. The therapeutic antibody is typically administered at a dose of about 1-20 mg / kg, although doses above or below this amount are contemplated within the above ranges. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or at any other suitable time interval, or the dose may be reduced proportionally as indicated by the requirements of the situation.
In some embodiments, the antagonist is given about 1 mg to 100 mg / kg systemically daily or weekly, or in smaller amounts for local delivery.
It is usually administered for a time and under conditions sufficient to mitigate or delay the progression of clinical signs of NDD such as MS. Clinical signs depend on the neurodegenerative disease to be treated, but include signs of reduced cognitive, sensory or motor skills. Common clinical signs include optic neuritis, transverse myelitis, brainstem or cerebellar defects.

Administration of the drug by convenient methods, such as oral, intravenous (if water soluble), intraperitoneal, intramuscular, subcutaneous, intraperitoneal, intrathecal or suppository route or implantation (e.g., using delayed release molecules) can do. Systemic or local administration may be used, but systemic administration is more convenient. Systemic reference includes intravenous, intraperitoneal, subcutaneous injection, infusion and administration by oral, rectal and nasal routes or by advantageous inhalation. Other possible routes of administration are by patch, cell transfer, implantation, sublingual, intraocular, topical or transdermal. Depending on the severity or stage of the disease and the integrity of the blood brain barrier, appropriate antagonists need to cross the blood brain barrier. In some embodiments, the agent is administered directly to the PNS or CNS, brainstem, or cerebellum.
The pharmaceutical composition is conveniently prepared according to conventional pharmaceutical compounding methods. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Company, Easton, PA, USA, 1990. The composition may contain an active agent or a pharmaceutically acceptable salt of the active agent. These compositions may contain, in addition to one active substance, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other substances well known in the art. The material should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, eg, intravenous, oral or parenteral.
"Pharmaceutically acceptable carrier" and / or diluent is a pharmaceutical vehicle comprised of a substance that is otherwise undesirable, i.e., unlikely to cause substantial side reactions with the active agent by itself. It is. As a carrier, all solvents, dispersion media, coating agents, antibacterial agents, antifungal agents, tonicity adjusting agents, agents that increase or decrease absorption rate or purification rate, buffers to maintain pH, chelating agents, membranes Or a barrier crossing agent. Pharmaceutically acceptable salts are otherwise undesirable salts. The drug or composition comprising the drug can be administered in the form of a pharmaceutically acceptable non-toxic salt, such as an acid addition salt or a metal complex.

For oral administration, the compounds can be prepared into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. When preparing an oral dosage form composition, for example, in the case of oral liquid preparations (eg, suspensions, elixirs and liquids), water, glycol, oil, alcohol, flavoring agent, preservative, coloring Conventional pharmaceutical media such as agents, suspensions; or in the case of oral solid preparations (eg powders, capsules and tablets) starch, sugar, diluents, granulating agents, lubricants, binders, disintegrants Any carrier such as can be used. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage form, in which case solid pharmaceutical carriers are obviously employed. Tablets may contain binders such as gum tragacanth, corn starch or gelatin; disintegrants such as alginic acid; and lubricants such as magnesium stearate. If desired, tablets may be sugar coated or enteric coated by standard techniques. The active agent can be encapsulated and stably passed through the gastrointestinal tract. See, for example, International Patent Application No. WO96 / 11698. For parenteral administration, the compound can be dissolved in a pharmaceutical carrier and administered as a solution or suspension. Examples of suitable carriers are water, saline, dextrose solution, fructose solution, ethanol, or oil of animal, vegetable or synthetic origin. The carrier may contain other ingredients such as preservatives, suspending agents, solubilizers, buffers and the like.
For parenteral administration, the antagonist can be dissolved in a carrier and administered as a solution or suspension. When the drug is administered intrathecally, the drug may be dissolved in cerebrospinal fluid. For transmucosal or transdermal delivery (including patch delivery), suitable penetrants known in the art are used to deliver the antagonist. For inhalation, delivery is accomplished using any convenient system such as a dry powder aerosol, liquid delivery system, air jet nebulizer, propellant system, and the like. For example, the formulation can be administered in the form of an aerosol or mist. The drug may be delivered in a sustained or sustained release manner. For example, biodegradable microspheres or capsules or other polymeric structures capable of sustained delivery may be included in the formulation. The formulation can be modified to alter pharmacokinetics and biodistribution. For a general discussion of pharmacokinetics, see, for example, Remington's. In some embodiments, the formulation may be incorporated into a lipid monolayer or bilayer such as a liposome or micelle.

Targeted therapies well known in the art can be used to deliver the antagonist, and more particularly, to certain types of cells, such as neurons, oligodendrocytes, astrocytes. Targeting methods may be useful if the antagonist is unacceptably toxic, or if the dosage is otherwise too high or not appropriate for other sites. Apart from this, targeting methods can help cross the blood brain barrier. Several strategies are known in the art to increase the accessibility of the nervous system to administered antagonists (Misra et al., J Pharm Sci 6: 252-273, 2003).
In some embodiments, the antagonist is administered to or adjacent to the neural tissue. In some embodiments, it is administered locally to the brain, spinal cord or optic nerve.
The actual amount of active agent administered and the rate and time course of administration will depend on the nature and severity of the disease. Determination of treatment prescriptions, such as dosage, timing, etc., is within the responsibility of a general practitioner or expert, typically individual patient status, delivery site, method of administration and other well known to the practitioner Consider factors. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences (supra).
Sustained release preparations that can be prepared are particularly convenient because of the long-term administration of appropriately stable antagonists. Examples of sustained release formulations include semi-permeable substrates of solid hydrophobic polymers containing antagonists, which are in the form of molded articles such as films or microcapsules. Examples of sustained-release substrates include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene -Vinyl acetate, degradable lactic acid-glycolic acid copolymer, and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules over 100 days, certain hydrogels release proteins in a shorter time. Liposomes that are small (about 200-800 cm) monolayer type and with a lipid content of more than about 30% cholesterol can be used, and the proportion of cholesterol is selected for optimal therapy.
Stabilize antagonists by modifying sulfhydryl residues, lyophilizing from acidic solutions, adjusting moisture content, using appropriate additives and developing unique polymer matrix compositions Can be achieved. For example, the in vivo half-life of the antagonist can be increased using techniques well known in the art, such as by attaching other elements such as polyethylene glycol (PEG) groups.

The present invention further targets combination therapy, e.g. targeting EphA4 signaling to reduce clinical signs of NDD such as MS, e.g. nerve damage and immune regulation or immunosuppression or Therapies that also provide inflammatory drugs or procedures are provided.
The present invention further provides immunomodulation or immunosuppression or combination therapy, such as targeting EphA4 signaling to reduce MS or other clinical signs of NDD, such as nerve damage, and to improve the immune or inflammatory component of the disease. Therapies that also provide anti-inflammatory drugs or procedures are provided. Immunomodulators used in the art include β-interferon, glatiramer acetate and natalizumab, which are considered in combination therapy.
Accordingly, the present invention contemplates a method of treating NDD, such as MS, comprising administering an EphA4 antagonist or EphA4 ligand antagonist together with at least one other therapeutic agent or procedure. In a similar embodiment, the present invention provides at least one other therapeutic agent or procedure together with an EphA4 antagonist or EphA4 ligand antagonist for use in the treatment of NDD such as MS. In another related embodiment, the invention provides an EphA4 antagonist or EphA4 ligand antagonist for use in the treatment of NDD, such as MS, administered with at least one other therapeutic agent or procedure. Similarly, the present invention provides at least one therapeutic agent or procedure for use in the treatment of NDD, such as MS, administered with an EphA4 antagonist or an EphA4 ligand antagonist. In some embodiments, the antagonist reduces the clinical progression of the disease. Reference to “both” includes sequential or simultaneous administration.
In some embodiments, the antagonist is administered in combination with an anti-inflammatory drug and other active compounds currently used for the treatment of the target disease. The compounds include immunomodulators such as glatiramer acetate and natalizumab, corticosteroids such as prednisolone, nonsteroidal anti-inflammatory drugs such as aspirin, ibuprofen, and COX-2 inhibitors, disease modifying antirheumatic drugs such as methotrexate, Leflunomide, sulfasalazine, azathioprine, cyclosporine, hydroxychloroquine, and D-penicillamine; and biological response modifiers such as cytokines (eg, IFNβ) or cytokine inhibitors.

The term “neural injury” or “axon (neuron) injury” includes cell death (apoptosis), cell degeneration, cell fragmentation, and a decrease in nerve conductivity or responsiveness. In some embodiments, neuronal damage occurs in acute or chronic demyelinating lesions, but damage can also occur in myelinated axons. In certain embodiments, markers of axonal damage can be associated with clinical progression. In particular, a low level of axonal damage compared to controls suggests that the subject antagonists can sufficiently protect the axons from damage to reduce clinical progression in NDD such as MS.
Neurological dysfunction is CNS or PNS dysfunction. In some embodiments, the neurological dysfunction is spinal cord function, cognitive motor or sensory function, or optic nerve function dysfunction associated with clinical progression of NDD, such as MS.
In some embodiments, the neurological dysfunction is limb weakness. In some embodiments, an amount of antagonist is provided that is effective to reduce progressive neurological dysfunction.
In some embodiments, the antagonist protects the function or integrity of the subject's neural tissue or reduces the rate, occurrence, or amount of nerve damage that occurs in the subject.
The term “injury” includes nerve (axon) changes that indicate a loss of cellular integrity or loss of function. The term includes cell death, degeneration and fragmentation. In some embodiments, the term extends to reduced nerve conductivity or responsiveness or demyelination.
In some embodiments, the antagonist protects the function or integrity of the subject's neural tissue during the chronic progression stage of NDD, such as MS, or reduces the rate, occurrence, or amount of axonal damage that occurs in the subject. Decrease. In some embodiments, administration is at or before the occurrence of clinical symptoms or before or at the occurrence of recurrence of clinical symptoms.

