A method of treating brain tissue damage, including administering to a subject in need thereof an effective amount of secretoneurin. Disclosed are methods of promoting angiogenesis or neurogenesis in the brain of subject. Also disclosed are a method of homing of stem cells to the brain of a subject and a method of protecting a neuronal cell from cell death.

Description of the Invention

BACKGROUND

Brain tissue damage, resulting either from injuries or disorders (e.g., neurodegenerative and cerebrovascular diseases), is a leading cause of long-term disability. Due to their pluripotency, embryonic stem cells (ES cells) hold a great promise for treating brain tissue damage (Taguchi et al., 2004, J. Clin. Invest.; 114(3):330-338). However, ethical and logistical considerations have hampered their use (Barinaga, 2000, Science, 287(5457):1421-1422). Use of non-ES pluripotent cells has also been exploited. They include adult bone marrow mesenchymal stem cells or stromal cells (Sanchez-Ramos et al., 2000, Exp. Neurol., 164(2):247-256 and Woodbury et al., 2000, J. Neurosci. Res., 61(4):364-370) and umbilical cord blood cells (Galvin-Parton et al., 2003, Pediatr. Transplant. 2003; 7(2):83-85 and Ha et al., 2001 Neuroreport., 2(16):3523-3527). Nonetheless, requirements for in vitro expansion and HLA-matching have limited clinical applications of these cells. Thus, there is a need for an alternative method of treating brain tissue damage.

SUMMARY

This invention is based, at least in part, on the discovery that brain tissue damage can be repaired by administration of secretoneurin.

Accordingly, one aspect of this invention features a method of treating brain tissue damage. The method includes administering to a subject in need thereof an effective amount of secretoneurin. The method can be used to treat brain tissue damage caused by a cerebral ischemia, e.g., stroke, or a neurodegenerative disease, e.g., Alzheimer's disease, epilepsy, Huntington's disease, Parkinson's disease, or Spinocerebellar disease. The method may further include administering to the subject an effective amount of stem cells. Suitable stem cells include hematopoietic stem cells, umbilical cord-derived mesenchymal stem cells, and stem cells that are c-kit.sup.+ or CD34.sup.+. Preferably, the stem cells are autologous to the subject. For example, the cells can be enriched from the blood or bone marrow from the subject.

"Treating" refers to administration of a compound or composition to a subject, who is suffering from or is at risk for developing brain tissue damage or a disorder causing such damage, with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate the damage/disorder, the symptom of the damage/disorder, the disease state secondary to the damage/disorder, or the predisposition toward the damage/disorder. An "effective amount" refers to an amount of the compound or composition that is capable of producing a medically desirable result, e.g., as described above, in a treated subject. The treatment method can be performed alone or in conjunction with other drugs or therapies.

This invention also features a method of promoting cerebral angiogenesis in a subject. The method includes administering intracerebrally to a subject in need thereof an effective amount of secretoneurin. The method further includes measuring a level of cerebral angiogenesis in the subject before and after administering secretoneurin to confirm promotion of cerebral angiogenesis. The method can also include administering to the subject an effective amount of stem cells, such as those described above.

Also within the scope of this invention is a method of promoting neurogenesis in the brain of a subject. The method includes administering intracerebrally to a subject in need thereof an effective amount of secretoneurin. The method further includes measuring a level of neurogenesis in the brain of the subject before and after administering promotion to confirm promotion of neurogenesis. For this purpose, one can use standard neurological behavioral measurements including those described in the examples below. The method can also include administering to the subject an effective amount of stem cells, such as those mentioned above.

This invention further features a method of promoting homing of stem cells to the brain of a subject by administering intracerebrally to a subject in need thereof an effective amount of secretoneurin. Suitable stem cells include those described above.

A method of preserving a neuronal cell is also contemplated in this invention. The method of claim includes contacting the cell with secretoneurin.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

DETAILED DESCRIPTION

The present invention relates to treating brain tissue damage using secretoneurin). Secretoneurin, a 33-amino acid neuropeptide (TNEIVEEQYTPQSLATLESVFQELGKLTGPNNQ: SEQ ID NO: 1), is derived from endoproteolytic processing of chromogranin/secretogranins (Cg/Sg) family protein. It is a abundant protein within large dense core vesicles in the endocrine tissue and nervous system (Kirchmair et al. 1993, Neuroscience, 53, 359-365; Saria et al., 1993, Neuroscience, 54, 1-4; Kahler et al., 1996, Eur. J. Pharmacol., 304, 135-139; Fischer-Colbrie et al., 1995, Prog. Neurobiol. 46, 49-70; and Cozzi et al., 1989, Neuroscience 28, 423-41.) As described in the examples below, secretoneurin unexpectedly enhanced the targeting of stem cells to injured brain, protected neurons from cell death, and promoted neurogenesis and angiogenesis in injured brain. Not only did it protect existing neurons or glial cells in the brain, it also facilitated neural regeneration to replace damaged neurons glial cells.

