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Title:  (+)-Trans-4-(1-aminoethyl)-1-(4-pyridycarbamoyl)- cyclohexane and method for promoting neural growth in the central nervous system and in a patient at a site of neuronal lesion
United States Patent: 
7,169,783
Issued: 
January 30, 2007

Inventors: 
McKerracher; Lisa (Montreal, CA), Lehmann; Maxime (Marseille, FR)
Assignee: 
Universite de Montreal (CA)
Appl. No.: 
10/022,301
Filed: 
December 17, 2001


 

George Washington University's Healthcare MBA


Abstract

The invention relates to an antagonist of one or more of Rho family members having ability to elicit neurite outgrowth from cultured neurons in an assay method which includes culturing neurons on a substrate that incorporates a growth-inhibiting amount of Rho family member and exposing the cultured neurons to a candidate Rho family member antagonist agent to permit neuron growth. Candidates which elicit neurite outgrowth from the cultured neurons are thus identified as Rho family antagonists.

SUMMARY OF THE INVENTION

The present invention relates to antagonists and inhibitors to members of the Rho family of proteins and diagnostic, therapeutic, and research uses for each of these aspects. In particular, members of the Rho family of proteins serve as a therapeutic target to foster regrowth of injured or degenerating axons in the CNS.

In accordance with the present invention, a preferred embodiment relates to antagonists and inhibitors of members of the Rho family of proteins and their use as a means of blocking a common signaling pathway used by the diverse growth inhibitory molecules. The antagonists and inhibitors may be mutated forms of Rho and biologically active (Rho family-inhibitory) fragments, peptides, C3 and biologically active (Rho family-inhibitory) fragments, or small molecules such as Y-27632.

In yet a further aspect of the present invention, Rho family member proteins can be used to design small molecules that antagonize and inhibit Rho family proteins, to block inhibition of neurite outgrowth. In another aspect of the present invention Rho family members can be used to design antagonist agents that suppress the myelin growth inhibitory system. These antagonist agents can be used to promote axon regrowth and recovery from trauma or neurodegenerative disease.

In a further aspect of the present invention, inhibitors of the Rho family of proteins can be used to block inhibition of neurite outgrowth and to suppress the myelin growth inhibitory system. Such inhibitors could block exchange of the GTP/GDP cycle of Rho activation/inactivation.

A further embodiment involves a method of suppressing the inhibition of neuron growth, comprising the steps of delivering to the nerve growth environment, antibodies directed against Rho family members in an amount effective to reverse said inhibition.

In accordance with another aspect of the present invention, there is provided an assay method useful to identify Rho family member antagonist agents that suppress inhibition of neuron growth, comprising the steps of: a) culturing neurons on a growth permissive substrate that incorporates a growth-inhibiting amount of a Rho family member; and b) exposing the cultured neurons of step a) to a candidate Rho family member antagonist agent in an amount and for a period sufficient prospectively to permit growth of the neurons; thereby identifying as Rho family antagonists the candidates of step b) which elicit neurite outgrowth from the cultured neurons of step a).

In accordance with another aspect of present invention, there is provided a method to suppress the inhibition of neuron, comprising the steps of delivering, to the nerve growth environment, a Rho family antagonist in an amount effective to reverse said inhibition.

In another embodiment, kinases activated by Rho, such as Rho-associated kinase, are antagonist candidates. Thus, compounds such as Y-27632 (U.S. Pat. No. 4,997,834), that block Rho-associated kinase activity, thereby inactivating the Rho signaling pathway, are also embodiments of this invention. Thus, the use of other compounds within this family of compounds as described in U.S. Pat. No. 4,997,834 that inhibit Rho kinase are also considered within the scope of this invention.

In another embodiment, the nucleic acids encoding Rho family members can be used in antisense techniques and therapies.

In yet another embodiment, a kit is provided comprising components necessary to conduct the assay method useful to screen Rho family antagonist agents.

DETAILED DESCRIPTION OF THE INVENTION

This invention arises from the discovery that Rho family members are key molecules in regulating inhibition by myelin proteins, and by MAG. Thus, this invention provides the advantage of identifying an intracellular target, Rho family members, for all of the multiple inhibitory proteins that must be inactivated to allow for growth on myelin. The method of this invention provides for inactivation of Rho family members, thereby stimulating neurite growth on growth inhibitory substrates. Therefore, antagonists that inactivate Rho family members in vivo should allow axon regeneration in the injured or diseased CNS.

