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Link:  Pharm/Biotech Resources


Title:  Neurogenic compositions and methods

United States Patent:  6,903,188

Issued:  June 7, 2005

Inventors:  Lukanidin; Eugene (Copenhagen, DK); Bock; Elisabeth Marianne (Charlottenlund, DK); Berezin; Vladimir (Copenhagen, DK)

Assignee:  Prolifia Inc. (Tucson, AZ)

Appl. No.:  781509

Filed:  February 12, 2001

Abstract

The present invention has found that the Mts1 protein is expressed in white matter astrocytes in the spinal cord. Such expression is significantly increased following sciatic nerve injury or dorsal root injury, particularly in astrocytes surrounding dorsal funiculus containing the central processes of the injured primary sensory neurons. The present invention has further demonstrated that Mts1 proteins administered extracellularly promote neurite outgrowth from neuronal cells. Based on these surprising findings, the present invention provides compositions and methods that are useful for the treatment of various neurological conditions characterized by death, degeneration or injury of neuronal cells.

Description of the Invention

FIELD OF INVENTION

The present invention relates to the discovery of the role of the Mts1/S100A4 protein in the neural system. Compositions and methods are provided that are useful for stimulating growth of neuronal cells and treating neuronal damage caused by disease or trauma.

BACKGROUND OF THE INVENTION

The S100 proteins comprise a large family of calcium-binding proteins, some of which are expressed at high levels in the nervous system. The S100 proteins have been implicated in a wide variety of functions, such as modulation of enzyme function, alteration of cytoskeletal dynamics, cell adhesion and control of cell cycle progression (Schafer et al., Trends Biochem Sci 21: 134-140, 1996). Expression of S100 protein has been shown to be associated with invasive potential and metastatic spread of tumor cells (Inoue et al., Virchows Arch A422: 351-355, 1993).

The primary structure of S100 proteins is highly conserved (Kligman et al., TIBS 13: 437-443, 1988; and Schaefer et al., TIBS 21: 134-140, 1996). In solutions S100 proteins easily form dimers and cystein residues are not necessary for the noncovalent dimerization of S100 (Mely et al., J. Neurochemistry 55: 1100-1106, 1990; Landar et al., Biochim. Biophys. Acta 1343: 117-129, 1997; and Raftery et al., J Am. Soc. Mass Spectrom. 9: 533-539, 1988). The tertiary structure of S100 proteins has been characterized (Kilby et al., Structure 4: 1041-1052, 1996; Smith et al., Structure 6: 211-222, 1998; Sastry et al., Structure 15: 223-231, 1998; and Matsumura et al, Structure 6: 233-241, 1998). Each S100 monomer contains two EF-hand calcium binding domains (Schafer et al., TIBS 21: 134-140, 1996). Calcium binding results in a conformational alteration and exposure of a hydrophobic patch via which S100 proteins interact with their targets (Smith et al, Structure 6: 211-222, 1998; Sastry et al, Structure 15: 223-231, 1998; Matsumura et al, Structure 6: 233-241, 1998; and Kilby et al., Protein Sci. 6: 2494-2503, 1997).

Intracellular and extracellular activities of S100 proteins have also been described (McNutt, J Cutan. Pathol. 25: 521-529, 1988). Intracellular S100 proteins interact with numerous target proteins and modulate multiple cellular processes regulating cell growth, differentiation, metabolism and cytoskeletal structure (Zimmer et al., Brain Res. Bulletin 37: 417-429, 1995; Schafer et al., TIBS 21: 134-140, 1996; Donato, Cell Calcium 12: 713-726, 1991; and Lukanidin et al., In: Gunter U, Birchmeier W, eds. Current Topics in Microbiology and Immunology: Attempts to Understand Metastasis Formation II. Berlin, Heidelberg: Springer-Verlag 213/II, 171-195, 1996). Extracellular disulfide-linked dimers of S100B protein have been reported to stimulate neurite outgrowth in primary cultures of cerebral cortex neurons (Kligman et al., TIBS 13: 437-443, 1988). Such activity has also been reported for oxidized form of the recombinant S100B protein (Winningham-Major et al., J. Cell Biol. 109: 3063-3071, 1989).

