JP2011173916A - Therapeutic agent of amyotrophic lateral sclerosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new therapeutic agent for amyotrophic lateral sclerosis (ALS) and an inhibitor of the progress of ALS. <P>SOLUTION: The therapeutic agent of ALS and the inhibitor of the progress of ALS contain a hepatocyte growth factor (HGF) as an active ingredient and are administered into the spinal cord or the cerebral ventricle. The HGF is preferably administered in an amount of 0.01-100 mg per day. Further, the HGF is preferably administered in an amount that allows the HGF expression level in the spinal cord to be twice or more than that before administration. The HGF is a known substance, and the HGF prepared by various methods can be used if it is purified to such an extent that it can be used as a medicine. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for amyotrophic lateral sclerosis (hereinafter ALS). More specifically, the present invention relates to an ALS therapeutic agent containing HGF (Hepatocyte Growth Factor) and / or HGF gene as an active ingredient.

Amyotrophic lateral sclerosis (ALS) is a serious neurodegenerative disease characterized by progressive loss of motor neurons and axonal degeneration of motor nerves, resulting in motor dysfunction and shortening of lifespan. About 15% of ALS patients have familial ALS (FALS), and about 15% to 25% of FALS have mutations in the gene encoding Cu2 ⁺ / Zn2 ⁺ superoxide dismutase (SOD1) Yes. Transgenic mice with high levels of mutant SOD1 protein and its activity show disease progression similar to both familial and sporadic ALS, so this mutant SOD1 overexpressing transgenic mouse was used as a model for ALS. Yes. In the present invention, G93A mice (Science, 264, 1772-1775 (1994)), which are transgenic mice that overexpress mutant SOD1 (G93A), were used.

  Since motor neuron degeneration is considered the first event of disease, many attempts have focused on directly helping motor neurons survive. However, none of these attempts were satisfactory. On the other hand, Brujin et al. Reported that inclusion bodies containing prominent SOD1 in astrocytes appear before clinical signs and are visibly increased during disease progression, with mutant SOD1 (G85R ) Pointing to astrocytes as the primary target for mutation-SOD1 mediated damage in overexpressing transgenic mice (Neuron, 18, 327-338 (1997)). Therefore, a bifunctional substance that can restore the function of astrocytes and directly prevent cell death of motoneurons is considered optimal by treatment with ALS. However, no such substance has been reported yet.

  Hepatocyte growth factor (HGF) was first identified as a potent mitogen for mature hepatocytes and its gene cloning was done in 1989 (Biochem. Biophys. Res. Commun., 122, 1450-1459 (1984), Nature, 342, 440-443 (1989)). Although HGF was discovered as a hepatocyte growth factor, numerous recent studies in expression and functional analysis, including knockout / knock-in mouse techniques, revealed that HGF is a novel neurotrophic factor (Ciba Symp., 212, 198-211 (1997), Nat. Neurosci., 2, 213-217 (1999)). HGF exhibits neurotrophic factor activity on hippocampus, cerebral cortex, dopaminergic mesencephalon, cerebellar granule, sensory, motor neuron, sympathetic neuroblast (Ciba. Found. Symp., 212, 198-211 (1997), Brain Res. Mol. Brain Res., 32, 197-210 (1995)). In particular, HGF is known as one of the most potent survival promoting factors for motor neurons, comparable to glial cell line-derived neurotrophic factor (GDNF) in vitro (Neuron, 17, 1157-1172 (1996)) . HGF neurotrophic factor activity has been demonstrated in vivo on developing fetal spinal motoneurons and mature motor neurons after axotomy of the hypoglossal nerve (J. Neurosci., 20, 326-337 (2000), Eur. J. Neurosci., 11, 4139-4144 (1999)). However, there has been no report regarding the involvement of HGF in ALS, and the fact that HGF expression has actually shown an effect on ALS. Therefore, it has been clarified whether HGF can be a therapeutic agent for ALS. There was no situation.