In some embodiments, the invention is a method of treating NDD, such as MS, in a subject comprising the following steps:
i) screening a biological sample from said subject for axonal damage;
ii) administering an EphA4 antagonist or EphA4 ligand antagonist under conditions sufficient to protect the function or integrity of the subject's neural tissue or to reduce the rate, occurrence or amount of nerve damage that occurs in the subject A method comprising the steps is contemplated.
In some embodiments, the antagonist is administered to or adjacent to neural tissue. Neural tissue may be in the brain, CNS, or spinal cord, but is not limited thereto. In some embodiments, it is administered locally to the brain, spinal cord or optic nerve.
In some embodiments, the subject is a mammal such as a human.
In some embodiments, the antagonist binds to EphA4 and down-regulates its level or activity. In an exemplary embodiment, the antagonist is ephrin that binds to EphA4 and down-regulates its level or activity, including soluble forms or functional variants or analogs thereof. Soluble ephrin, an Eph receptor antagonist, is well known in the art and is described, for example, in Gale et al., Neuron. 17 (1): 9-19, 1996 and Davis et al., 1994 (above). . These ephrins typically do not contain any membrane anchors and / or transmembrane domains and therefore have a soluble form in that they do not bind to the membrane. Other forms may be soluble in that they are bioavailable at concentrations sufficient to produce an effect. These forms include, for example, forms that have a membrane anchor or transmembrane domain but lack signal transduction capabilities. These forms include micelle or protein liposome forms. Suitable ephrins for use in the present invention are ephrin A ephrins such as A1, A2, A3, A4, A5, A6, or ephrin B ephrins such as ephrin B2 or B3. In some embodiments, the ephrin is A4, A5, B2 or B3. Ephrin may be monomeric, dimeric, tetrameric or multimeric. In some embodiments, the soluble ephrin is an Fc or His fusion protein. In particular, Fc fusion is contemplated.

In other embodiments, the antagonist binds to the EphA4-binding ephrin molecule and inhibits its level or activity. In an exemplary embodiment, the antagonist is an Eph receptor, such as a soluble Eph receptor polypeptide or a functional variant or analog thereof. Again, soluble Eph receptors are well known in the art, for example as described in Brantley et al., 2002 (supra). These Eph receptors typically have a soluble form in that they do not bind to the membrane because they do not contain any membrane anchors and / or transmembrane domains. Other forms may be soluble in that they are bioavailable at concentrations sufficient to produce an effect. These forms include, for example, forms that have a membrane anchor or transmembrane domain but lack signal transduction capabilities. These forms include micelle or protein liposome forms. Typically, the antagonist is an EphA4 receptor, such as a soluble EphA4 receptor polypeptide or a functional variant or analog thereof. In some embodiments, EphA4 is a monomer, dimer, tetramer or multimer. In some embodiments, the EphA4 receptor is an EphA4-Fc or His fusion protein. In particular, Fc fusion is contemplated.
In some embodiments, the antagonist is a protein, polypeptide or peptide, such as but not limited to a structure-fixing peptide or peptidomimetic. Various peptide antagonists are well known in the art. These and other suitable peptide antagonists can be used in the present invention. For example, the antagonists described in Murai et al., 2004 (supra) may be used. Methods for identifying peptide antagonists are disclosed, for example, in Koolpe et al., J Biol Chem., 280 (17): 17301-17311, 2005. In other embodiments, antagonists are (typically small or medium) organic molecules, such as, but not limited to, molecules derived from libraries of pharmaceutically acceptable small organic molecules. Various organic molecule antagonists are well known in the art. Reference can be made to Noberini et al., J Biol Chem., 283 (43): 29461-29472, 2008, which discloses small molecule antagonists. Kolb et al., Proteins, 73 (1): 11-18, 2008; Chrencik et al., Structure, 14 (2): 321-330, which also discloses methods for selecting and purifying small molecule antagonists. See also 2006; and Chrencik et al., J Biol Chem., 282 (50): 36505-36513, 2007. These and other suitable organic molecule antagonists may be used in the present invention. For example, the antagonists described in Qin et al. 2008 (supra) may be used.

Isolated recombinant or synthetic polypeptides are prepared from genetically engineered host cells, including, for example, expression systems, by methods well known in the art. The practitioner will refer in particular to Sambrook et al., 1989 (above) and Ausubel et al., 1999 (above) for definitions and terms in the art and other methods known to those skilled in the art.
The nucleotide and amino acid sequences of EphA4 and EphA4 binding ephrins are disclosed under appropriate accession numbers in electronically accessible databases. The appropriate nucleotide sequence is inserted into the expression system by any of a variety of well-understood routine procedures, for example as shown in Sambrook et al., 1989 (supra) and described in the Examples. And can be expressed in a suitable host cell. Polypeptides can be recovered and purified from recombinant cultures or other biological sources, generally by routine methods including chromatography or filtration.
DNA encoding EphA4 or ephrin polypeptide can be recombinantly expressed as a fusion protein with the Fc region of human IgG heavy chain. For example, soluble forms of ephrin A4, A5 or ephrin B3 are prepared by recombinantly expressing ephrin A5-Fc, ephrin A3-Fc or ephrin B3-Fc fusion protein. The extracellular domain gives a soluble form of the protein, but does not contain any part of the domain (e.g. amino acids 20-546 of human EphA4) including the ligand binding domain (amino acids 20-274) or functional fragments or variants. Consider. Once expressed, the signal peptide may be cleaved and no signal peptide is shown in FIG. The mouse and human homologues of EphA4 shared 98.2% amino acid sequence identity (536/546). The Fc portion of the fusion protein facilitates the purification of the protein using chromatography and enhances the function, stability, half-life, structure or solubility of the fusion partner. Mouse Fc from IgG1 lacks complement and effector functions, whereas human homologues have these functions that can interfere with action or activity. Thus, different IgG isoforms that modify Fc or do not interfere with effector cells may be utilized. In an exemplary embodiment, an IgG4 isoform is utilized. Eph or ephrin may be used in the form of a monomer, dimer, tetramer or multimer as required.
In some embodiments, if necessary, nucleic acids and viruses or other vectors containing nucleic acids are used to deliver or induce silencing of EphA4 or EphA4 ligand gene antagonists. DNA (gDNA, cDNA), RNA (sense RNA, antisense RNA, mRNA, tRNA, rRNA, small interfering RNA (SiRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), piwi interaction Nucleic acids such as piwi-interacting RNA (PiRNA), microRNA (miRNA), small nuclear RNA (SnoRNA), small nuclear (SnRNAs) ribozymes, aptamers, DNAzymes or other ribonuclease-type complexes Methods for generating chimeric or fusion constructs capable of producing DNA or RNA in eukaryotes are disclosed in the art.

In some embodiments, the antagonist is an antibody or antigen-binding fragment or aptamer of an antibody that binds to EphA4 or EphA4 ligand.
As used herein, the term “antibody” refers to a whole antibody that exhibits the desired binding and blocking functions, or fragments thereof, such as F (ab ′) 2, Fab, Fab ′, Fv, VH or VK fragments, single fragments. Including single chain antibodies, multimeric monospecific antibodies or fragments thereof, or bispecific or multispecific antibodies or antigen binding fragments thereof. Reference to binding means that the fragment binds to the antigen with sufficient affinity and specificity to be therapeutically useful. In addition, antibodies or fragments thereof are selected for their ability to bind and block target activities such as intracellular signaling. The antibody used herein may be a polyclonal or monoclonal antibody. The antibody may belong to any immunoglobulin class, for example IgG, eg IgG1, IgG2, IgG3, IgG4, IgE, IgM or IgA antibody. The antibody may be of animal, eg mammalian origin, for example a mouse, rat or human antibody. Alternatively, the antibody may be a chimeric antibody. As used herein, the term chimeric antibody is used to mean any antibody that contains portions from different animal species. A specific non-limiting example is the antibody having a mouse-derived variable region and a human-derived antibody constant region. Antibodies in which one or more CDR sequences are derived from a mouse and the remaining variable and constant regions are derived from human immunoglobulins are generally described as “humanized” antibodies. An antigen-binding fragment of an antibody includes all or part or portions of a variable region sufficient to exhibit the desired binding to EphA4 or EphA4 ligand.