The mts1/S100A4 gene, a member of the S100 gene family, was isolated as a gene specifically expressed in metastatic murine tumor cell lines (Ebralidze et al., Genes Dev. 3: 1086-1092, 1989). Studies of Mts1-transfected non-metastatic murine cell lines and Mts1 transgenic mice both indicate that Mts1 plays an important role in tumor progression (Grigorian et al., Gene 135: 229-238, 1993; Takenaga et al., Oncogene 14: 331-337, 1997; Ambartsumian et al., Oncogene 13: 1621-1630, 1996; and Davies et al., Oncogene 13: 1631-1637, 1996). Mts1 has also been shown to affect the cytoskelton and cell motility (Takenaga et al., Jpn. J Cancer Res. 85: 831-839, 1994) via association with stress fibers (Gibbs et al., J. Biol. Chem. 269: 18992-18999, 1994). The heavy chain of non-muscle myosin (MHC) has been identified as a target for the Mts1 protein (Kriajevska et al., J. Biol. Chem. 239: 19679-19682, 1994).

The present invention identifies, for the first time, the neurogenic function of the Mts1 protein. Accordingly, the present invention provides novel compositions and methods useful for stimulating neurite growth in the treatment of neural damage caused by disease or physical trauma.

SUMMARY OF THE INVENTION

One embodiment of the present invention provides an isolated functional derivative of an Mts1 protein. A preferred functional derivative of an Mts1 protein is Mts1 del75.

Another embodiment of the present invention provides an isolated multimeric Mts1 protein complex. Such complex includes at least three Mts1 protein molecules or functional derivatives thereof.

In another embodiment, the present invention provides pharmaceutical compositions which include an isolated functional derivative of an Mts1 protein, or a multimeric Mts1 protein complex, and a pharmaceutically acceptable carrier. The pharmaceutical compositions can also include one or more neurotropic factors.

In a further embodiment, the present invention provides methods of stimulating growth of neuronal cells by administering an Mts1 protein or a functional derivative thereof.

In a further embodiment, the present invention provides methods of treating neurological conditions in a subject by administering to the subject a therapeutically effective amount of an Mts1 protein or a nucleotide sequence encoding an Mts1 protein. The methods of the present invention can be employed in the treatment of a variety of neurological conditions characterized by neuronal degeneration, neuronal death or injury caused by disease, physical trauma or ischemic conditions. Such neurological conditions include Parkinson's disease, Down's Syndrome, Alzheimer's disease, stroke, cardiac arrest, sciatic crush, spinal cord injury, damaged sensory neurons in dorsal root ganglia and other tissues, as well as degenerative diseases of the retina.

DETAILED DESCRIPTION OF EMBODIMENTS

The Mts1/S100A4 protein is known in the art to be involved in the control of cell proliferation and metastasis of tumor cells. The present inventor has surprisingly discovered a function of the Mts1/S100A4 protein that is associated with the nervous system.

Specifically, it has been discovered by the present inventor that the Mts1 protein is expressed in white matter astrocytes in the spinal cord. In accordance with the present invention, it has also been found that sciatic nerve injury as well as dorsal root injury induces a marked and prolonged increase in the level of the Mts1 protein, particularly in astrocytes surrounding dorsal funiculus containing the central processes of the injured primary sensory neurons. Additionally, the present invention demonstrates that Mts1 proteins administered extracellularly promote neurite outgrowth from neuronal cells.

Accordingly, the present invention employs the neurogenic activity of the Mts1 protein and provides compositions and methods that are useful for the treatment of various neurological conditions characterized by the death, degeneration or injury of neuronal cells.

By "neurogenic activity" is meant a biological activity that induces, stimulates, or enhances the growth, maintains the survival, or prevents the death of the neuronal cells of the central and peripheral nervous system of a mammal. The activity can manifest as differentiation of neurons, extension of neuritic processes (i.e., outgrowth or elongation of neurites), or innervation of neuritic processes into a tissue.

One embodiment of the present invention provides an isolated functional derivative of an Mts1 protein.

"An Mts1 protein" as used herein, refers to a wild type Mts1 protein of a mammalian origin, such as human, murine and the like. Preferred Mts1 proteins of the present invention include human Mts1 (SEQ ID NO: 1) and murine Mts1 (SEQ ID NO: 2), which are also described in U.S. Pat. No. 5,801,142 and Ebralidze et al., Genes Dev. 3: 1086-1092, 1989, respectively.

"A functional derivative of an Mts1 protein" refers to a modified Mts1 protein having one or more amino acid substitutions, deletions or insertions, which retains substantially the neurogenic activity of a wild type Mts1 protein. By substantially is meant at least about 35%, preferably, at least about 40%.

