WO2006006337A1 - Preventive or therapeutic agent for neurological disorder - Google Patents

Abstract

[PROBLEMS] To provide a preventive or therapeutic drug for neurological disorders, such as motor neuron degenerative disease and spinal damage. [MEANS FOR SOLVING PROBLEMS] As a result of study of the effect of HDGF on the culture of vertebral tissue of rat, it has been found that HDGF markedly accelerates the extension of neuroaxon and dendrite and promotes the living of vertebral motor neuron in culture system. Thus, there is provided a preventive or therapeutic drug for neurological disorders, comprising hepatoma-derived growth factor (HDGF) as an active ingredient.

Description

 Specification

 Drugs for preventing or treating neurological diseases

 Technical field

 [0001] The present invention relates to a drug for preventing or treating neurological diseases such as motor neuron degenerative diseases and spinal cord injury.

 Background art

 [0002] A hepatoma-derived growth factor (HDGF) is a molecule that has been purified from the culture medium of the hepatoma cell line HuH-7 and subsequently cleaved (Non-patent Document 1) and is present in the nucleus. It is a unique nutritional factor. So far, it has been known that vascular endothelial cysts, smooth muscle cysts, fibroblasts, and hepatoma cells have a mitogenic effect (Non-Patent Documents 2 to 4). It is also known that HDGF acts as a growth factor in many cells, HDGF is mainly expressed in neurons, and HDGF protein is localized in the nucleus (Non-patent Document 2). In addition, hepatoma-induced growth factor-like protein (HDGFX), which is similar to HDGF, has the effect of promoting neuronal development, and can be used to treat and diagnose neurological diseases such as Parkinson's disease and Alzheimer's disease. (Patent Document 1). On the other hand, a method for obtaining a large amount of HDGF protein by genetic engineering has also been attempted (Patent Document 5).

 [0003] Patent Document 1: Special Table 2003-528583

 Patent Document 2: JP-A-11-103859

 Non-Patent Document 1: Nakamura H. et al., JBC 269: 25143-25149 (1994)

 Non-Patent Document 2: Nakamura H. et al "Clin. Chim. Acta. 183: 273-284 (1989)

 Non-Patent Document 3: Everett A.D. et al., J. Clin. Invest. 105: 567-575 (2000)

 Non-Patent Document 4: Oliver J.A. et al., J. Clin. Invest. 102: 1208-1210 (1998)

 Non-Patent Document 5: Zhiwei Zhou et.al., JBC (2004) 279: 27320-27326

 Disclosure of the invention

 Problems to be solved by the invention

[0004] The present invention relates to prevention or treatment of neurological diseases such as motor neuron degenerative diseases and spinal cord injury. Provide drugs for.

 Means for solving the problem

[0005] The present inventors created a transgenic mouse of PQBP-polygnoretamine chain binding protein, which is considered to be involved in the pathology of polyQ disease (Hum. Mol. Genet. 8, 977-987 (1999) candidate molecule). and was examined, late-onset motor neuron disease similar expressions system to show that the force ^s I ChikaraTsuta (Okuda et al., Human Molecular Genetics 2003, vol.12, page 71 1-725,). PQBP- 1 is a nuclear protein that interacts with the mutant ataxin-1 and colocalizes with the mutant huntingtin or ataxin-1 in the aggregate.

 In order to investigate the molecular mechanism of neuronal cell death that exhibits symptoms similar to those of neurodegeneration in human patients, the present inventors have investigated the anterior horn of the spinal cord from this transgenic mouse and the mouse before the onset of symptoms as a control. Microarray analysis using tissue was performed. As a result, it was found that half of the spots changed 1.5 times or more were mitochondrial genome genes. Furthermore, as a result of analysis using an electron microscope, morphological abnormalities were revealed in the mitochondria of spinal motor neurons. Furthermore, overexpression of PQBP-1 in neurons in primary cultures induced hyperpolarization of the mitochondrial membrane potential known to precede apoptosis death (results not shown above). These results indicate that overexpression of PQBP-1 leads to mitochondrial dysfunction, which changes spinal motor neurons and facilitates cell death. It was also found that the expression of HDGF, which has a trophic factor effect in cancer cell lines, has been promoted.

 [0006] Therefore, the present inventor examined the effect of HDGF on spinal cord tissue culture in rats, and HD GF significantly accelerated the extension of neurites and promoted the survival of spinal motor neurons in the culture system. As a result, the present invention has been completed. The accelerated regulation of HDGF plays a role in protecting spinal neurons in the degenerative process, and the function of HDGF in protecting these motor neurons may be useful in the treatment of motor neuron degeneration.

That is, the present invention is a drug for treating or preventing a neurological disease comprising hepatoma cell-derived growth factor (HDGF) as an active ingredient.