While many secretoneurin preparations can be used to practice this invention, highly purified secretoneurin is preferred. Examples include mammalian secretoneurin (e.g., human, mouse, and rat secretoneurins) or non-mammalian secretoneurin having substantially the same biological activity as mammalian secretoneurin. Naturally occurring secretoneurin, chemically synthesized secretoneurin, and genetically engineered secretoneurin all can be used. Secretoneurin obtained by chemical synthesis or genetic engineering may be that having the same amino acid sequence as naturally occurring secretoneurin or an functionally equivalent there of. A "functional equivalent" refers to a polypeptide derivatives of a naturally occurring secretoneurin (SEQ ID NO: 1), e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It possesses one or more of the activities of secretoneurin, e.g., the ability to protect cells from cell death, to promote angiogenesis or neurogenesis, and to mobilize stem cells from bone marrow into the peripheral blood or into the brain. The term "secretoneurin" also covers chemically modified secretoneurin. Examples of chemically modified secretoneurin include secretoneurin subjected to conformational change, addition or deletion of a sugar chain. Once purified and tested by standard methods, secretoneurin can be administered to a subject as described below.

To practice the treatment method of this invention, one can administer secretoneurin via intracerebral injection. A sterile injectable composition (e.g., aqueous or oleaginous suspension) can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or di-glycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents.

A carrier in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the active ingredient of the formulation (and preferably, capable of stabilizing it) and not deleterious to the subject to be treated. For example, solubilizing agents, such as cyclodextrins (which form specific, more soluble complexes with one or more of active compounds of the extract), can be utilized as pharmaceutical excipients for delivery of the active compounds. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.

Suitable in vitro assays can be used to preliminarily evaluate the efficacy of a secretoneurin preparation. For example, one can measure the neuroprotective effect of the preparation. More specifically, the preparation can be added to a suitable cell culture (e.g., a primary cortical cell culture of rat or mouse neuron-glial cells as described in Example 1 below) and the level of protection is determined. One then compares the level with a control level obtained in the absence of the preparation. If the level of interest is higher than the control, the preparation is identified as being active for treating brain tissue damage. One can also evaluate the efficacy of a secretoneurin preparation by examining the preparation's effects on cell death according to standard methods. For example, one can measure the level of a protein involved in cell-death (e.g., caspase). If the level is lower than that obtained in the absence of the preparation, the preparation is determined to be active.

The preparation can further be examined for its efficacy in mobilizing bone marrow-derived stem cells to the peripheral blood or the brain by an in vivo assay. For example, the preparation can be evaluated in an animal (e.g., a mouse or rat model). The level of mobilized stem cells in the peripheral blood or the brain is determined by standard methods.

The preparation can also be administered to an animal model having brain tissue damage or a disorder causing such damage. The therapeutic effects of the preparation are then evaluated according to standard methods (e.g., those described in Examples 2-8 below). To confirm efficacy in promoting cerebrovascular angiogenesis, one can examine the animal before and after the treatment by standard brain imaging techniques, such as computed tomography (CT), Doppler ultrasound imaging (DUI), magnetic resonance imaging (MRI), and proton magnetic resonance spectroscopy (.sup.1H-MRS).

One can also measure the expression level of a neuronal marker, a vacular marker, a glial marker, a trophic factor, or a cell death-related protein in a sample (e.g., cerebrospinal fluid) obtained from the animal before or after administration of secretoneurin to confirm efficacy. The expression level can be determined at either the mRNA level or the protein level. Methods of measuring mRNA levels in a tissue sample or a body fluid are well known in the art. To measure mRNA levels, cells can be lysed and the levels of mRNA in the lysates, whether purified or not, can be determined by, e.g., hybridization assays (using detectably labeled gene-specific DNA or RNA probes) and quantitative or semi-quantitative RT-PCR (using appropriate gene-specific primers). Alternatively, quantitative or semi-quantitative in situ hybridization assays can be carried out on tissue sections or unlysed cell suspensions using detectably (e.g., fluorescent or enzyme) labeled DNA or RNA probes. Additional mRNA-quantifying methods include the RNA protection assay (RPA) method and the serial analysis of gene expression (SAGE) method, as well as array-based technologies.

Methods of measuring protein levels in a tissue sample or a body fluid are also well known in the art. Some of them employ antibodies (e.g., monoclonal or polyclonal antibodies) that bind specifically to a target protein. In such assays, the antibody itself or a secondary antibody that binds to it can be detectably labeled. Alternatively, the antibody can be conjugated with biotin. Its presence can be determined by detectably labeled avidin (a polypeptide that binds to biotin). Combinations of these approaches (including "multi-layer sandwich" assays) can be used to enhance the sensitivity of the methodologies. Some protein-measuring assays (e.g., ELISA or Western blot) can be applied to body fluids or to lysates of cells, and others (e.g., immunohistological methods or fluorescence flow cytometry) can be applied to histological sections or unlysed cell suspensions. Appropriate labels include radionuclides (e.g., .sup.125I, .sup.131I, .sup.35S, .sup.3H, or .sup.32P) enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or .beta.-glactosidase), fluorescent/luminescent agents (e.g., fluorescein, rhodamine, phycoerythrin, GFP, BFP, and Qdot.TM. nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.). Other applicable methods include quantitative immunoprecipitation or complement fixation assays.