  The present invention relates to a novel therapeutic agent for ALS. More specifically, the present invention relates to an ALS therapeutic agent containing HGF and / or HGF gene as an active ingredient.

  The present inventors first examined the involvement of HGF in ALS using G93A transgenic mice, which are ALS model mice. As a result, it was found that c-Met / HGF receptor-like immunoreactivity (c-Met-IR) is localized in motor neurons of G93A transgenic mice as well as wild-type littermates. . Furthermore, we found that the expression level of c-met and HGF mRNA in the anterior horn of spinal cord (motor neurons were localized) increased with the growth of the mouse as ALS progressed in G93A transgenic mice. These results suggest that HGF plays some role in ALS motor neurons.

Next, the present inventors created a transgenic mouse that expresses rat HGF in a neuron-specific manner using a neuron-specific enolase promoter (NSE), and using that mouse, HGF actually had an effect on ALS. Whether to show. As a result, we found for the first time that HGF reduces motor neuron death and suppresses axonal degeneration of motor neurons. We also found that HGF exerts a neuroprotective effect not only on motor neurons but also on DRG sensory nerves related to ALS-related neurotoxicity. Furthermore, HGF has been shown to have the effect of delaying muscle loss in ALS through a neuroprotective effect.
Furthermore, the present inventors examined whether the progression of ALS can actually be suppressed (delayed) by HGF using the transgenic mice. The results show that very small amounts of HGF expression (approximately twice that of wild-type and G93A mice) surprisingly delays the onset of paralysis, extends life span, and improves exercise capacity. It was. The above findings revealed for the first time that the expression of HGF has a therapeutic effect on ALS.

  Next, as a result of examining the mechanism for suppressing ALS progression by HGF, the present inventors have shown that HGF brings about an ALS improvement effect by at least the following three new mechanisms.

(1) Inhibition of caspase-1 induction in motor neurons It is shown that caspase-1 is activated and / or induced in motor neurons of transgenic mice overexpressing mutant SOD1 in the middle stage of ALS (Proc. Natl. Acad. Sci. USA, 95, 1563-15768 (1998), Science, 288, 335-339 (2000)), and introduction of dominant negative inhibitors against caspase-1 into G93A mice is about Caspase-1 played an important role in the progression of the disease, as it successfully delayed mortality for 2 weeks (Nature, 388, 31 (1997), J. Exp. Med., 185, 933-940 (1997)). It is believed that Therefore, the present inventors examined whether HGF changes the induction of caspase-1, and as a result, it became clear that HGF has an effect of suppressing the induction of caspase-1 in ALS motoneurons.

(2) Phosphorylation of Akt in the spinal cord Akt phosphorylation has been shown to be involved in HGF survival-promoting activity in cerebral cortical neurons and kidney epithelial cells. It has been shown to induce Bcl-xL expression and block large amounts of apoptosis (Biochem. Biophys. Res. Commun., 244, 683-690 (1998), Hepatology, 30, 151-159 (1999)). Thus, the present inventors examined whether or not Akt phosphorylation occurs by HGF expression, and as a result, Akt phosphorylation was specifically observed in the spinal cord of ALS.

(3) Suppression of glial cell-specific glutamate transporter (EAAT2 / GLT1) down-regulation in reactive astrocytes It has been pointed out that glutamate-mediated excitotoxicity is involved in motor neuron degeneration of ALS by reducing glutamate clearance Yes. Consistent with this hypothesis, glutamate transport activity was clearly reduced in the spinal cord and motor cortex of patients with sporadic ALS (N. Engl. J. Med., 326, 1464-1468 (1992)) and astrocytes The immune response to the glial cell-specific glutamate transporter (EAAT2 / GLT-1), which is thought to be a major transporter to suppress glutamatergic neurotoxicity localized in (Ann. Neurol., 38, 73-84 (1995)) has been reported. In addition, EAAT2 decreased in G85R-type transgenic mice (Neuron, 18, 327-338 (1997)), and inactivation of glial glutamate transporter associated with SOD1 mutations (A4V and I113T) in ALS (Nat. Neurosci., 2,427-433 (1999)) has also been reported. As described above, since glutamatergic excitotoxicity is thought to play a role in ALS motor neuron degeneration, the present inventors examined the involvement of HGF in the EAAT2. As a result, it was clarified that HGF has a function of suppressing down-regulation of EAAT2 in ALS and retaining functional astrocytes even at the end of ALS.