This invention provides for the use of Rho, or proteins related to Rho as therapeutic targets for agents designed to block growth inhibition by myelin or myelin proteins. One embodiment pertains to the use of Rho antagonists that foster axon regeneration in the central nervous system. The therapeutic agent or antagonist can be small molecules, proteins or peptides, or any agent that binds to Rho or its family members to inactivate this pathway.

Another embodiment pertains to the use of the Rho regulatory pathway as a target for Rho antagonists. This pathway involves the GDP/GTP exchange proteins (GEPs). Rho has two interconvertible forms, GDP-bound inactive, and GTP-bound active forms. The GEPs promote the exchange of nucleotides and thereby constitute targets for regulating the activity of Rho. In another embodiment GDP dissociation inhibitors (GDPs) inhibit the dissociation of GDP from Rho, and thereby prevent the binding of GTP necessary for the activation of Rho. Therefore, GDIs are targets for agents that regulate Rho activity. The GTP-bound active Rho can be converted to the GDP-found inactive form by a GTPase reaction that is facilitated by its specific GTPase activating protein (GAP). Thus, another embodiment pertains to the use of GAPs as targets for the regulation of Rho activity. Another embodiment pertains to the fact that Rho is found in the cytoplasm complexed with a GTPase inhibiting protein (GDI). To become active, Rho binds GTP and is translocated to the membrane. Thus, agents that affect Rho binding to the plasma membrane are also considered within the scope of this invention. Yet another embodiment pertains to the observation that a bacterial mon-ADP ribosyltransferase, C3 transferase, ribosylates Rho to inactivate the protein. Thus this embodiment pertains to the use of C3 transferase to inactivate Rho and stimulate axon growth. Likewise, other bacterial toxins, such as toxins A and B, with related Rho-inhibitory activity are considered to be within the scope of this invention. Moreover, various mutations of the Rho protein can create dominant negative Rho, which can interfere with the biological activity of endogenous Rho in neurons. Thus, yet a further embodiment of this invention pertains to the use of dominant negative forms of Rho, used to inactivate Rho, to foster axon growth.

"Antagonist" refers to a pharmaceutical agent which in accordance with the present invention which inhibits at least on biological activity normally associated with Rho family 25 members, that is blocking or suppressing the inhibition of neuron growth. Antagonists which may be used in accordance with the present invention include without limitation, one or more Rho family members fragment, a derivative of Rho family members or of a Rho family members fragment, an analog of Rho family members or of a Rho family members fragment or of said derivative, and a pharmaceutical agent, and is further characterized by the property of 30 suppressing Rho family members-mediated inhibition of neurite outgrowth. Preferred antagonists include: mutated forms of Rho, such as Rho wherein the effector Domain, A-37, has been mutated to prevent GTP exchange; the ADP-ribosyl transferase C3 and biologically effective fragments that antagonize Rho family members in one of the assays of this invention; and compounds such as Y-27632 that antagonise Rho-associated kinase (Somiyo, 1997, Nature, 389:908 910; Uehata, et al., 1997, Nature 389:990 994; U.S. Pat. No. 4,997,834).

The antagonist of Rho family members in accordance with the present invention is not limited to Rho family members or its derivatives, but also includes the therapeutic application of all agents, referred herein as pharmaceutical agents, which alter the biological activity of the Rho family members protein such that inhibition of neurons or their axon is suppressed.

The term "effective amount" or "growth-promoting amount" refers to the amount of pharmaceutical agent required to produce a desired antagonist effect of the Rho family members biological activity. The precise effective amount will vary with the nature of pharmaceutical agent used and may be determined by one or ordinary skill in the art with only routine experimentation.

As used herein, the Rho family of proteins comprises, but is not limited to rho, rac, cdc42 and their isotypes, such as RhoA, RhoB, RhoC, as well as Rho-associated kinases that are expressed in neural tissue. Other members of the Rho family that are determined and whose inhibition of activity allows for neurite outgrowth are contemplated to be part of this invention. (See, for example, Katoh, H., et al., J. Biol. Chem., 273:2489 2492, 1998; van Leeuwen, F., et al., J. Cell Biol., 139:797 807, 1997; Matsui et al., EMBO J. 15:2208 2216, 1996; Amano et al., Science, 275:1308; Ishizaki, T. et al., (1997) FEBS Lett., 404:118 124).