In accordance with the present invention, a preferred functional derivative of a wild type Mts1 protein is Mts1-del75, i.e., deletion of the Tyr residue at the position 75 in human or murine Mts1 protein, or the corresponding Tyr in any other mammalian Mts1 proteins. It has been determined by the present inventor that Mts1-del75 is able to form polymers and confers about 70% neurogenic activity compared to a wild type Mts1 protein. Another Mts1 mutant which has all four Cysteine residues mutated to Serine (designated herein as "4S") retains about 40% of the neurogenic activity of a wild type Mts1 protein.

Those skilled in the art can use any of the well-known molecular cloning techniques to generate Mts1 derivatives having one or more amino acid substitutions, deletions or insertions. See, for example, Current Protocols in Molecular Cloning (Ausubel et al., John Wiley & Sons, New York). Once a modified Mts1 protein is made, such protein can be tested in functional assays to determine whether such modified protein exhibits neurogenic activity.

In accordance with the present invention, the neurogenic activity of an Mts1 protein or protein complex can be determined by a number of assays. A typical functional assay is described in Example 2 hereinbelow. Briefly, an Mts1 protein is added in various doses in the culture medium of neuronal cells, such as hippocampal neuronal cells, or PC-12 cells. The cells can be kept exposed to the protein for a certain period of time and the outgrowth of neurites from the cultured cells are monitored. Parameters such as the length of the longest neurite extension, the number of neurite branches per cell, and the total neurite length per cell, are measured. The determination as to whether a modified Mts1 protein possesses neurogenic activity can be made by comparing these parameters with those values of a wild type Mts1 protein and those values of a control protein without neurogenic activity. Other assays which can be employed for such determination include, e.g., the standard assay of endothelial cell motility in Boyden Chamber.

Another embodiment of the present invention provides an isolated multimeric Mts1 protein complex.

In accordance with the present invention, it has been found that the neurogenic activity of Mts1 is associated with the polymeric forms composed of three or more Mts1 protein molecules. Not intending to be bound by any theory, it is proposed herein that the Mts1 protein mediates its neurogenic effects via a cell surface receptor which recognizes polymeric forms of the Mts1 protein.

According to the present invention, the terms "a multimeric Mts1 protein complex" and "a polymeric Mts1 protein complex" as used herein refer to a complex having at least three, i.e., three or more, molecules of an Mts1 protein or a functional derivative of an Mts1 protein. The complex can have a Mw of at least about 30 kd, more preferably, at least about 100 kd, and up to about 200 kd, as determined by, e.g., size-exclusion chromatography.

In accordance with the present invention, the Mts1 protein molecules in the complex can be held together by covalent and/or non-covalent interactions among Mts1 protein molecules. For example, there are four Cys residues in both human and murine Mts1, which can form intramolecular disulfide bonds under appropriate conditions thereby leading to formation of polymeric Mts1 complexes. The present invention also contemplates polymeric Mts1 complexes formed by chemical cross-linking reagents. Chemical cross-linking reagents and use thereof in making multimeric protein complexes are well known in the art. In accordance with the present invention, a Mts1 protein complex having neurogenic activity can be formed through non-covalent interactions among Mts1 molecules as well. For example, the present invention provides that Mts1-4S, while unable to form any intramolecular or intermolecular disulfide bonds, is able to form polymers and confers neurogenic activity at a level of about 40% of that of a wild type Mts1 protein.

The Mts1 complexes of the present invention can be isolated by a variety of methods. For example, an Mts1 protein can be dissolved in solution under conditions that favor the formation of polymers, e.g., a saline solution of about 0.15 M NaCl, pH7.5 with a Mts1 concentration higher than, preferably, 1 mg/ml. Afterwards, the solution can be subjected to an appropriate chromatography procedure using, e.g., Size-Exclusion-Column euqilibrated with a TND buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.5). The Mts1 protein can be eluted using the same TND buffer, and fractions containing polymers can be collected and separated from the fractions containing dimers. Such procedure is described in Example 3 hereinbelow. An Mts1 protein can also be subjected to chemical cross-linking prior to chromatography or fraction procedures. Those skilled in the art can make modifications when appropriate and necessary. in another embodiment, the present invention provides pharmaceutical compositions which include a functional derivative of an Mts1 protein, or an isolated multimeric Mts1 protein complex composed of at least three Mts1 protein molecules.

The pharmaceutical compositions of the present invention can be employed to promote neuronal cell growth or maintain the survival of neuronal cells in the treatment of neurological conditions characterized by the death, degeneration or injury of neuronal cells.