  Thus, HGF prevents glial cell-specific glutamate transporter (EAAT2 /) by preventing at least three mechanisms: caspase-1 induction in motor neurons, by phosphorylation of Akt, and in reactive astrocytes. It has been shown to improve ALS by preventing down-regulation of GLT1).

  As described above, HGF has two effects, ALS, through direct neurotrophic factor activity on motor neurons and indirect improvement of glutamate cytotoxicity on motor neurons by maintaining the level of glutamate transporter in astrocytes. It has the effect of improving the motor function and life expectancy. Such a bifunctional growth factor has not been known so far, and HGF is the first example. These functions of HGF indicate that HGF (gene or protein) can be used therapeutically in ALS and related motor neuron diseases. The present invention has been completed based on the above findings.

  That is, the present invention includes (1) an ALS therapeutic agent containing HGF and / or HGF gene as an active ingredient, (2) an ALS progression inhibitor containing HGF and / or HGF gene as an active ingredient, (3) An inhibitor of caspase-1 induction in motor neurons, containing HGF and / or HGF gene as an active ingredient, (4) an Akt phosphorylation promoter in spinal cord containing HGF and / or HGF gene as an active ingredient, and (5) It relates to a decrease inhibitor of glial cell-specific glutamate transporter (EAAT2 / GLT-1) in astrocytes, which contains HGF and / or HGF gene as an active ingredient.

  The present invention provides a therapeutic agent (progression inhibitor) for ALS comprising HGF and / or HGF gene as an active ingredient. HGF has two functions: ALS, through direct neurotrophic factor activity on motor neurons and indirect improvement of glutamate cytotoxicity on motor neurons by maintaining glutamate transporter levels in astrocytes. Has the effect of improving life. Such a bifunctional growth factor has not been known so far, and therefore HGF and / or HGF gene can be effectively used as an unprecedented effective therapeutic agent.