Based on the results from the assays described above, an appropriate dosage range and administration route can be determined. The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.001-100 mg/kg. Dosage variations are necessary in view of the variety of compounds available and the different efficiencies of various routes of administration. The variations can be adjusted using standard empirical routines for optimization as is well understood in the art. In general, secretoneurin can be administered at 10 to 500 .mu.g/day/kg body weight for 2-10 days; preferably, 20 to 200 .mu.g/day/kg body weight for 3-8 day; and, more preferably, 50 to 100 .mu.g/day/kg body weight for 4 to 6 days.

The treatment method of this invention optionally includes administering to a subject an effective amount of stem cells. Both heterologous and autologous stem cells can be used. In the former case, HLA-matching should be conducted to avoid or minimize host reactions. In the latter case, autologous cells are enriched and purified from a subject to be treated before the cells are introduced back to the subject.

In both cases, secretoneurin can be used as the active ingredient to mobilize hematopoietic stem cells (HSCs) out of bone marrow so as to increase the number of stem cells in the peripheral blood, which home to the brain (HSCs, once in the peripheral blood, are called peripheral blood stem cells or PBSC).

Other factors, such as granulocyte-colony stimulating factor (G-CSF), can also be used to mobilize HSCs. In one embodiment, PBSCs are obtained from a subject as follows: A subject is first administered G-CSF to mobilize HSCs from bone marrow into the peripheral blood. After this enriching step, peripheral blood are collected and PBSCs purified. To prepare PBSCs, G-CSF is administered at 10 to 200 .mu.g/day/kg body weight for 2-10 days. The G-CSF can be administered to a subject via any suitable routes. Examples include injection subcutaneously, intramuscularly, and intraperitoneally. PBSCs are generally purified based on their physical and biochemical properties. For example, peripheral blood cells may be concentrated for hematopoietic stem cells by centrifugation, counter-current elutriation, selection with cell surface markers (e.g., CD34.sup.+ or stem cell related antibodies), or removal of lineage positive (committed) hematopoietic cells. Such methods are well known in the art. See e.g., U.S. Pat. Nos. 5,061,620; 5,087,570; 5,061,620; 4,714,680; 4,965,204; and 5,035,994. Details of using G-CSF to obtain greater than 90% purity of PBSCs can be found U.S. application Ser. No. 11/134,613.

Stem cells other than PBSC can be used as long as they possess the potential to differentiate into cells of vascular or neural-glial lineage. Methods of preparing and testing such cells are well-known in the art. Examples includes embryonic stem cells (Thomson et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:7844-7844; 1996, Biol. Reprod. 55:254-259; Shamblott et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:13726; and U.S. Pat. Nos. 6,921,632 and 6,875,607), bone marrow-derived mesenchymal stem cells (Tse, et al., 2000, Journal of the American Society of Hematology, vol. 96, No. 11; Woodbury et al., 2002, J. Neuroscience Res. 96:908-917; Sanchez-Ramos et al., 2000, Exp. Neurol., 164(2):247-256; Woodbury et al., 2000, J. Neurosci. Res., 61(4):364-370); and U.S. Pat. Nos. 5,486,359, 6,355,239), and umbilical cord-derived stem cells (Mitchell et al., 2003, Stem Cells. 2003;21(1):50-60; Galvin-Parton et al., 2003, Pediatr. Transplant. 2003;7(2):83-85; Ha et al., 2001 Neuroreport., 2(16):3523-3527; and U.S. Provisional Application No. 60/706,377.

Purified stem cells are tested and stored by standard techniques. They can be administered intracerebrally to a subject in need thereof. In general, 1.times.10.sup.4 and 1.times.10.sup.6 (e.g., 5.times.10.sup.4 to 8.times.10.sup.6 and more preferably 1.times.10.sup.5 to 6.times.10.sup.5) cells are administered. Multiple sites can be used depending on the site and nature of particular damage. Examples below describes approximate coordinates for administering cells in a rat or a mouse. Coordinates for other disorders in other species can be determined accordingly based on comparative anatomy. Before or after the treatment, a subject can be examined to confirm treatment efficacy. To this end, one can use suitable standard tests or techniques described above and in the examples below (see Original Patent).

Claim 1 of 9 Claims

1. A method of treating brain tissue damage, comprising administering to a subject in need thereof an effective amount of secretoneurin, wherein the secretoneurin is administered intracerebrally into an affected area and the brain tissue damage is caused by cerebral ischemia or stroke.

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