As used herein, the terms "Rho family member biological activity" refers to cellular events triggered by, being of either biochemical or biophysical nature. The following list is provided, without limitation, which discloses some of the known activities associated with contact-mediated growth inhibition of neurite outgrowth, adhesion to neuronal cells, and promotion of neurite out growth from new born dorsal root ganglion neurons.

As used herein, the term "biologically active", or reference to the biological activity of Rho family members, or polypeptide fragment thereof, refers to a polypeptide that is able to produce one of the functional characteristics exhibited by Rho family members or its receptors described herein. In one embodiment, biologically active proteins are those that demonstrate inhibitory growth activities central nervous system neurons. Such activity may be assayed by any method known to those of skill in the art.

The term C3 refers to C3 ADP-ribosyltransferase, a specific Rho inactivator. A preferred representative example is C3 ADP-ribosyltransferase, a 23 KDa exoenzyme secreted from certain strains of types C and D from Clostridium botulinum, which specifically ADP-ribosylates the rho family of these GTP-binding proteins. This ADP-ribosylation occurs at a specific asparagine residue in their putative effector domain, and presumably interferes with their interaction with a putative effector molecule downstream in signal transduction. Numerous references describing these compounds can be found in Methods in Enzymology, Vol 256, Part B, Eds.; W. E. Balch, C. H. Der, and A. Hall; Academic Press, 1995, for e.g. Pgs. 196 206, 207 et seq, 184 189 and 174 et seq.

Based on the present evidence that Rho family members can affect growth inhibitory protein signals in myelin, the means exist to identify agents and therapies that suppress myelinmediated inhibition of nerve growth. Further, one can exploit the growth inhibiting properties of Rho family members, or Rho family members antagonists, to suppress undesired nerve growth. Without the critical finding that a Rho family member has growth inhibitory properties, these strategies would not be developed.

Rho Family Member Antagonists and Assay Methods to Identify Rho Family Members Antagonists

In one embodiment, Rho family member antagonists will be inhibitors of GTPase activity. The GTP/GDP cycle of Rho family members activation/inactivation is regulated by a number of exchange factors. Compounds that block exchange, thereby inactivating Rho family members are preferred embodiments of this invention.

In another embodiment suitable Rho family member antagonist candidates are developed comprising fragments, analogs and derivatives of Rho family members. Sequences for Rho family members are known, such as those described: Chardin, P., et al., (1988) Nucleic Acids Research, 16:2717; Yeramian, et al., (1987) Nucleic Acids Research, 15: 1869). Such candidates may interfere with Rho family members-mediated growth inhibition as competitive but non-functional mimics of endogenous Rho family members. From the amino acid sequence of Rho family members and from the cloned DNA coding for it, it will be appreciated that Rho family members fragments can be produced either by peptide synthesis or by recombinant DNA expression of either a truncated domain of Rho family members, or of intact Rho family members could be prepared using standard recombinant procedures, that can then be digested enzymically in either a random or a site-selective manner. Analogs of Rho family members or Rho family members fragments can be generated also by recombinant DNA techniques or by peptide synthesis, and will incorporate one or more, e.g. 1 5, L- or D-amino acid substitutions. Derivatives of Rho family members, Rho family members fragments and Rho family members analogs can be generated by chemical reaction of the parent substance to incorporate the desired derivatizing group, such as N-terminal, C-terminal and intra-residue modifying groups that have the effect of masking or stabilizing the substance or target amino acids within it.

In specific embodiments of the invention, candidate Rho family member antagonists include those that are derived from a determination of the functionally active region(s) of Rho family member. The antibodies mentioned above and any others to be prepared against epitopes in Rho family members, when found to be function-blocking in in vitro assays, can be used to map the active regions of the polypeptide as has been reported for other proteins (for example, see Fahrig, et al., (1993) Europ. J Neurosci., 5 1118 1126; Tropak, et al., (1994) J. Neurochem., 62, 854 862). Thus, it can be determined which regions of Rho family members GTPases recognized by substrate molecules that are involved in inhibition of neurite outgrowth. When those are known, synthetic peptides can be prepared to be assayed as candidate antagonists of the Rho family members effect. Derivatives of these can be prepared, including those with selected amino acid substitutions to provide desirable properties to enhance their effectiveness as antagonists of the Rho family members candidate functional regions of Rho family members can also be determined by the preparation of altered forms of the Rho family members domains using recombinant DNA technologies to produce deletion or insertion mutants that can be expressed in various cell types as chimeric proteins. All of the above forms of Rho family members, and forms that may be generated by technologies not limited to the above, can be tested for the presence of functional regions that inhibit or suppress neurite outgrowth, and can be used to design and prepare peptides to serve as antagonists.