Figure 1 shows the results of immunohistological analysis of c-Met expression (c-Met-IR) in motor neurons of G93A transgenic mice (G93A in the figure) and wild-type littermates (wildtype in the figure). FIG. In the figure, 2mo indicates 2 months of age, and 8mo indicates 8 months of age. FIG. 2 is a graph showing the results of quantitative quantification of HGF and c-met RNA in the spinal cord by competitive RT-PCR in the progression of disease in G98A mice and comparison with that of wild-type mice of the same age. The horizontal axis indicates the age, and the vertical axis indicates the amount of c-met RNA (left graph) or the amount of HGF RNA (right graph). In the figure, the arrowhead indicates the c-met or HGF band, and the bar indicates the competitor band. FIG. 3 is an electrophoretogram showing the results of analyzing the expression of exogenous HGF in each tissue of an HGF transgenic mouse produced using a neuron-specific enolase promoter by an RNase protection assay. In the figure, the arrow indicates a band corresponding to exogenous HGF, and the bar indicates a band corresponding to endogenous HGF. In the figure, Br is brain, Lu is lung, Ht is heart, Li is liver, Kd is kidney, Sp is spleen, St is stomach, Sc is spinal cord, Ts is testis, Ms. Refers to muscle, In refers to intestine, and Sk refers to skin. FIG. 4 is a graph showing plasma HGF levels in 2 month old wild type and HGF transgenic mouse littermates (n = 6). Analysis was performed by enzyme linked immunoassay (ELISA). FIG. 5 is a graph showing the results of ELISA analysis of the HGF content in the whole spinal cord of wild-type and HGF transgenic mouse littermates at the postnatal developmental stage. In the figure, P2 is the second day after birth, P14 is the 14th day after birth, 1mo is 1 month old, and 6mo is 6 months old. FIG. 6 is a photomicrograph showing the results of histological analysis of the number and morphology of motor neurons in the anterior lumbar spinal horn. In the figure, wildtype is the result of a wild type mouse, HGF is the result of an HGF single transgenic mouse, G93A is the G93A transgenic mouse (ALS terminal stage), and G93A / HGF is the double transgenic mouse (ALS terminal stage). FIG. 7 is a graph showing the results of quantitative comparison of the number of lumbar motoneurons in each transgenic mouse. In the figure, ◯ indicates a wild type, ● indicates an HGF-only transgenic mouse, Δ indicates a G93A transgenic mouse, and ▲ indicates a double transgenic mouse. FIG. 8 is a graph showing the wet weight of the gastrocnemius muscle in each transgenic mouse. In the figure, ◯ indicates a wild type, ● indicates an HGF-only transgenic mouse, Δ indicates a G93A transgenic mouse, and ▲ indicates a double transgenic mouse. FIG. 9 is a photomicrograph showing the morphology of anterior root (V, upper panel) and dorsal root (D, lower panel) sections (1 μm) of each 8-month-old transgenic mouse. FIG. 10 is a graph showing the results of ELISA analysis of HGF levels in spinal cords of 2, 6 and 8 months of age in each transgenic mouse. FIG. 11 is a graph showing the results of comparing the onset of paralysis in G93A transgenic mice (Δ, n = 15 in the figure) and G93A / HGF double transgenic mice (Δ, n = 16 in the figure). FIG. 12 is a graph showing the results of comparing the onset of death in G93A transgenic mice (Δ in the figure, n = 15) and G93A / HGF double transgenic mice (Δ in the figure, n = 16). FIG. 13 shows wild-type (○ in the figure, n = 14), HGF single transgenic mouse (● in the figure, n = 15), G93A transgenic mouse (Δ in the figure, n = 15), and double transgenic It is a graph which shows the result of having measured the hind-limb extension reflex of a mouse (▲ in the figure, n = 16). FIG. 14 shows wild-type (○ in the figure, n = 14), HGF-only transgenic mouse (● in the figure, n = 15), G93A transgenic mouse (Δ in the figure, n = 15), and double transgenic It is a graph which shows the result of having measured the retention time on the rotor rod of a mouse (▲ in the figure, n = 16). FIG. 15 shows wild type (circle in the figure, n = 14), HGF single transgenic mouse (● in the figure, n = 15), G93A transgenic mouse (Δ in the figure, n = 15), and double transgenic It is a graph which shows the result of having measured the stride of the mouse (▲ in the figure, n = 16). FIG. 16 is a photomicrograph showing the result of analysis by immunostaining of aggregation of mutant SOD1 in the lumbar spine of G93A and G93A / HGF littermates during disease progression. In the figure, 4mo indicates 4 months of age, and 8mo indicates 8 months of age. FIG. 17 shows the amount of mutant SOD1 in the lumbar spinal cord of each mouse of wild type (W), HGF alone (H), G93A (G), and double transgenic (G / H) was analyzed by immunoblotting. It is an electrophoresis photograph which shows a result. In the figure, 2mo represents 2 months of age, 6mo represents 6 months of age, and 8mo represents 8 months of age. FIG. 18 shows phosphorylated Akt (p-Akt) in the 8-month-old lumbar spine of wild type (W), HGF alone (H), G93A (G), and double transgenic (G / H) mice. ) Electrophoresis photographs showing the results of immunostaining of Akt, Bcl-xL, and Bcl-2. FIG. 19 is a photomicrograph showing the results of immunohistochemical analysis by GFAP of the 8-month-old anterior lumbar spinal horn of four types of mice. FIG. 20 is a graph quantitatively showing the immunohistochemical analysis by GFAP of FIG. In the figure, ○ indicates wild type, ● indicates HGF single transgenic, Δ indicates G93A transgenic, and ▲ indicates double transgenic (n = 3 for each group). FIG. 21 is an electrophoresis photograph showing the results of immunoblotting of spinal cord EAAT2, GFAP and c-Met at 2, 6 and 8 months in 4 mice. In the figure, W is wild type, H is HGF single transgenic, G is G93A transgenic, and G / H is double transgenic. FIG. 22 is a graph showing the relative level of EAAT2 after standardization (dividing EAAT2 level by GFAP level for standardization) (n = 4 mice for each group). FIG. 23 is an electrophoretogram showing immunoblotting of EAAT2 and c-Met in primary cultured astrocytes after HGF treatment. FIG. 24 is a graph quantitatively showing the results of immunoblotting in FIG. 23 (n = 2 in each group).