This hepatoma cell-derived growth factor (HDGF) consists of the amino acid sequence shown in SEQ ID NO: 1. A protein, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and having a nutritional effect on motor neurons, or a salt thereof. May be.

 The present invention also relates to the use of the prophylactic or therapeutic agent for the manufacture of a prophylactic or therapeutic agent for neurological diseases.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0008] “Hepatoma cell-derived growth factor (HDGF)” refers to a factor that has been purified and cloned from the culture supernatant of the liver cancer cell line HuH-7 using the mitogenic effect on swiss3T3 cells as an indicator.

Five.

 HDGF is derived from (1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or (2) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. Or a protein having a nutritional effect on motor neurons, or (3) a salt thereof.

 [0009] "Motor neurons" constitute the nervous system that moves the muscles directly, and upper motor neurons (those that make pyramidal tracts) and lower motor neurons (large neurons in the anterior horn of the spinal cord project axons onto the muscles. Are). Upper motor neurons project to cerebral cortical power lower motor neurons.

 [0010] The "nutrient effect" refers to an effect that has both the maintenance of survival of nerve cells and the extension of neurites. This trophic effect can be achieved, for example, by culturing rat fetal spinal cord slices in a medium without serum or nutrient factors (Dulbecco's modified eagles medium (DMEM)) in the presence or absence of HDGF (3-30 ng / mL). The effect can be confirmed by observing spinal cord tissue on the 10th day and observing the extension of neurites of motor neurons.

 [0011] The "neurological disorder" that is the target of the preventive or therapeutic agent of the present invention refers to a disease that causes a disorder or a decrease in function of nerve cells, glial cells, neurites, synapses, myelin, etc. due to various pathological conditions. For example, diseases caused by motor neuron degeneration or spinal cord injury.

 “Motor neuron degenerative disease” refers to progressive neurological disease accompanied by motor neuron death.

“Spinal cord injury” refers to tissue damage to the spinal cord and spinal cord root due to trauma. Examples of such neurological diseases include amyotrophic lateral sclerosis (ALS), bulbar spinal muscular atrophy (Kennedy disease), spinocerebellar degeneration (including familial), spinal muscular atrophy (spinal muscula) ratrophy), spinal cord injury (traffic trauma, etc.), muscle atrophy associated with deformed spondylosis, and the like.

 [0012] The preventive or therapeutic agent for neurological diseases of the present invention includes, as necessary, usual pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffering agents, Various preparation ingredients such as a disintegrant, a solubilizer, a solubilizer, and an isotonic agent may be added.

 In addition, the dose of the preventive or therapeutic agent for neurological diseases of the present invention is appropriately selected depending on the purpose of administration (for example, the type of disease), the age and weight of the individual to be administered, the type of active ingredient, and the like. The dosage is such that the active ingredient (HDGF) is, for example, 0.01-100 mg, preferably 3-100 mg.

 Examples of the method for administering the preventive or therapeutic agent for neurological diseases of the present invention include an in vivo method for direct introduction into the body. The dosage form is appropriately selected depending on the purpose of administration (for example, the type of disease), the age and weight of the individual to be administered, the type of active ingredient, and the like.

Nasal mucosa inhalation, cerebrospinal fluid injection and the like. As this dosage form, for example, it may be orally administered in the form of powder, granule, capsule, syrup, lj, suspension or the like, or in the form of solution, emulsion, suspension, etc. These may be administered parenterally in the form of injections or intranasally in the form of sprays.

[0013] In addition, an expression vector may be used to express HDGF in tissues or cells. Examples of such vectors include viral vectors such as lentiviral vectors, herpes virus (HSV) vectors, adenoviral vectors, and human immunodeficiency virus (HIV) vectors. Expression vectors can be constructed based on conventional genetic engineering methods. The expression vector thus constructed can be used as a preventive or therapeutic agent for neurological diseases.

[0014] A peptide having a smaller molecular weight using only an effective domain based on HDGF, a fusion substance of a peptide and a carrier, and a substance mimicking the peptide may be used as a therapeutic agent. While the invention is illustrated, it is not intended to limit the invention Absent.

[0015] Test Example 1

 First, a PQBP_1 (polyglutamine chain binding protein) transgenic mouse was prepared as follows.

 pCAGGS (Niwa et al, Gene 1991, vol. 108, 193-200) donated with human PQBP-1 full-length cDNA (Hum. Mol. Genet. 1999, vol. 8, page 977-987 Waragai et al.) The Sall-Hindm fragment 3.1 kb of the resulting plasmid was excised and injected into fertilized eggs of C57BL / 6.