Methods for preparing monoclonal and polyclonal antibodies are well known to those skilled in the art and are described, for example, in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, Chapters 5 and 6. For example, the antibodies of the present invention can be prepared by conventional immunization and recombinant DNA techniques. Thus, for example, polyclonal antibodies can be obtained from the sera of animals immunized with the target protein or fragment thereof. If it is desired to obtain a mouse polyclonal antibody, the immunogen can be injected into any appropriate host, such as BALB / c mice, the serum collected, and the antibody recovered from the serum. Monoclonal antibodies are obtained from hybridomas derived from spleen cells of animals immunized in this way and fused to appropriate “immortal” B-tumor cells. In each case from serum or hybridomas using standard purification and / or enrichment techniques, eg, by chromatography, with protein A, or by other affinity chromatography using the protein of the invention or fragments thereof. Antibodies can be recovered.
Once a cell line expressing an appropriate antibody, such as a hybridoma, is obtained, a variable region gene encoding the desired antibody, including sequences encoding CDRs, can be identified. From here, at least one DNA sequence encoding the variable domain of the heavy or light chain of the antibody, and optionally one or more other DNA sequences encoding the remainder of the heavy and / or light chain Obtaining a chimeric antibody by preparing a replicable expression vector and transforming an appropriate cell line in which production of the antibody will occur, for example, a non-producing myeloma cell line such as the mouse NSO line it can. Certain methods of producing antibodies in this way are generally well known and routinely used. Antibody production includes not only stimulating immune responses by injection into animals, but also synthetic antibody production, screening of recombinant immunoglobulin libraries for specific binding molecules or in vitro stimulation of lymphocyte populations Processes are also included.

Antibodies are particularly useful drugs when the target has a binding site accessible from outside the cell. Thus, the drug does not need to cross the cell membrane or completely traverse the cell membrane. Antibodies specific for EphA4 or ephrin polypeptides can be used directly, for example, as antagonists. Antibodies can be generated using methods well known in the art, including, for example, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments, and fragments produced from Fab expression libraries. .
In some embodiments, the antibodies of the invention are CDR grafted antibodies. As used herein, the term “CDR grafted antibody” refers to a heavy and / or light chain grafted into the framework of the heavy and / or light chain variable region of an acceptor antibody (eg, a human antibody). Means an antibody molecule comprising one or more CDRs from a donor antibody (eg, mouse monoclonal antibody). The construction of CDR grafted antibodies is fully described in European patent application EP-A-0239400, which is hereby incorporated by reference. Some criteria for selecting which framework residues need to be changed are described in International Patent Application WO 90/07861, which is incorporated herein by reference.
Antigen binding agents capable of intracellular transmission, or functionally active fragments thereof, include camel and llama antibodies, scFv antibodies, intracellular antibodies or nanobodies, such as antibodies such as scFv intracellular antibodies and _VHH intracellular antibodies. It is done. Harmsen and De Haard, Appl. Microbiol. Biotechnol. 77 (1): 13-22, 2007; Tibary et al., Soc. Reprod. Fertil. Suppl. 64: 297-313, 2007; Muyldermans, J. Biotechnol. 74 : 277-302, 2001; and the literature cited therein, such antigen binding agents can be made. In one embodiment, scFv intracellular antibodies that can interfere with protein-protein interactions are used in the present invention. For the production method, see, for example, Visintin et al., J. Biotechnol, 135: 1-15, 2008 and Visintin et al, J. Immunol. Methods, 290 (1-2): 135-53, 2008. I want.

For use in the present invention, the modulator is a cell penetrating peptide sequence or a nuclear localized peptide sequence such as the sequence disclosed in Constantini et al., Cancer Biotherm. Radiopharm. 23 (1): 3-24, 2008. May be included.
In some embodiments, the agent that down-regulates EphA4 receptor formation, expression or activity is derived from an EphA4 polypeptide or coding sequence thereof, or a variant or analog thereof. Thus, for example, the drug may be a carbohydrate-structured peptide or a microprotein that is α-resin, cell permeable, and capable of interfering with protein-protein interactions (see, eg, Wilder et al., ChemMedChem. 2 (8): 1149-1151, 2007: see Henchey et al., Curr Opin Chem Biol. 12 (6): 692-697, 2008) for review.
In some embodiments, published nucleotide sequences of EphA4 or binding ligands, ephrins, such as ephrin A or B ephrin; ephrin A4, A5, B2 or B3 or other EphA4 ligand genes or related variants or variants thereof A drug is derived from a nucleic acid molecule having Variants are sufficient so that selective hybridization is achieved under conditions of moderate or high stringency with naturally occurring forms of these molecules or their complementary forms over all or part thereof. A nucleic acid molecule that is similar or has about 60% to 90% or 90% to 98% sequence identity with a nucleotide sequence that defines a natural antagonist polypeptide sequence over a comparison window comprising at least about 15 nucleotides Is included. Preferably, the hybridization region is about 12 to about 18 or more nucleobases in length. Preferably, the percent identity between a particular nucleotide sequence and a reference sequence is at least about 80%, or 85%, more preferably about 90% or greater similarity, such as about 95%, 96%, 97%, 98 %, 99% or more. A percent identity of 80% to 100% is included. The length of the nucleotide sequence depends on its proposed function. For example, small interfering RNAs are typically about 20-24 nucleotides in length, and molecules designed to provide dominant negative function require full-length or substantially full-length molecules There is. As already mentioned, homologues are also included. The term “homolog” (s) broadly refers to molecules that are functionally and structurally related, including those from other tumors. Homologues and orthologs are examples of mutants.

As used herein, the expression “hybridization” is used to denote pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or DNA-RNA hybrid. Complementary base sequences are those sequences that are related by base pairing rules. In DNA, A pairs with T and C pairs with G. In RNA, U pairs with A and C pairs with G. In this regard, the terms “match” and “mismatch” as used herein refer to the potential for hybridization of paired nucleotides in complementary nucleic acid strands. Matched nucleotides such as the classical AT and GC base pairs described above hybridize efficiently. Mismatches are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing involves hydrogen bonding between complementary nucleosides or nucleotide bases (nucleobase) of the oligomeric compound strand. This hydrogen bond may be a Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bond. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. As is well known to those skilled in the art, hybridization can occur in a variable environment.
As used herein, the expression “stringency” refers to the temperature and ionic strength conditions during hybridization and the presence or absence of certain organic solvents. The higher the stringency, the higher the degree of complementarity observed between sequences.
As used herein, “stringent conditions” or “moderate or high stringency conditions” hybridize only to polynucleotides having a high percentage of complementary bases, preferably perfect complementarity. Represents temperature and ion conditions. The required stringency is dependent on the nucleotide sequence and depends on the various components present during hybridization and varies greatly with the use of nucleotide analogs. Generally, stringent conditions are selected to be about 10-20 ° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) when 50% of the target sequence hybridizes with the complementary probe.

  Of course, the polynucleotide hybridizes to the target sequence under at least low stringency conditions, preferably at least moderate stringency conditions, and more preferably under high stringency conditions. Reference herein to low stringency conditions refers to at least about 1% v / v to at least about 15% v / v formamide and at least about 1M to at least about 2M for hybridization at 42 ° C. Salts, as well as at least about 1 M to at least about 2 M salts for washing at 42 ° C. Low stringency conditions include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65 ° C., as well as for washing at room temperature ( It may also include i) 2x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. Moderate stringency conditions include at least about 16% v / v to at least about 30% v / v formamide and at least about 0.5M to at least about 0.9M salt for hybridization at 42 ° C, and 42 ° C. At least about 0.5M to at least about 0.9M salt. Moderate stringency conditions include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65 ° C, and 42 ° C for washing. (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS may also be included. High stringency conditions include at least about 31% v / v to at least about 50% v / v formamide and at least about 0.01M to at least about 0.15M salt for hybridization at 42 ° C, and 42 ° C. Includes at least about 0.01M to at least about 0.15M salt for washing. High stringency conditions include 1% BSA for hybridization at 65 ° C, 1 mM EDTA, 0.5M NaHPO4 (pH 7.2), 7% SDS, and (i) 0.2 for washing at temperatures above 65 ° C. xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS may also be included. Other stringent conditions are well known in the art. One skilled in the art will recognize that various factors can be manipulated to optimize the specificity of hybridization. Optimization of the final wash stringency can help to ensure a high degree of hybridization. For detailed examples, see pages 2.10.1 to 2.10.16 of Ausubel et al. (Supra) and sections 1.101 to 1.104 of Sambrook et al. (1989) (supra).

  The terms used to describe the sequence relationship between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence identity”, “percent sequence identity” and “substantially Identity ”. A “reference sequence” is at least 12, frequently 15-18, and often at least 25 monomeric units, the monomeric unit being a length including nucleotides and amino acid residues. Each of the two polynucleotides can contain (1) a similar sequence between the two polynucleotides (ie, only part of the complete polynucleotide sequence), and (2) a sequence that differs between the two sequences, so that two ( Sequence comparison between (or more than two) polynucleotides is typically done by comparing the sequences of two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity . A “comparison window” compares at least 6, usually about 50 to about 100, more usually about 100 to 150 flanks, after optimally aligning the two sequences, comparing the sequence with a reference sequence at the same number of flanking positions. Means a conceptual segment of position. The comparison window may contain about 20% or less additions or deletions (ie, gaps) relative to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. By computer implementation of the algorithm (GAS, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or in any of a variety of selected methods The best alignment created (ie, yielding the highest percentage of homology over the comparison window) can lead to an optimal alignment of the sequences to align the comparison windows. Reference may also be made, for example, to the BLAST family of programs disclosed by Altschul et al., Nucleic Acids Research 25: 3389-3402, 1997. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, Chapter 15, 1994-1998.