In accordance with an aspect of the invention, the Rho family member antagonist is formulated as a pharmaceutical composition which contains the Rho family member antagonist in an amount effective to suppress Rho family member-mediated inhibition of nerve growth, in combination with a suitable pharmaceutical carrier. Such compositions are useful, in accordance with another aspect of the invention, to suppress Rho family member-inhibited nerve growth in patients diagnosed with a variety of neurological disorders, conditions and ailments of the PNS and the CNS where treatment to increase neurite extension, growth, or regeneration is desired, e.g., in patients with nervous system damage. Patients suffering from traumatic disorders (including but not limited to spinal cord injuries, spinal cord injuries, spinal cord lesions, surgical nerve lesions or other CNS pathway lesions) damage secondary to infarction, infection, exposure to toxic agents, malignancy, paraneoplastic syndromes, or patients with various types of degenerative disorders of the central nervous system can be treated with such Rho family members antagonists. Examples of such disorders include but are not limited to Strokes, Alzheimer's disease, Down's syndrome, Creutzfeldt-Jacob disease, kuru, Gerstman-Straussler syndrome, scrapie, transmissible mink encephalopathy, Huntington's disease, Riley-Day familial dysautonomia, multiple system atrophy, amylotrophic lateral sclerosis or Lou Gehrig's disease, progressive supranuclear palsy, Parkinson's disease and the like. The Rho family members antagonists may be used to promote the regeneration of CNS pathways, fiber systems and tracts. Administration of antibodies directed to an epitope of Rho family member, or the binding portion thereof, or cells secreting such antibodies can also be used to inhibit Rho family member function in patients. In a particular embodiment of the invention, the Rho family members antagonist is used to promote the regeneration of nerve fibers over long distances following spinal cord damage.

In another embodiment, the invention provides an assay method adapted to identify a Rho family member antagonists, that is agents that block or suppress the growth-inhibiting action of Rho family members. In its most convenient form, the assay is a tissue culture assay that measures neurite out-growth as a convenient end-point, and accordingly uses nerve cells that extend neurites when grown on a permissive substrate. Nerve cells suitable in this regard include neuroblastoma cells of the NG108 lineage, such as NG108 15, as well as other neuronal cell lines such as PC12 cells (American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA, ATCC Accession No. CRL 1721), human neuroblastoma cells, and primary cultures of CNS or PNS neurons taken from embryonic, postnatal or adult animals. The nerve cells, for instance about 10.sup.3 cells-microwell or equivalent, are cultured on a growth permissive substrate, such as polylysine or laminin, that is over-layed with a growth-inhibiting amount of Rho family members. The Rho family members incorporated in the culture are suitable myelin-extracted Rho family members, although forms of Rho family members other than endogenous forms can be used provided they exhibit the Rho family members property of inhibiting neuron growth when added to a substrate that is otherwise growth permissive.

In this assay, candidate Rho family member antagonists, i.e., compounds that block the growth-inhibiting effect of Rho family members, are added to the Rho family member containing tissue culture preferably in amount sufficient to neutralize the Rho family member growth-inhibiting activity, that is between 1.5 and 15 .mu.g of Rho family members antagonist per well containing a density of 1000 NG108-15 cells/well cultured for 24 hr. in Dulbecco's minimal essential medium. After culturing for a period sufficient for neurite outgrowth, e.g. 3 7 days, the culture is evaluated for neurite outgrowth, and antagonists are thereby revealed as those candidates, which elicit neurite outgrowth. Desirably, candidates selected as Rho family members antagonists are those which elicit neurite outgrowth to a statistically significant extent, e.g., in at least 50%, more desirably at least 60%, e.g. 70%, per 1,000 cultured neurons.