The functional derivative or the protein complex of an Mts1 protein for use in the pharmaceutical compositions can be modified according to procedures known in the art in order to enhance penetration of the blood-brain barrier. For example, U.S. Pat. No. 5,604,198 discloses that a molecule can be conjugated to a hydrophobic carrier which enhances the permeability of the blood brain barrier (BBB). WO 90/14838 teaches chemical modifications of a protein by increasing lipophilicity, altering glycosylation or increasing the net positive charge in order to enhance the BBB permeability of the protein.

According to the present invention, the pharmaceutical compositions can also include one or more neurotropic factors.

Neurotropic factors are proteins which promote the survival of neurons, some of which are also capable of promoting neurite outgrowth and glial cell restoration or inducing cells to secrete other neurotropic factors. Preferred neurotropic factors for use in the present pharmaceutical compositions are those to which a broad range of cell types respond. Examples of preferred neurotropic factors include members of the BDNF/NGF family, such as bFGF (basic fibroblast growth factor), aFGF (acidic fibroblast growth factor), CNTF (ciliary neurotrophic factor), NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), GDNF (glial cell line-derived neurotrophic factor), NT-3 (neurotrophin-3), NT-4/5 (neurotrophin 4/5), IGF-1 (insulin growth factor-I), IGF-II (insulin growth factor-II), and functional peptide fragments thereof. Human neurotropic factors and functional derivatives are preferred.

The active ingredients of the pharmaceutical compositions are preferably provided in a pharmaceutically acceptable carrier. The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the active ingredients contained therein, its use in the pharmaceutical compositions of the present invention is appropriate. Examples of carriers include oils, water, saline solutions, gel, lipids, liposomes, resins, porous matrices, binders, fillers and the like, or combinations thereof. The carrier can also be a controlled release matrix which allows a slow release of the active ingredients mixed or admixed therein. Examples of such controlled release matrix material include, but are not limited to, sustained release biodegradable formulations described in U.S. Pat. No. 4,849,141 to Fujioka et al., U.S. Pat. No. 4,774,091 to Yamashira, U.S. Pat. No. 4,703,108 to Silver et al., and Brem et al. (J. Neurosurg. 74: 441-446, 1991), all of which are incorporated herein by reference.

In accordance with the present invention, a Mts1 functional derivative or an Mts1 polymeric complex can be combined with the carrier in solutions or in solid phase, preferably in a manner that favors the stablization of the polymeric conformation of the Mts1 protein. If the mixing step is to be performed in liquid phase, Mts1 proteins can be dissolved together with a carrier in solutions such as saline (about 0.15 M NaCl pH7.5) with an Mts1 concentration of higher than, preferably, 1 mg/ml. If the mixing is to be performed in solid phase, the Mts1 polymeric proteins can be freeze-dried first to preserve the polymeric conformation, then admixed with the carrier. The mixture can be made in formulations suitable for injections, implantations, inhalations, ingestions and the like.

In a further embodiment, the present invention provides methods of stimulating growth of neuronal cells by administering an Mts1 protein, a functional derivative of an Mts1 protein, or a protein complex thereof, to such neuronal cells.

According to the present invention, an Mts1 protein or a functional derivative or complex thereof, can be administered to neuronal cells that are cultured in vitro. This aspect of the invention is particularly useful in regeneration of neurons for autotransplantation or neuron replacement as an alternative treatment procedure to brains of patients with neurological disorders. Techniques of culturing neurons in vitro fare known in the art and are described in, e.g., U.S. Pat. Nos. 5,483,892, 5,753,506, 5,898,066, and 5,667,978, Mou et al. J. Comp. Neurol. 386: 529 (1997), and Tan et al. Cell Transplant 5: 577 (1996), the teachings of which are incorporated herein by reference.

In a further embodiment, the present invention provides methods of treating neurological conditions in a subject by administering to the subject a therapeutically effective amount of an Mts1 protein, a functional derivative thereof, or a nucleotide sequence encoding an Mts1 protein.

The methods of the present invention can be employed in the treatment of a variety of neurological conditions characterized by neuronal degeneration, neuronal death or injury caused by disease, physical trauma or ischemic conditions. Such neurological conditions include Parkinson's disease, Alzheimer's disease, Down's Syndrome, stroke, cardiac arrest, sciatic crush, spinal cord injury, multiple sclerosis, peripheral neuropathies associated with diabetes, motorneuron diseases, damaged sensory neurons in dorsal root ganglia and other tissues, as well as degenerative diseases of the retina.

By "treating" is meant prevent or inhibit neuronal degeneration or neuronal death, promoting or stimulating neuronal growth such that the symptoms of the disease condition are prevented or alleviated.