In some embodiments, the present invention contemplates the use of full-length polypeptides or one or more biologically active portions or peptides of these molecules, such as structure-fixing peptides, as antagonists. A biologically active moiety or peptide includes one or more binding domains that contribute to the activity of the target antagonist, eg, a ligand or receptor binding domain. In some embodiments, the peptide antagonist blocks the GH loop and prevents high or moderate affinity binding between the eph and ephrin binding domains. Another binding domain is shown in FIG. A biologically active portion or peptide of a full-length polypeptide, such as a structure-fixing peptide is for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 350, 400, 450, 500, 550, 600 ~ It may be a polypeptide or peptide that is about 640 or about 700, 800, 900, 1000, 1200, or more amino acid residues long. Typically, the ligand / receptor binding moiety is about 150-300 amino acid residues. An example is shown in FIG.
The amino acid sequences of exemplary antagonists are presented in SEQ ID NOs: 2-10 as shown in Table 1.

As a “mutant” antagonist polypeptide, a soluble EphA4, ephrin A or ephrin B, eg from ephrin A4, A5, B2 or B3 to the N-terminus and / or C-terminus of the native protein A deletion (cut) or addition of one or more amino acids; a deletion or addition of one or more amino acids at one or more sites of the natural protein; or one or more amino acids at one or more sites of the natural protein And proteins derived by substitution of. Variant proteins encompassed by the present invention are biologically active, i.e. they continue to be inhibitors of EphA4 or ephrin-mediated signaling. Variants include soluble forms lacking membrane anchors and / or transmembrane domains. Antagonist variants are selected on the basis that they suppress or antagonize the biological activity of EphA4 or its cell-bound ligand. Such mutants can arise, for example, from genetic polymorphisms or by artificial manipulation. A biologically active variant of a natural polypeptide is at least 40%, 50%, 60%, 70% common with the amino acid sequence of the natural protein as determined by modern sequence alignment programs using default parameters. Typically at least 75%, 80%, 85%, preferably about 90% to 95% or more, more preferably about 98% or more. Biologically active variants of an antagonist polypeptide generally have as few as 100, 50 or 20 amino acid residues, preferably as few as 1-15 amino acid residues, 1-10. As many as, for example, 6-10, as many as 5, 4, 3, 2 or even 1 amino acid residue may differ from the polypeptide.
In one example, a functionally active splice variant of ephrin-A4 is disclosed in Aasheim et al., Blood, 95 (1): 221-230, 2000. The splice variant lacks 146 nucleotides at the 3 ′ end of the open reading frame of the first part of exon IV. As a result, the carboxy terminus is changed and there is no GPI-signal sequence. COS cells transfected with cDNA produced soluble ephrin-A4.
EphA4 or EphA4-binding ephrin signaling antagonist polypeptides or peptides can be altered in various ways, such as amino acid substitutions, deletions, truncations, and insertions. Such operating methods are generally known in the art. For example, amino acid sequence variants of ephrin B or ephrin A polypeptides can be prepared by introducing mutations into the encoded DNA. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. For example, Kunkel, Proc. Natl. Acad. Sci. USA, 82: 488-492, 1985; Kunkel et al., Methods in Enzymol., 154: 367-382, 1987; U.S. Pat.No. 4,873,192; Watson et al. , Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987; and references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein in question can be found in the model of Dayhoff et al. (Natl. Biomed. Res. Found, 5: 345-358, 1978). Fusion proteins such as Fc fusion have been particularly proposed. The fusion protein typically includes a linked sequence.

Methods for screening combinatorial library gene products generated by point mutations or truncations and methods for screening cDNA libraries for gene products having selected properties are well known in the art. The method is applicable to rapid screening of gene libraries created by combinatorial mutagenesis of polypeptides. Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in libraries, can be used in conjunction with screening assays to identify useful polypeptide variants (Arkin et al., Proc. Natl. Acad. Sci. USA, 89: 7811-7815, 1992; Delgrave et al., Protein Engineering, 6: 327-331, 1993). Conservative substitutions such as exchanging one amino acid for another with similar properties may be desirable.
A variant polypeptide may contain conservative amino acid substitutions at various positions along its sequence when compared to a reference amino acid sequence. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains has been defined in the art. Amino acid residues can be further subclassified as cyclic or acyclic, and aromatic or non-aromatic trivial classifications for the side chain substituents of the residues, and as small or large. A residue is considered small if it contains a total of 4 or fewer carbon atoms provided that there are additional polar substituents, including the carboxyl carbon. A residue is considered small if it contains no more than 3 carbon atoms in the absence of additional polar substituents. Small residues are, of course, always non-aromatic. Amino acid residues are divided into two or more classes according to their structure. The subclassification according to this scheme is shown in Table 2 for naturally occurring protein amino acids.

Conservative amino acid substitutions include groupings based on side chains. Whether an amino acid change results in a functional antagonist can be easily determined by analyzing its activity when binding to its target and suppressing EphA4-mediated signaling, receptor activation, phosphorylation, etc. . Easily assessable activities are known to those skilled in the art, e.g. binding or dimer detected by nuclear magnetic resonance spectroscopy (NMR) observing heteronuclear single quantum coherence (HSQC) spectra. Assays to determine incorporation or oligomerization, Biacore, kinetics, affinity and pull-down analysis. Table 3 below shows conservative substitutions under the heading Typical substitutions. Further preferred substitutions are indicated under the heading Preferred substitutions. Amino acid substitutions included within the scope of the present invention generally include (a) the structure of the peptide backbone within the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Is achieved by selecting substitutions whose effects on maintaining are not significantly different. After introducing substitutions, mutants are screened for biological activity.
One skilled in the art can modify an antagonist to enhance its ability to cross the blood brain barrier using activity, stability, or, if necessary, an endogenous or introduced transport molecule or carrier.
In some embodiments, an ephrin or analog of an Eph polypeptide has increased stability and activity or reduced undesirable pharmacological properties. Analogs can also be designed to improve the ability to cross biological membranes, the blood brain barrier, or interact only with specific substrates. Thus, an analog may retain some functional attributes of the parent molecule, but may have modified specificity, or achieve a new function that is useful in this context, ie, for administration to a subject. can do. Polypeptide drug analogs contemplated herein include, but are not limited to, side chain modifications, incorporation of unnatural amino acids and / or derivatives thereof during peptide, polypeptide or protein synthesis and use of cross-linking agents. As well as other methods of imposing structural constraints on proteinaceous molecules or analogs thereof.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t- The use of butyl glycine, norvaline, phenyl glycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and / or the D-isomer of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 4.
Using a cross-linking agent, for example, a homo-bifunctional cross-linking agent, such as a bifunctional imide ester having (CH ₂ ) _n spacer groups (n = 1 to n = 6), glutaraldehyde, N-hydroxysuccinimide ester and Stabilizing the 3D structure using a hetero-bifunctional reagent that usually contains an amino reaction component such as N-hydroxysuccinimide and another group specific reaction component such as maleimide or dithio component (SH) or carbodiimide (COOH) Can do. Furthermore, peptides can be structurally constrained, for example, by incorporation of C _α and N _α -methyl amino acids and introduction of double bonds between the C _α and C _β atoms of amino acids.

In another aspect, the invention provides an EphA4 antagonist or EphA4 ligand antagonism in the manufacture of a composition for protecting the function or integrity of a subject's neural tissue or reducing the rate, occurrence or amount of nerve damage in a subject. Provide drug use. Examples of nerve tissues include CNS and PNS tissues, brain, spinal cord, and optic nerve. In some embodiments, the subject has or is at risk of having a condition associated with nerve injury.
In a similar embodiment, a method for protecting the function or integrity of a subject's neural tissue or for reducing the rate, occurrence or amount of nerve damage in a subject, wherein said subject is an EphA4 antagonist or EphA4 ligand antagonist A method comprising the step of administering a drug is provided. In some embodiments, the subject has or is at risk of having a condition associated with nerve injury.
Similarly, EphA4 antagonists or EphA4 ligand antagonists are provided that are used to protect the function or integrity of a subject's neural tissue or to reduce the rate, occurrence, or amount of axonal damage in a subject. In some embodiments, the subject has or is at risk of having a condition associated with nerve injury.
Similarly, a pharmaceutical composition comprising an EphA4 antagonist or EphA4 ligand antagonist for use in protecting the function or integrity of a subject's neural tissue or for reducing the rate, occurrence or amount of axonal damage in a subject is contemplated. . In some embodiments, the subject has or is at risk of having a condition associated with nerve injury.
Nerve damage can result from a disease or disorder that results in axonal disconnection, neuronal degeneration, or demyelination. Examples of conditions that cause nerve damage include traumatic lesions (eg, caused by physical or surgery and compression injury); ischemic lesions (eg, cerebral or spinal cord obstruction and ischemia); malignant lesions; (For example, from abscess or concomitant with infection by human immunodeficiency virus, Lyme disease, pulmonary tuberculosis, syphilis, or herpes infection); Associated with chorea or amyotrophic lateral sclerosis; lesions associated with nutritional disorders or disorders (eg vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marquia furva vinyami disease ( Marchiafava-Bignami disease) and alcoholic cerebellar degeneration); neurological lesions associated with systemic disease (eg, diabetes, systemic disease) Associated with tematodes, cancer or sarcoidosis); lesions caused by toxic substances (eg alcohol, lead or neurotoxin); and other demyelinating lesions (eg human immunodeficiency virus-related myelopathy, transverse myelopathy of various etiologies, progressive Multifocal leukoencepholopathy and central pontine myelinolysis).