Other assay tests that could be used include without limitation the following: 1) The growth cone collapse assay that is used to assess growth inhibitory activity of collapsin (Raper, J. A., and Kapfhammer, J. P., (1990) Neuron, 2, 21 29; Luo, L., et al., (1993) Cell 75, 217 227) and of various other inhibitory molecules (Igarashi, M., et al., (1993) Science 259, 77 79) whereby the test substance is added to the culture medium and a loss of elaborate growth cone morphology is scored. 2) The use of patterned substrates to assess substrate preference (Walter, J. et al., (1987) Development 101, 909 913; Stahl, et al, (1990) Neuron 5, 735 743) or avoidance of test substrates (Ethell, D. W., et al., (1993) Dev. Brian Res. 72, 1 8). 3) The expression of recombinant proteins on a heterologous cell surface, and the transfected cells are used in co-culture experiments. The ability of the neurons to extend neurites on the transfected cells is assessed (Mukhopadhyay et al., (1994) Neuron 13, 757,767). 4) The use of sections of tissue such as sections of CNS white matter, to assess molecules that may modulate growth inhibition (Carbonefto, S., et al., (1987) J. Neuroscience 7, 610 620; Salvo, T. and Schwab, M. E., (1989) J. Neurosci., 9:1126 1133). 5) Neurite retraction assays whereby test substrates are applied to differentiated neural cells for their ability to induce or inhibit the retraction of previously extended neurites (Jalnink, et al., (1994) J. Cell Bio. 126, 801 810; Sudan, H. S., et al., (1992) Neuron 8, 363 375; Smalheiser, N., (1993) J. Neurochem. 61, 340 342). 6) The repulsion of cell-cell interactions by cell aggregation assays (Kelm, S., et al., (1994) Current Biology 4, 965 972; Brady-Kainay, S., et al., (1993) J. Cell Biol. 4, 961 972). 7) The use of nitrocellulose to prepare substrates for growth assays to assess the ability of neural cells to extend neurites on the test substrate (Laganeur, C. and Lemmon, V., (1987) PNAS 84, 7753 7757; Dou, C-L and Levine, J. M., (1994) J. Neuroscience 14, 7616 7628).

Diagnostic, Therapeutic and Research Uses for Rho Family Member Antagonists

Rho family member antagonists have uses in diagnostics. Such molecules can be used in assays to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting neurite growth extension, invasiveness, and regeneration. Alternatively, the Rho family member antagonists may be used to monitor therapies for diseases and conditions which ultimately result in nerve damage; such diseases and conditions include but are not limited to CNS trauma, (e.g. spinal cord injuries), infarction, infection, malignancy, exposure to toxic agents, nutritional deficiency, paraneoplastic syndromes, and degenerative nerve diseases (including but no limited to Alzheimer's disease, Parkinson's disease, Huntington's Chorea, amyotrophic lateral sclerosis, progressive supra-nuclear palsy, and other dementias). In a specific embodiment, such molecules may be used to detect an increase in neurite outgrowth as an indicator of CNS fiber regeneration. For example, in specific embodiments, altered levels of Rho family members activity in a patient sample containing CNS myelin can be diagnostic marker for the presence of a malignancy, including but not limited to glioblastoma, neuroblastoma, and melanoma, or a condition involving nerve growth, invasiveness, or regeneration in a patient.

Useful for nerve growth suppression are pharmaceutical compositions that contain, in an amount effective to suppress nerve growth, Rho family member antagonist in combination with an acceptable carrier. Candidate Rho family members antagonists include fragments of Rho family members that incorporate the ectodomain, including the ectodomain per se and other N- and/or C-terminally truncated fragments of Rho family members or the ectodomain, as well as analogs thereof in which amino acids, e.g. from 1 to 10 residues, are substituted, particularly conservatively, and derivatives of Rho family members or Rho family members fragments in which the N- and/or C-terminal residues are derivatized by chemical stabilizing groups.

In a preferred embodiment, mutated forms of Rho family members are used as antagonists. One key example is Rho with a mutated effector domain, A-37, which prevents GTP exchange. Various other mutations of the Rho protein that create dominate negative Rho which can interfere with the biological activity of endogenous Rho in neurons are considered as antagonists within the scope of this invention to inactivate Rho, thereby fostering growth of neurons. In another preferred embodiment GDP dissociation inhibitors (GDIs), which inhibit the dissociation of GDP from Rho, and thereby prevent the binding of GTP necessary for the activation of Rho are used as antagonists.