In accordance with the methods of the present invention, an Mts1 protein can be first treated to enrich the polymeric forms, or can be used directly, as certain percentage of the molecules spontaneously associate with each other to form polymers in solution. An Mts1 protein or a functional derivative thereof can be modified in order to enhance penetration of the blood-brain barrier as described hereinabove.

Nucleic acid sequences encoding an Mts1 protein can also be employed in the methods of the present invention. Such sequences are preferably provided in an expression vector. Expression vectors for use in the present methods include any appropriate gene therapy vectors, such as nonviral (e.g., plasmid vectors), retroviral, adenoviral, herpes simplex viral, adeno-associated viral, polio viruses and vaccinia vectors. Examples of retroviral vectors include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV)-derived recombinant vectors. Multiple teachings of gene therapy are available to those skilled in the art, e.g., W. F. Anderson (1984) "Prospects for Human Gene Therapy" Science 226: 401-409; S. H. Hughes (1988) "Introduction" Current Communications in Molecular Biology 71: 1-12; T. Friedman (1989) "Progress Toward Human Gene Therapy" Science 244: 1275-1281 and W. F. Anderson (1992) "Human Gene Therapy" Science 256: 608-613. Preferred vectors include neurotropic vectors such as herpes simplex viral vectors (U.S. Pat. No. 5,673,344 to Kelly et al. and adenoviral vectors (Barkats et al., Prog. Neurobiol. 55: 333-341, 1998).

Mts proteins or Mts1-encoding nucleic acid molecules can be used alone or in conjunction with one or more neurotropic factors described hereinabove, including members of the BDNF/NGF family such as bFGF, aFGF, CNTF, NGF, BDNF, GDNF, NT3, NT4/5, IGF-1 and IGF-II, as well as the functional peptide fragments identified thereof. Human neurotropic factors are preferred for treating a human subject.

The therapeutically active ingredients, i.e., Mts1 proteins or nucleic acid molecules, alone or in conjunction with neurotropic factors, can be combined with a pharmaceutically acceptable carrier and prepared in formulations suitable for injections, implantations, inhalations, ingestions and the like. Pharmaceutically acceptable carriers are described hereinabove and include oils, water, saline solutions, gel, lipids, liposomes, resins, porous matrices, binders, fillers and the like, or combinations thereof.

According to the present invention, these therapeutic compositions can be administered to the subject being treated by standard routes, including the oral, ophthalmic nasal, topical, transdermal, parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular), intracranial, intracerebral, intraspinal, intravaginal, intrauterine, or rectal route. Depending on the condition being treated, one route may be preferred over others, which can be determined by those skilled in the art. For example, topical route can be chosen when the target area includes tissues or organs readily accessible by topical application, such as neurological conditions of the eye or the facial tissue. For certain conditions, direct injection or surgical implantation in the proximity of the damaged tissues or cells may be preferred in order to avoid the problems presented by BBB. Successful delivery to CNS (Central Nervous System) by direct injection or implantation has been documented. See, e.g., Otto et al., J. Neurosci. Res. 22: 83-91 (1989); Goodman & Gilman's The Pharmacological Basis of Therapeutics, 6th ed, pp244; Williams et al., Proc. Natl. Acad. Sci. USA 83: 9231-9235 (1986); and Oritz et al., Soc. Neurosci. Abs. 386: 18 (1990).

According to the present invention, the therapeutic ingredients are preferably administered to the subject in need thereof as early as possible after the neuronal injury or death occurs in order to achieve the best therapeutic efficacy.

The amount of an Mts1 protein, a functional derivative, or an Mts1-encoding nucleic acid molecule to be therapeutically effective depends on the disease state or condition being treated and other clinical factors, such as weight and physical condition of the subject, the subject's response to the therapy, the type of formulations and the route of administration. The precise dosage to be therapeutically effective and non-detrimental to the subject can be determined by those skilled in the art. As a general rule, the therapeutically effective amount of Mts1 protein can be in the range of about 0.01 mg to about 10 mg per kilogram of body weight; preferably, in the range of about 0.1 mg to about 5 mg per kilogram of body weight. The therapeutically effective dosage of an Mts1 protein can be in the range of about 0.5 μg to about 2 mg per unit dosage form. A unit dosage form refers to physically discrete units suited as unitary dosages for mammalian treatment: each unit containing a pre determined quantity of the active material calculated to produce the desired therapeutic effect in association with any required pharmaceutical carrier. The methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.

Claim 1 of 13 Claims

1. An isolated multimeric Mts1 protein complex, comprising at least three molecules of an Mts1 protein.

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