[0016] In order to examine the protein expression of HDGF in this transgenic mouse, two types of antibodies against human and rat C-terminal sequences were prepared by the following procedure. Immunize rabbits with the peptide KEEAEAPGVRDHESL (C-terminal 15 amino acids of mouse H DGF, SEQ ID NO: 2) or peptide KEDAEAPGIRDHESL (C-terminal 15 amino acids of human HDG F, SEQ ID NO: 3) conjugated with a carrier protein via cysteine Serum was obtained. Antibodies for mice were designated as mC15-l and mC15-2, and antibodies for humans were designated as hC15_l and hCl5-2.

 [0017] In order to test the specificity of this antibody and examine the effect of HDGF on neurons, recombinant mouse HDGF was prepared by the following procedure.

 To express GST-HDGF fusion protein, PCR-amplified mouse full length HDGF cDNA (SEQ ID NO: 5) using HDGF-F primer: AAAGGGATC CGATCCAACCGGCAGAAAGAG (SEQ ID NO: 4) and HDGF-R primer: AAAGAA TTCTACAGGCTCTCATGATCTCT (SEQ ID NO: 5). 6, BC005713) was subcloned between the BamHI and EcoRI restriction enzyme sites of pGEX-3X (Amersham Biosciences). This subcloning changed the first two N-terminal amino acids of HDGF from M-S-to G-H. After induction for 5 hours with 0.1 mM ITPG, E. coli was collected by centrifugation and sonicated. Recombinant HDGF (GST-HDGF) was recovered from the lysate using a glutathione 'Sepharose 4B column (Amersham Biosciences).

HDGF is cleaved from the fusion protein by factor Xa and purified by Hei'rap Heparin Hp. Amersham Biosciences. [0018] Four antibodies against HDGF were characterized by Western plots using extracts of bacterial or non-expressing butterflies expressing the fusion protein. The results are shown in Fig. 1. It was confirmed that all four antibodies mentioned above detected HDGF. The specificity of these antibodies was repeatedly confirmed. Since mC15-2 and hC15_2 antibodies detected a weak non-specific band in the Bacteria extract by prolonged exposure of the film, mC15-1 or hC15-1 was used thereafter.

 [0019] Western blotting with mC15-l was performed using spinal cord tissue of a transgenic mouse. The result is shown in Fig.2. It can be seen that the 37 kD band corresponding to HDGF is increased in the spinal cord of transgenic mice (Tg). That is, HDGF protein expression is increased in the anterior horn of the spinal cord of Transgene mice.

 [0020] Test Example 2

 To further verify whether HDGF protein is increased in motor neurons, we next performed immunohistochemistry (IHC) of lumbar spinal cord tissue in PQBP-1 transgenic mice and control mice as follows: .

 Mice were perfused via the heart with chilled 4% paraformaldehyde (PFA), and the brain, spinal cord and muscle were fixed after treatment with 4% PFA. Paraffin sections of 5-10 μm were deparaffinized with xylene and dehydrated using an ethanol dilution series. Endogenous peroxidase was inactivated by treatment with 0.3% hydrogen peroxide in PBS for 30 minutes. Paraffin sections were treated with 3% goat serum for 30 minutes for HDGF staining. As primary antibodies, anti-HDGF ′ C-terminal polyclonal rabbit heron serum (diluted 1: 1000) and / or anti-GFAP rabbit rabbit antibody (Chem icon, 1: 500) were used. HRP-conjugated anti-rabbit antibody (Envision, DAKO) was used as a secondary antibody and developed with diaminobenzidine (DAB, Sigma).

The results are shown in FIG. In the high-magnification image (second and fourth lines in Fig. 3), HDGF is strongly stained in the nucleus of the motor neuron (white arrow) in the transgenic mouse, whereas the cytoplasm is mostly in the control mouse. Strongly stained in motor neurons (black arrow). HDGF is expressed in the nucleus of neurons and glial cells. The percentage of motor neurons that are nucleus (stained) positive in the anterior horn neurons is 17% (4/24) in the control mouse (control) and 89% (17/19) in the transgenic mouse (Tg). At It was. In other parts of the spinal neurons, staining differences were not clear between the two groups. The staining was similar for the Darial cells.

 These data forces show that HDGF is specifically regulated by PQBP_1 in the motor neurons of the degenerative spinal cord.

 Example 1

 [0022] In this example, the ability of HDGF to have a trophic factor effect on spinal motor neurons was examined. Since HDGF is mitogenic for non-neuronal cells (Clin. Chim. Acta. 18 3, 273-284 (1989), etc.), it is speculated that HDGF protects the survival of motor neurons under metamorphic stress. In order to verify this hypothesis, the following experiment was conducted.

 [0023] The spinal cord of an E14Sprague-Dawley rat fetus was dissected, and the dorsal root of the spinal ganglion and the meninges were excised. The ventral half of the spinal cord was separated under a microscope and cut into 1 mm cubes, while the dorsal half of the spinal cord was discarded. The abdominal tissue was covered with poly-L-lysine and inoculated overnight with DMEM, seeded in a 35 mm dish (Falcon), and cultured at 37 ° C under 5% CO.