Also provided are medical kits comprising the antagonists described herein together with instructions for use in the treatment of NDD diseases described herein. Also provided is a medical kit comprising an EphA4 antagonist or EphA4 ligand antagonist together with instructions for neuroprotection in the treatment of NDD such as MS.
The present invention is a therapeutic protocol for treating NDD, such as MS, in a subject, which in turn is sufficient to screen a biological sample from said subject for axonal damage, further reducing nerve damage Further provided is a treatment protocol comprising administering an EphA4 antagonist or an EphA4 ligand antagonist under the proposed time and conditions and re-screening said subject for axonal injury. In some embodiments, the sample is tested for evidence of autoimmunity, inflammation, or demyelination separately or in addition.
Reference to “sample” refers to any biological fluid, cell, tissue, organ or portion thereof that the target nucleic acid molecule or polypeptide contains or may contain. The term encompasses not only samples present in an individual, but also samples obtained or derived from an individual. For example, the sample may be a pathological tissue section of a specimen obtained by biopsy or a cell that has been placed in or adapted to tissue culture. The sample may be a subcellular fraction or extract, or a purified or crude nucleic acid or polypeptide specimen.

Similarly, various aspects of affinity binding formats that can be used in the diagnostic methods of the present invention are known. Affinity binding methods are described in common laboratory manuals such as Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1999.
In one assay that utilizes immunoaffinity, the marker is attached using a magnetic antibody that binds to a neurodegenerative marker, and a free and bound antibody, and thus the presence or absence of the marker, using a high _Tc superconducting quantum interference device. Measure the level. Liposome immunotransfer and liquid phase competitive strip immunoassay are described, for example, in Glorio-Paulet et al., J Agric Food Chem 48 (5): 1678-1682, 2000.
In another embodiment, the present invention provides a method of screening any one or more Eph or ephrin modulators for the ability to reduce the clinical progression of NDD, such as MS disease.
In another embodiment, a screen for identifying an agent for the treatment of NDD, such as MS, comprising a screening agent capable of modulating the level or activity of a polypeptide having a functional activity of EphA4 or EphA4 ligand I will provide a.

  Antagonists can be developed from natural products, combinatorial synthetic organic or inorganic compounds, peptides / polypeptides / proteins, nucleic acid molecules. Libraries or phage or other display technologies containing all of these can be used to screen or test suitable agents. Natural products include coral, soil, plants, or the product from the marine or Antarctic environment. A library of small organic molecules can be generated and screened using high throughput techniques known to those skilled in the art. See, for example, US Patent No. 5,763,623 and US Patent Application No. 20060167237. Combinatorial synthesis provides a very useful approach in which a large number of related compounds with different substitutions of a common or subset of parent structures are synthesized. The compounds are usually not oligomers and are similar in basic structure and function, for example, with varying chain length, ring size or number or substitution. Virtual libraries are also contemplated and can be constructed to test compounds by computer simulation (see, eg, US Publication No. 20060040322) or in vitro or in vivo assays well known in the art. Small molecule libraries suitable for testing are available in the art (see, for example, Amezcua et al., Structure (London) 10: 1349-1361, 2002). Yeast SPLINT antibody libraries are available to test for intracellular antibodies capable of disrupting protein-protein interactions (see Visintin et al., (Supra)). Examples of suitable synthetic methods for molecular libraries can be found, for example, in the literature: DeWitt et al., Proc. Natl. Acad. Sci. USA 90: 6909, 1993; Erb et al., Proc. Natl. Acad. USA 91: 11422, 1994; Zuckermann et al., J. Med. Chem. 37: 2678, 1994; Cho et al., Science 261: 1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33: 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33: 2061, 1994; and Gallop et al., J. Med. Chem. 37: 1233, 1994.

Thus, use of biological libraries; parallel solid or liquid phase libraries that can be handled spatially; synthetic library methods that require deconvolution integration; “one bead one compound” library methods; and affinity chromatography selection Drugs can be obtained using any of a number of approaches of combinatorial library methods well known in the art, such as synthetic library methods. The biological library approach is suitable for peptide libraries, while the other four approaches can be applied to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam, Anticancer Drug Des. 12: 145, 1997; US patents) No. 5,738,996; and US Pat. No. 5,807,683). Compound libraries can be obtained, for example, in solution (eg Houghten, Bio / Techniques 13: 412-421, 1992) or beads (Lam, Nature 354: 82-84, 1991), chips (Fodor, Nature 364: 555- 556, 1993), bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. USA 89: 1865-1869, 1992) or phage (Scott and Smith, Science 249: 386-390, 1990; Devlin, Science 249: 404-406, 1990; Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378 -6382, 1990).
The invention is further illustrated by the following non-limiting examples.

Example 1: Generation of soluble EphA4 receptor Recombinant mouse EphA4 mouse Fc (mEphA4mFc) or human EphA4 human Fc (hEphA4hFc) fusion proteins were produced by transient transfection in mammalian cells. FreeStyle (TM) 293-F cells and mammalian expression vector pcDNA3.1 were obtained from Invitrogen for cell culture. Cells were cultured in FreeStyle ™ expression medium (Invitrogen). All tissue culture media were supplemented with penicillin / streptomycin / fungizone reagent (Invitrogen) and cells were maintained at 37 ° C. in an incubator with 8% CO ₂ atmosphere.
Transient transfection of mEphA4mFc or hEphA4hFc-encoding expression plasmid (see FIG. 6) using FreeStyleTM 293-F cells was performed using 293fectin transfection reagent (Invitrogen) according to the manufacturer's instructions. . Transfect cells (1000 ml) at a final concentration of 1 x 10 ⁶ viable cells / ml and 2/10 Wave Bioreactor system in Cellbag 2L (GE Healthcare) for 5 days at 37 ° C in 8% CO ₂ atmosphere Incubated on 2/10 or 20/50 (GE Healthcare). The culture conditions were rocked 35 times per minute at an angle of 8 °. Four hours after transfection, Pluronic F68 (Invitrogen) was added to a final concentration of 0.1% v / v. Twenty-four hours after transfection, cell cultures were supplemented with Tryptone N1 (Organotechnie, France) to a final concentration of 0.5% v / v. Cell culture supernatant was collected by centrifugation at 2500 rpm, passed through a 0.45 μM filter (Nalgene) and purified.
CDNA expression plasmids encoding mouse EphA4 mouse Fc and human EphA4 human Fc were prepared. Synthetic genes encoding mouse EphA4 (mEphA4mFc) fused to mouse IgG1Fc and human EphA4 (hEphA4hFc) fused to human IgG4Fc were constructed by GenScript Corporation (Piscataway, New Jersey) and Geneart AG (Regensburg, Germany), respectively. A Kozak sequence (GCCACC) was introduced just before the N-terminus of each protein to increase translation initiation. The codon usage of the mouse EphA4 gene, mouse IgG1Fc gene, human EphA4 gene and human IgG4Fc gene was adapted to the codon bias of the homosapiens gene. An NheI restriction enzyme cleavage site was introduced at the 5 ′ end of the cDNA, an XhoI restriction enzyme cleavage site was introduced at the 3 ′ end, and the synthetic cDNA was ligated into NheI-XhoI digested pcDNA3.1. The cDNA was digested with NheI and XhoI and ligated into pcDNA3.1. Large scale preparation of plasmid DNA was performed using Qiagen Maxi or Giga kit according to manufacturer's instructions. The nucleotide sequence of the plasmid construct was verified by sequencing both strands using the Big Dye Terminator v3.1 Cycle Sequencing and Applied Biosystems Automated Sequencer.
For analysis of protein expression of mEphA4mFc or hEphA4hFc protein, 20 μl aliquots of culture supernatant from transfection of expression constructs encoding mEphA4mFc or hEphA4hFc were electrophoresed on a 4-20% Tris-Glycine SDS polyacrylamide gel. Visualization was performed by staining with Coomassie Blue reagent. Cell culture supernatant containing mEphA4mFc or hEphA4hFc protein was collected by centrifugation at 2500 rpm, passed through a 0.45 μM filter (Nalgene) and purified.
For purification, conditioned media containing EphA4Fc was concentrated using target flow filtration. The concentrated medium was purified using protein A by affinity chromatography. Endotoxin was removed by ion exchange chromatography. Aggregates were removed using size exclusion chromatography. That is, Fc-binding dimeric EphA4 proteins derived from other molecular weight species were isolated. Protein quantification was performed based on absorbance units of 280 and 1.0, equivalent to 1.3 mg / ml. Purity was based on protein visualization by SDS-PAGE and Coomassie blue staining.