In yet another preferred embodiment, GTPase activating protein (GAP) which facilitates the conversion of the GTP-bound active Rho to the GDP-bound inactive form forms the target for regulation of Rho activity. Thus, compounds that activate GAP, thereby facilitating the conversion of active Rho into inactive Rho.

In still another preferred embodiment, compounds that affect Rho binding to the plasma membrane, thereby decreasing the activity of Rho are also considered Rho antagonists of this invention. In this case, the target design is based on the knowledge that Rho is found in the cytoplasm complexed with GTPase inhibiting protein (GDI). To become active, Rho binds GTP and is translocated to the membrane. Thus, agents that induce Rho binding to the plasma membrane would decrease Rho activity.

In specific embodiments of the invention, candidate Rho family members antagonists include specific regions of the Rho family members molecule, and analogs or derivatives of these. These can be identified by using the same technologies described above for identification of Rho family members regions that serve as inhibitors of neurite outgrowth.

The Rho family members related derivatives, analogs, and fragments of the invention can be produced by various methods known in the art. The manipulations, which result in their production can occur at the gene or protein level. For example, Rho family members-encoding DNA can be modified by any of numerous strategies known in the art (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982), such as by cleavage at appropriate sites with restriction endonuclease(s), subjected to enzymatic modifications if desired, isolated, and ligated in vitro.

Additionally, the Rho family members-encoding gene can be mutated in-vitro or in-vivo for instance in the manner applied for production of the ectodomain, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in-vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in-vitro site directed mutagenesis (Hutchinson, et al., (1978) J. Biol. Chem. 253, 6551), use of TAB.TM. linkers (Pharmacia), etc.

For delivery of Rho family members antagonists, various known delivery systems can be used, such as encapsulation in liposomes or semipermeable membranes, expression in suitably transformed or transfection glial cells, oligodendroglial cells, fibroblasts, etc. according to the procedure known to those skilled in the are (Lindvall, et al., (1994) Curr. Opinion Neurobiol. 4, 752 757). Linkage to ligands such as antibodies can be used to target delivery to myelin and to other therapeutically relevant sites in-vivo. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal routes, and transfusion into ventricles or a site of operation (e.g. for spinal cord lesions) or tumor removal. Likewise, cells secreting Rho family members antagonist activity, for example, and not by way of limitation, hybridoma cells encapsulated in a suitable biological membrane may be implanted in a patient so as to provide a continuous source of Rho family members inhibitor.

Therapeutic Uses of Rho family Antagonists

In an embodiment, antagonists, derivatives, analogs, inhibitors of Rho family members can be used in regimens where an increase in neurite extension, growth, or regeneration is desired, e.g., in patients with nervous system damage. Patients suffering from traumatic disorders (including but not limited to spinal cord injuries, spinal cord lesions, or other CNS pathway lesions), surgical nerve lesions, damage secondary to infarction, infection, exposure to toxic agents, malignancy, paraneoplastic syndromes, or patients with various types of degenerative disorders of the central nervous system can be treated with such inhibitory protein antagonists. Examples of such disorders include but are not limited to Alzheimer's Disease, Parkinson's Disease, Huntington's Chorea, amyotrophic lateral sclerosis, progressive supranuclear palsy and other dementias. Such antagonists may be used to promote the regeneration of CNS pathways, fiber systems and tracts. Administration of antibodies directed to an epitope of, (or the binding portion thereof, or cells secreting such as antibodies) can also be used to inhibit Rho family members protein function in patients. In a particular embodiment of the invention, antibodies directed to Rho family members may be used to promote the regeneration of nerve fibers over long distances following spinal cord damage.

Various delivery systems are known and can be used for delivery of antagonists or inhibitors of Rho family members and related molecules, e.g., encapsulation in liposomes or semipermeable membranes, expression by bacteria, etc. Linkage to ligands such as antibodies can be used to target myelin associated protein-related molecules to therapeutically desirable sites in vivo. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal routes, and infusion into ventricles or a site of operation (e.g. for spinal cord lesions) or tumor removal.

In addition, any method which results in decreased synthesis of Rho family members may be used to diminish their biological function. For example, and not by way of limitation, agents toxic to the cells which synthesize Rho family members and/or its receptors (e.g. oligodendrocytes) may be used to decrease the concentration of inhibitory proteins to promote regeneration of neurons.
 

Claim 1 of 8 Claims

1. A method of promoting neural growth, the method comprising delivery to a central nervous system tissue of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)-cyclohexane.
 

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