 Recombinant HDGF (GST-HDGF) was added at three HDGF concentrations of 3, 10, and 30 ng / ml, and nerve axon promoting activity was assayed. Six to ten transplants were used for each concentration. The medium was changed every other day.

 As a control, a purified motta solution with unfused GST protein strength was also added.

 Transplant axon elongation was measured 7 days later using WinROOF software (Mitani Corporation). For anti-ChAT antibody staining, tissues were fixed with 0.1% paraformaldehyde and incubated with goat anti-ChAT polyclonal antibody (Chemicon) at a dilution of 1: 500 at 4 ° C for 12-24 hours.

 Staining was visualized with donkey anti-goat IgG labeled with Alexa Fluor488 (Molecular Probes) and measured with a fluorescence microscope (OLYMPUS, IX-71) and soft AQUACOSMOS (HAMAMATSU).

 In addition, the sections were stained with anti-ChAT (choline acetyltransferase) antibody to distinguish them from motor neuron neurites and elongated fibers of these cells.

[0024] The results are shown in FIG. When observed with a phase-contrast microscope (PC), axon elongation was clearly promoted. Among these axons, ChAT-positive fibrils (ct-ChAT) also become HDGF It can be seen that the axons of motor neurons are more responsive to HDGF. It is clear that HDGF has a marked effect on the extension of neuronal neurites.

[0025] Next, the length of the neurites was quantified in the unstained image and the ChAT-stained image, and the values were compared in the presence and absence of HDGF.

 Axon length was quantified using the software WinROOF (Mitani Cooporation) and compared to controls for various concentrations of HDGF. The purified HDGF was diluted with a heparin column eluate.

 The results are shown in Fig. 5. The star and double star are significantly different from the control by the Duunet test (p 0.05 and p 0.01 respectively).

 These results indicate that HDGF has a nerve neurite promoting effect in spinal neurons and spinal motoneurons.

[0026] Next, it was examined whether HDGF promoted the survival of ChAT-positive neurons in a medium without other nutrient factors.

Spinal cord slices were cultured in medium without serum or nutrient factors (DMEM), and the number of ChAT positive neurons per unit area (0.02 mm ² ) was counted for 10 visual fields surrounding the spinal cord tissue on the 10th day of culture. The values were compared in the presence and absence of HDGF.

 The result is shown in Fig. 6. The double asterisk shows a significant difference (p 0.01) from the control by t-test. Addition of HDGF increased the number of surviving motor neurons, confirming that HDGF acts as a trophic factor for spinal motor neurons.

 Brief Description of Drawings

 [0027] FIG. 1 is a view showing a Western blot using four types of antibodies against HDGF. The arrow indicates the specific band of recombinant HDGF.

 FIG. 2 is a diagram showing a Western plot analysis using spinal cord tissue of a transgenic mouse. Tg represents a 12-month-old transgenic mouse, and Cont represents an age-matched litter. Each shows the result of n = 3. The numbers below indicate the intensity ratio corrected by GAPDH.

FIG. 3 shows immunohistochemistry (IHC) of lumbar spinal cord tissue of PQBP_1 transgenic mice. The top two lines show the immunohistochemistry of anti-HDGF antibody (mC15-l) and the bottom The two lines show double staining with anti-HDGF antibody (brown) and anti-GFAP (blue). The length of the horizontal bar in the figure is 50 μm.

4] This figure shows the effect of SDGF on spinal motor neurons. PC shows a phase contrast microscopic image, and ChAT shows ChAT staining.

 FIG. 5 is a diagram showing axonal elongation in motor neuron transplant culture. Cont indicates the concentration of HDGF in the absence of HDGF.

FIG. 6 shows the number of surviving ChAT-positive cell bodies on the 10th day of culture. The vertical axis shows the number per unit area (0.02mm ² ) of ChAT-positive neurons on day 10 of culture

Claims

The scope of the claims  [1] A preventive or therapeutic agent for neurological diseases comprising hepatoma cell-derived growth factor (HDGF) as an active ingredient. [2] The preventive or therapeutic agent for a neurological disease according to claim 1, wherein the neurological disease is a disease caused by motor neuron degeneration or spinal cord injury. [3] The hepatoma cell-derived growth factor (HDGF) force A protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 are deleted, substituted or added. The preventive or therapeutic agent for a neurological disease according to claim 1 or 2, which is a protein comprising an amino acid sequence and having a nutritional effect on motor neurons, or a salt thereof.  [4] A protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and A vector for expressing a protein having a nutritional effect is a preventive or therapeutic agent for a neurological disease comprising one as an active ingredient.  [5] Use of the prophylactic or therapeutic agent according to any one of claims 1 to 4 for the manufacture of a prophylactic or therapeutic agent for a neurological disease.