Example 2: Assay The severity of disease based on histological analysis of the degree of inflammatory infiltrate is based on the area of inflammatory wet foci per spinal cord section as reported (Butzkueven et al., Glia 53: 696-703, 2006). Briefly, in some embodiments, the total inflammatory lesion area of a DAPI stained section is calculated as the total percentage of the area of all inflammatory wet foci per total area of the spinal cord within the same section. Count on 15-20 sections per mouse. Sections are graded using a semi-quantitative scale (Soilu-Hanninen et al., J Neurosci Res 59: 712-721, 2000). 0 is no inflammatory cells; 1 is several inflammatory cells; 2 is moderate perivascular cuffing; 3 is dense inflammatory cell infiltration, parenchymal necrosis. Quantify the number of lesions per section.
Perform immunohistochemical analysis of immune cells in the test subjects. A wide range of T cell (CD4, CD8, T cell receptors), B cell (CD19) and bone marrow cell (CD11b, IBA1) markers can be investigated. If both populations change, more specific subpopulation markers are utilized. These markers are also used in FACS analysis of spleen cells and lymphocytes to determine whether there is a significant difference in the immunological status of EphA4-/-mice after EAE induction or after antagonist administration.
Quantify the number of mature and immature oligodendrocytes in lumbar dorsal horny white matter and optic nerve sections. For comparison of mature oligodendrocyte density, in sections immunostained with mouse anti-CC1 antibody (APC Ab-7, Oncogene Research Products, Germany) or in sections from mice hybridized to the transgenic PLP-dsRed system Count the number of mature oligodendrocytes. PLP-expressing (mature) oligodendrocytes express the marker dsRed; these mice are on the C57BL / 6 background. The oligodendrocyte precursor is labeled with a rabbit anti-NG2 antibody (Chemicon). For analysis of oligodendrocyte apoptosis, sections are TUNEL labeled using the TMR In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions (Butzkueven et al., 2002 (supra)). Sections are co-stained with CC-1 antibody. Confirm apoptosis of TUNEL positive cells by nuclear fragmentation or condensation using DAPI. For each animal, for the spinal cord and optic nerve, count the total number of apoptotic cells and the number of apoptotic oligodendrocytes per section in five 100 μm apart 10 μm sections. Assess statistical significance using the Mann Whitney U-test. As described in the literature (Butzkueven et al., 2006 (supra)), Luxol Fast Blue staining of sections is used to assess the extent of demyelination and remyelination over the course of the disease.

  For example, axonal damage can be assessed by immunostaining of β-amyloid precursor protein (βAPP) and hyperphosphorylation and aggregation of tau. APP-positive axon terminal spheres and spheroids, which are recently useful axon indicators, can be detected within the active boundary of acute MS lesions and chronic active lesions, but detection within chronic inactive lesions is low. Yes (Kornek et al., 2000 (above); Ferguson et al., 1997 (above)), and immunostaining of phosphorylated tau reveals areas of axonal damage within MS / EAE plaques (Schneider et al. J Biol Chem. 279 (53): 55833-95583, 2004). Some methods conveniently use image analysis (Image Pro) based on methylene blue staining of resin-embedded sections and automatic quantification of the number and area of axons within a defined spinal cord region. The hind cable is used as a comparison area between animals. In some embodiments, ELISA-based analysis of phospho-NF in blood is an accurate marker of axonal damage. In the mouse neuroinflammatory context, phospho-NF-H levels are highly related to axonal loss, as reviewed by Gresle et al., J Neurosci Res. 86 (16): 3548-3555, 2008 There is. In humans, CSF pNF-H levels increased during the relapse of MS subjects, and this level may be a clinically relevant marker. The higher level was also associated with a faster conversion to a secondary progression over a three-year period and predicted poor clinical outcome after relapse.

Axonal loss of the optic nerve has been reported in the MOE model of EAE before the onset of clinical symptoms (Hobom et al., Brain Pathol 14: 148-157, 2004) and is before the onset of clinical symptoms in the EAE model 6 Examine the tissue from day to day (Butzkueven et al., 2002 (above)). Sections from test subjects are immunostained for phosphorylated tau and βAPP and counterstained with H & E to highlight the plaque area. Count the number of plaques in the longitudinal and transverse sections of the lumbar and optic nerves and βAPP ending spheres / spheroids in the adjacent normal appearing white matter (NAWM). The total axon density in and adjacent to the plaque in the spinal cord and the entire optic nerve is determined by automatic counting of the methylene blue stained cross-sectional areas of these tissues. At least five 50 μm-separated sections of each tissue are counted for each animal and the results are expressed as the average density of axons per mm ² . Assess statistical significance in the Mann Whitney U-test.

Plaques within and in adjacent NAWM glial fibrillary acidic protein (GFAP) positive astrocytes counted the number and immunostaining, expressed as astrocytes per 1 mm ² at each time point in the course of the study. Image analysis (NIH Image J) is used to determine the amount of GFAP staining per field as a percentage of the total number of pixels in the field (as in FIG. 3). Digital images of GFAP-stained tissue are taken at high magnification (x100) at multiple sites (5-10) in and around the lesion in at least 5 sections (10 μm) per tissue per mouse. To examine any differences in GFAP expression levels, spinal cord homogenates are processed for Western analysis. Densitometry is performed with an autoradiogram using NIH Image J software to determine the relative level of GFAP bands and normalize to β-actin levels.
The level of EphA4 expression is measured over the course of disease in wild type mice. Spinal cord and optic nerve sections are immunostained for EphA4. The level of EphA4 expression and phosphorylation is also measured in spinal cord tissue. Homogenize the lumbar spinal cord tissue and collect an aliquot of lysate for Western analysis. The rest is used for immunoprecipitation with anti-EphA4 antibody and then probed for phosphotyrosine and EphA4 expression.

Example 3: EphA4 deficiency alters disease progression and functional outcome in MS model (EAE induction and clinical evaluation)
To elucidate the role EphA4 plays in the severity and progression of EAE and to determine whether blocking EphA4 results in the potential for MS treatment, EphA4-/-(Dottori et al., Proc Natl Acad Sci USA 95: 13248-53, 1998) and wild-type mice, the clinical course and severity of EAE, and pathological and histological features were investigated.
Since EphA4-/-(Dottori et al., 1998 (above)) mice are backcrossed to the C57BL / 6 background, the MOE model of EAE is used with these mice. C57BL / 6 mice are MOG 35-55 (MEVGWRSPFSRVVHLYRNGK (SEQ ID NO: 1)) peptide (Butzkueven et al., 2002 (supra); Slavin et al., Autoimmunity 28: 109-20, 1998; Gold et al. , (Above)) or susceptible to induction of EAE by passive transfer of MOG-specific T cells. The clinical course of both MOE models of EAE based on the C57BL / 6 background tends to be chronically progressive, characterized by immune cell infiltration and localized demyelination throughout the CNS, particularly within the spinal cord and optic nerve .
EAE disease severity was assessed daily for up to 1 month and for up to 3 months for additional age groups using the following standard 5-point paraplegia scale: 0 for no clinical symptoms; 1 for fur ruffling (fur ruffling) or distal tail weakness; 1.5 is tail ataxia or slight hind limb paralysis; 2 is complete tail paralysis; 2.5 is complete tail paralysis and partial paralysis acting on one or both hind limbs; 3 Is complete hind limb paralysis; 3.5 cannot return to its correct position when placed on its back; 4 is complete paralysis of both hind limbs and forelimb weakness or moribund; and 5 is dead. Mice that reach grade 4 are killed according to the requirements of the Ethics Committee and CNS tissue is harvested for histology. The occurrence of clinical symptoms using this model is typically around 10-12 days (Butzkueven et al., 2002 (supra)).
A study was performed using n = 10 EphA4-/-mice and n = 10 control wild-type littermates (see FIG. 1). Clinical signs began to develop in both age groups as well, but the disease was more severe in wild-type mice, with an average clinical grade of 2.3 ± 0.3 (maximum grade = 4; maximum of 60% EphA4 null mice averaged 1.7 ± 0.2 (max grade 2.75; max 60% grade reached 1.5). Therefore, the clinical course of EAE is less severe in EphA4-/-mice.

Example 4: Immunohistological characterization of tissues affected by EAE in EphA4-/-and wild type mice Analysis of bone marrow sections from mice described in Example 1 was performed using DAPI (not shown) and microglial cells. When assessed with the markers CD11b and IBA1 (not shown), both wild type and EphA4 knockout mice showed typical inflammatory infiltration. There was no significant difference in the number of lesions per section or the number of DAPI nuclei in the outer white matter.
The level of phospho-nerve fibers in the blood was assessed as a surrogate marker for axonal damage that was well associated with disease severity and the histological characteristics of axonal damage were also assessed (Gresle et al, 2008 ( the above)). EphA4 knockout mice showed decreased levels of phospho-NF, suggesting that the axonal damage in the mice was reduced (see FIG. 2).

Example 5: EphA4 is expressed on astrocytes of EAE lesions Astrocyte gliosis is characteristic of MS lesions. To evaluate whether EphA4 is expressed on astrocytes in EAE lesions, spinal cords from C57BL / 6 mice experimentally induced EAE by administration of MOG35-55 peptide (Butzkueven et al., 2002 (above)) Sections were immunostained for EphA4 and glial fibrillary acidic protein (GFAP) expression. EphA4 was expressed on all glial fibrillary acidic protein (GFAP) positive and thus reactive astrocytes around the lesion in the spinal cord of grade 3 EAE mice (FIG. 3).

Example 6: EphA4 blocker after CNS injury
After CNS injury, and within MS and EAE lesions, the blood-brain barrier is breached, which provides a pathway for access to antagonists only at the site of CNS injury. To detect the presence of EphrinA5-Fc, injured spinal cord sections were immunostained using biotin-labeled anti-human IgG, followed by vector ABC kit and DAB. PBS treated spinal cord showed no labeling, while EphrinA5-Fc treated spinal cord showed labeling at the lesion site. This suggests that EphrinA5-Fc was able to cross the blood brain barrier and enter the parenchyma (Figure 4). Since the blood brain barrier is broken within an EAE lesion, treatment of EAE with IP injection of an EphA4 blocker would be a viable way to deliver these molecules specifically to the CNS region affected by the lesion .

Example 7: Addition of uncomplexed ephrin A5-Fc to astrocytes blocks IFNγ-induced EphA4 activation Anti-cancer of wild-type astrocytes cultured from neocortex to produce an activated form of ephrin A5-Fc They were treated with ephrinA5-Fc that was complexed with human IgGFc or remained uncomplexed to block EphA4 activation. The effect on EphA4 activation under basal conditions and in response to cytokines was evaluated. Under basal conditions, complex (poly) ephrin A5-Fc increased phosphorylation of EphA4. Importantly, unconjugated (mono) ephrin A5-Fc decreased phosphorylation of EphA4 both under basal conditions and in response to IFNγ (FIG. 5). Thus, in addition to controlling neuronal growth cone responses, modulation of EphA4 signaling also has a direct effect on astrocytes.

Example 8: Further studies As described in Example 1, E57A induced EAE by MOG is administered EphA4 blockers such as soluble EphrinA5-Fc or EphA4-Fc. Using the assay of Example 2, to assess the effects of EphA4 antagonists before, during, and after clinical disease development, such as clinical severity, inflammatory infiltration and oligodendrocyte survival .
In some embodiments, a CHO cell transfectant system that expresses both Eph-human IgGFc recombinant protein and ephrin-human IgGFc recombinant protein is used (Coulthard et al., Growth Factors 18: 303-17 , 2001). Soluble ephrin A5-Fc or EphA4-Fc protein is produced from these systems. In studies using radiolabeled ephrin A5, 65% of IP injected ephrin A5 is rapidly cleared into the tissue, with similar uptake in all tissues tested. However, clearance from the blood is relatively slow, with 5% of the injected dose remaining in the 24-hour circulation. This suggests that a daily dosing regimen could maintain an effective circulatory inhibitor. The intact non-clustered ephrin A5-Fc or EphA4-Fc is tested in EAE affected animals.
Given the preliminary data that IP-injected EphrinA5-Fc enters the CNS parenchyma at the CNS injury site of spinal cord injury, where it breaks the blood-brain barrier, the first study of the EAE utilizes this route of administration. Invasion into the CNS parenchyma is assessed by immunostaining in preparation for human-IgG. Soluble anti-Ephrin is expected to cross the blood brain barrier at the site of EAE lesions, but if access is inadequate, use a mini-osmotic infusion pump (Alzet) with a catheter inserted into the fourth ventricle, and the pump Are implanted under the skin.
The first study utilizes a single IP injection of 500 μg / day / mouse ephrinA5-Fc or EphA4-Fc, which has been shown to be effective in spinal cord studies. Antagonists are administered from disease induction until tissue is collected for analysis. In some embodiments, administration occurs before the occurrence of clinical symptoms of the disease or induction of EAE. Subsequent studies improve the timing of treatment initiation, such as delaying administration until clinical symptoms occur or after assessment of axonal damage. Optimize dose as needed.

The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.
Throughout this specification, the goal was to describe preferred embodiments of the present invention without limiting the invention to any one embodiment or characteristic of a unique population. Accordingly, one of ordinary skill in the art appreciates that various modifications and changes can be made to the specific illustrated embodiment without departing from the scope of the invention in light of the present disclosure. All such modifications and changes are intended to be included within the scope of the appended claims.

table 1
Overview of array identifiers

Table 2
Subclassification of amino acids

Table 3
Typical and preferred amino acid substitutions

Table 4
Non-traditional amino acid codes
______________________________________________________________________________
Non-traditional code non-traditional code amino acid amino acid
______________________________________________________________________________
α-Aminobutyric acid Abu LN-Methylalanine Nmala
α-amino-α-
Methyl butyrate Mgabu LN-Methylarginine Nmarg
Aminocyclopropane-Cpro LN-Methylasparagine Nmasn
Carboxylate LN-Methylaspartic acid Nmasp
Aminoisobutyric acid Aib LN-Methylcysteine Nmcys
Aminonorbornyl-Norb LN-Methylglutamine Nmgln
Carboxylate LN-Methylglutamic acid Nmglu
Cyclohexylalanine Chexa LN Methylhistidine Nmhis
Cyclopentylalanine Cpen LN-Methylisoleucine Nmile
D-Alanine Dal LN-Methylleucine Nmleu
D-Arginine Darg LN-Methyllysine Nmlys
D-Aspartate Dasp LN-Methylmethionine Nmmet
D-cysteine Dcys LN-Methylnorleucine Nmnle
D-Glutamine Dgln LN-Methylnorvaline Nmnva
D-Glutamic acid Dglu LN-Methylornithine Nmorn
D-histidine Dhis LN-Methylphenylalanine Nmphe
D-Isoleucine Dile LN-Methylproline Nmpro
D-leucine Dleu LN-methylserine Nmser
D-Lysine Dlys LN-Methylthreonine Nmthr
D-methionine Dmet LN-methyltryptophan Nmtrp
D-Ornithine Dorn LN-Methyltyrosine Nmtyr
D-Phenylalanine Dphe LN-Methylvaline Nmval
D-proline Dpro LN-methylethylglycine Nmetg
D-serine Dser LN-methyl-t-butylglycine Nmtbug
D-threonine Dthr L-norleucine Nle
D-tryptophan Dtrp L-norvaline Nva
D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib
D-valine Dval α-methyl-γ-aminobutyrate Mgabu
D-α-Methylalanine Dmala α-Methylcyclohexylalanine Mchexa
D-α-Methylarginine Dmarg α-Methylcyclopentylalanine Mcpen
D-α-methyl asparagine Dmasn α-methyl-α-naphthylalanine Manap
D-α-Methyl Aspartate Dmasp α-Methylpenicillamine Mpen
D-α-Methylcysteine Dmcys N- (4-Aminobutyl) glycine Nglu
D-α-Methylglutamine Dmgln N- (2-Aminoethyl) glycine Naeg
D-α-Methylhistidine Dmhis N- (3-Aminopropyl) glycine Norn
D-α-Methylisoleucine Dmile N-Amino-α-methylbutyrate Nmaabu
D-α-Methylleucine Dmleu α-Naphthylalanine Anap
D-α-Methyllysine Dmlys N-Benzylglycine Nphe
D-α-Methylmethionine Dmmet N- (2-carbamylethyl) glycine Ngln
D-α-Methylornithine Dmorn N- (Carbamylmethyl) glycine Nasn
D-α-Methylphenylalanine Dmphe N- (2-carboxyethyl) glycine Nglu
D-α-Methylproline Dmpro N- (Carboxymethyl) glycine Nasp
D-α-Methylserine Dmser N-Cyclobutylglycine Ncbut
D-α-Methylthreonine Dmthr N-Cycloheptylglycine Nchep
D-α-Methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-α-Methyltyrosine Dmty N-Cyclodecylglycine Ncdec
D-α-Methylvaline Dmval N-Cyclododecylglycine Ncdod
DN-methylalanine Dnmala N-cyclooctylglycine Ncoct
DN-methylarginine Dnmarg N-cyclopropylglycine Ncpro
DN-methyl asparagine Dnmasn N-cycloundecylglycine Ncund
DN-methyl aspartate Dnmasp N- (2,2-diphenylethyl) glycine Nbhm
DN-methylcysteine Dnmcys N- (3,3-diphenylpropyl) glycine Nbhe
DN-Methylglutamine Dnmgln N- (3-Guanidinopropyl) glycine Narg
DN-methylglutamate Dnmglu N- (1-hydroxyethyl) glycine Nthr
DN-methylhistidine Dnmhis N- (hydroxyethyl)) glycine Nser
DN-methylisoleucine Dnmile N- (imidazolylethyl)) glycine Nhis
DN-Methylleucine Dnmleu N- (3-Indolylethyl) glycine Nhtrp
DN-methyllysine N-methyl-γ-aminobutyrate Nmgabu
N-methylcyclohexyl Dnmlys
Alanine Nmchexa DN-Methylmethionine Dnmmet
DN-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala DN-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib DN-methylproline Dnmpro
N- (1-methylpropyl)
Glycine Nile DN-Methylserine Dnmser
N- (2-methylpropyl)
Glycine Nleu DN-methylthreonine Dnmthr
DN-methyltryptophan Dnmtrp N- (1-methylethyl) glycine Nval
DN-methyltyrosine Dnmtyr N-methyla-naphthylalanine Nmanap
DN-Methylvaline Dnmval N-Methylpenicillamine Nmpen
γ-aminobutyric acid Gabu N- (p-hydroxyphenyl) glycine Nhtyr
Lt-Butylglycine Tbug N- (Thiomethyl) glycine Ncys
L-Ethylglycine Etg Penicillamine Pen
L-homophenylalanine Hphe L-α-methylalanine Mala
L-α-Methylarginine Marg L-α-Methylasparagine Masn
L-α-Methyl aspartate Masp L-α-Methyl-t-butylglycine Mtbug
L-α-Methylcysteine Mcys L-Methylethylglycine Metg
L-α-Methylglutamine Mgln L-α-Methylglutamate Mglu
L-α-Methylhistidine Mhis L-α-Methylhomophenylalanine Mhphe
L-α-Methylisoleucine Mile N- (2-Methylthioethyl) glycine Nmet
L-α-Methylleucine Mleu L-α-Methyllysine Mlys
L-α-Methylmethionine Mmet L-α-Methylnorleucine Mnle
L-α-Methylnorvaline Mnva L-α-Methylornithine Morn
L-α-Methylphenylalanine Mphe L-α-Methylproline Mpro
L-α-Methylserine Mser L-α-Methylthreonine Mthr
L-α-Methyltryptophan Mtrp L-α-Methyltyrosine Mtyr
L-α-Methylvaline Mval LN-Methylhomophenylalanine Nmhphe
N- (N- (2,2-diphenylethyl) Nnbhm N- (N- (3,3-diphenylpropyl) Nnbhe
Carbamylmethyl) glycine Carbamylmethyl) glycine
1-carboxy-1- (2,2-diphenyl-Nmbc
Ethylamino) cyclopropane
______________________________________________________________________________

Table 5
List of abbreviations

(Cited document)
Aasheim et al., Blood, 95 (1): 221-230, 2000
Altschul et al., Nucleic Acids Research 25: 3389-3402, 1997
Amezcua et al., Structure (London) 10: 1349-1361, 2002
Arkin et al., Proc. Natl. Acad. Sci. USA, 89: 7811-7815, 1992
Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999
Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, Chapter 15, 1994-1998
Barnes et al., Brain 114: 1271-80, 1991
Bjartmar et al., J Neurol Sci 206: 165-71, 2003
Bjartmar et al., Neurology 57: 1248-52, 2001
Brantley et al., Oncogene 21: 7011-26, 2002
Butzkueven et al. Nat Med 8: 613-9, 2002
Butzkueven et al., Glia 53: 696-703, 2006
Carell et al., Angew. Chem. Int. Ed. Engl. 33: 2061, 1994
Carrell et al., Angew. Chem. Int. Ed. Engl. 33: 2059, 1994
Cheng et al., Cell 82: 371-81, 1995
Cho et al., Science 261: 1303, 1993
Chrencik et al., J Biol Chem., 282 (50): 36505-36513, 2007
Chrencik et al., Structure, 14 (2): 321-330, 2006

Coligan et al., Current Protocols In Protein Science, John Wiley & Sons, Inc., 1995-1997
Constantini et al., Cancer Biotherm. Radiopharm. 23 (1): 3-24, 2008
Coulthard et al., Growth Factors 18: 303-17, 2001
Cull et al., Proc. Natl. Acad. Sci. USA 89: 1865-1869, 1992
Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378-6382, 1990
Davis et al., Science 266: 816-9, 1994
Dayhoff et al., Natl. Biomed. Res. Found, 5: 345-358, 1978
De Stefano et al., Brain 121 (Pt 8): 1469-77, 1998
Delgrave et al., Protein Engineering, 6: 327-331, 1993
Devlin, Science 249: 404-406, 1990
DeWitt et al., Proc. Natl. Acad. Sci. USA 90: 6909, 1993
Dobrzanski et al., Cancer Res 64: 910-9, 2004
Dottori et al., Proc Natl Acad Sci USA 95: 13248-53, 1998
Erb et al., Proc. Natl. Acad. Sci. USA 91: 11422, 1994
Ferguson et al., Brain 120: 393-9, 1997
Fodor, Nature 364: 555-556, 1993
Fu et al., Nature Neuroscience 10: 67-76, 2007
Gale et al., Neuron. 17 (1): 9-19, 1996
Gallop et al., J. Med. Chem. 37: 1233, 1994
Gold et al., Brain. 129 (Pt 8): 1953-1971, 2006
Goldshmit et al., Brain Res Brain Res Rev 52: 327-45, 2006
Goldshmit et al., J Neurosci 24: 10064-73, 2004
Gonen et al., Neurology 54: 15-9, 2000

Gresle et al., J Neurosci Res. 86 (16): 3548-3555, 2008
Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988
Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1999
Harmsen and De Haard, Appl. Microbiol. Biotechnol. 77 (1): 13-22, 2007
Henchey et al., Curr Opin Chem Biol. 12 (6): 692-697, 2008
Henkemeyer et al., Cell 86: 35-46, 1996
Hobom et al., Brain Pathol 14: 148-157, 2004
Horner and Gage, Nature.407 (6807): 963-970, 2000
Houghten, Bio / Techniques 13: 412-421, 1992
Kidd et al., Brain 122 17-26, 1999
Kolb et al., Proteins, 73 (1): 11-18, 2008
Koolpe et al., J Biol Chem., 280 (17): 17301-17311, 2005
Kornek et al., Am J Pathol 157: 267-76, 2000
Kunkel et al., Methods in Enzymol., 154: 367-382, 1987
Kunkel, Proc. Natl. Acad. Sci. USA, 82: 488-492, 1985
Lam, Anticancer Drug Des. 12: 145, 1997
Lam, Nature 354: 82-84, 1991
Liedtke et al., Am J Pathol 152: 251-9, 1998
Lo et al., J Neurophysiol 90: 3566-71, 2003
Misra et al., J Pharm Sci 6: 252-273, 2003
Monschau et al., Embo J 16: 1258-67, 1997
Murai et al., Mol Cell Neurosci. 24 (4): 1000-1011, 2004
Muyldermans, J. Biotechnol. 74: 277-302, 2001
Noberini et al., J Biol Chem., 283 (43): 29461-29472, 2008

Ohta et al., Mech Dev 64: 127-35, 1997
Pasquale, Nat Rev Mol Cell Biol. 6 (6): 462-75, 2005
Peterson et al., Ann Neurol 50: 389-400, 2001
PubChem Bioassay AID No. 689 (Colorimetric assay for HTS discovery of chemical inhibitors of EphA4 receptor antagonists)
Qin et al., J Biol Chem. 283 (43): 29473-29484, 2008
Raine et al., Lab Invest 31: 369-80, 1974
Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Company, Easton, PA, USA, 1990
Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor Laboratory, Cold Spring Harbour, NY, 1989
Schneider et al. J Biol Chem. 279 (53): 55833-95583, 2004
Scott and Smith, Science 249: 386-390, 1990
Shamah et al., Cell 105: 233-44, 2001
Smith et al., Brain Res 264: 241-53, 1983
Sobel, Brain Pathol. 15 (1): 35-45, 2005
Soilu-Hanninen et al., J Neurosci Res 59: 712-721, 2000
Stein et al., Genes Dev 12: 667-78, 1998
Tibary et al., Soc. Reprod. Fertil. Suppl. 64: 297-313, 2007
Trapp et al., Curr Opin Neurol 12: 295-302, 1999
Trapp et al., N Engl J Med 338: 278-85, 1998
Visintin et al, J. Immunol. Methods, 290 (1-2): 135-53, 2008
Visintin et al., J. Biotechnol, 135: 1-15, 2008
Wahl et al., J Cell Biol 149: 263-70, 2000
Watson et al., Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987

Wilder et al., ChemMedChem. 2 (8): 1149-1151, 2007
Wilkinson, Nat Rev Neurosci. 2 (3): 155-164, 2001
Wu et al., Neuroimage 37: 1138-47, 2007
Wujek et al., J Neuropathol Exp Neurol 61: 23-32, 2002
Zuckermann et al., J. Med. Chem. 37: 2678, 1994

Claims (21)

  A method of treating a neurodegenerative disease (NDD) in a subject in need of treatment comprising the step of administering an EphA4 antagonist or an EphA4 ligand antagonist to the subject.   2. The method of claim 1, wherein the antagonist binds to EphA4 and downregulates its level or activity.   3. The method of claim 2, wherein the antagonist is soluble ephrin or a functional variant or analog thereof.   4. The method of claim 3, wherein the antagonist is soluble ephrin A5, soluble ephrin B2, or soluble ephrin B3.   3. The method of claim 2, wherein the antagonist is an antibody or antigen-binding fragment that binds to EphA4 and downregulates its level or activity.   3. The method of claim 2, wherein the antagonist binds to EphA4-binding ephrin and down regulates its level or activity.   7. The method of claim 6, wherein the antagonist is soluble EphA4 or a functional variant or analog thereof.   8. The method of claim 7, wherein the soluble EphA4 is EphA4-Fc.   7. The method of claim 6, wherein the antagonist is an antibody or antigen-binding fragment that binds to EphA4-binding ephrin and down-regulates its level or activity.   2. The antagonist of claim 1, wherein the antagonist is a nucleic acid, polypeptide, peptide or organic molecule that binds to EphA4 or EphA4 ligand or a nucleic acid encoding EphA4 or EphA4 ligand and down-regulates its level or activity. Method.   11. The method of any one of claims 1-10, wherein the antagonist is administered with at least one other therapeutic agent or procedure.   12. The method according to any one of claims 1 to 11, wherein the antagonist reduces clinical progression of the disease.   13. The method of any one of claims 1-12, wherein the subject is at risk for NDD or has clinical signs of NDD.   The method according to any one of claims 1 to 13, wherein the neurodegenerative disease is multiple sclerosis (MS) or a variant thereof.   15. The method of any one of claims 1-14, wherein the method comprises screening a biological sample from the subject for axonal damage.   16. The method of any one of claims 1-15, wherein the subject exhibits or may exhibit less nerve damage or less progressive neurological dysfunction.   17. The method of claim 16, wherein the antagonist protects the function or integrity of the subject's neural tissue or reduces the rate, occurrence, or amount of nerve damage that occurs in the subject.   18. The antagonist protects the function or integrity of the subject's neural tissue or reduces the rate, occurrence, or amount of nerve damage that occurs in the subject during a chronic progression stage of NDD, such as MS. The method described in 1.   17. The method of claim 16, wherein the neurological dysfunction is limb weakness.   20. The administration according to any one of claims 1 to 19, wherein the administration is before or at the occurrence of one or more clinical symptoms of NDD or before or at the occurrence of a recurrence of one or more clinical symptoms of NDD. the method of.   The method according to any one of claims 1 to 20, wherein the method is locally administered to the brain, spinal cord, or optic nerve.