  HGF used in the present invention is a known substance, and its base sequence and amino acid sequence are Nature, 342, 440 (1989), Japanese Patent No. 2777678, Biochem. Biophys. Res. Commun., 163, 967 (1989), Biochem. Biophys. Res. Commun., 172, 321 (1990). If the said HGF was refine | purified to the grade which can be used as a pharmaceutical, what was prepared by the various method can be used. In addition, products already available on the market (for example, Toyobo Code No. HGF-101) can be used.

  As a method for producing HGF, for example, primary cultured cells or established cells that produce HGF can be cultured, separated from the culture supernatant, etc., and purified to obtain the HGF. In addition, the gene encoding HGF is incorporated into an appropriate vector by genetic engineering techniques, transformed into a suitable host, and the desired recombinant HGF is obtained from the culture supernatant of the transformant. (See, for example, Nature, 342, 440 (1989), Japanese Patent Laid-Open No. 5-111383, Biochem. Biophys. Res. Commun., 163, 967 (1989), etc.). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, yeast or animal cells can be used. Examples of animal cells include CHO cells, COS cells, and mouse C127 cells (all available from ATCC, etc.). The HGF thus obtained may have a structure similar to that of natural HGF as long as it has substantially the same action as natural HGF. That is, 1) a protein encoded by DNA that hybridizes with the HGF cDNA under stringent conditions, or 2) one or more (preferably several) amino acids are substituted for the amino acid sequence of the HGF. Of the proteins composed of deleted and / or added amino acid sequences, and the like, any protein having an action as HGF is included in the category of HGF of the present invention. Here, the DNA encoding the proteins 1) and 2) can be easily obtained by, for example, site-directed mutagenesis, PCR, or usual hybridization, and specifically, Molecular Cloning 2nd. This can be done with reference to basic books such as Edt., Cold Spring Harbor Laboratory Press (1989).

  The present invention is the first to clarify that HGF is a therapeutic agent for ALS, specifically, an ALS progression inhibitor. HGF has two functions: ALS, through direct neurotrophic factor activity on motor neurons and indirect improvement of glutamate cytotoxicity on motor neurons by maintaining glutamate transporter levels in astrocytes. Has the effect of improving life. Such a bifunctional growth factor has not been known so far, and HGF is the first example. By exerting these two actions, HGF can delay / suppress the progression of ALS and related motor neuron diseases.

As a mechanism of action of HGF on ALS, it is considered that HGF exhibits an ALS improving effect by exerting at least the following three actions.
(1) Inhibition of caspase-1 induction in motor neurons (2) Phosphorylation of Akt in the spinal cord (3) Inhibition of downregulation of glial cell-specific glutamate transporter (EAAT2 / GLT1) in reactive astrocytes Action

  Endogenous growth factors having such an action have not been known so far, and HGF is the first growth factor which has been shown to have the above action. By exerting these actions, HGF can delay or suppress the progression of ALS and related motor neuron diseases. Here, motor neuron diseases related to ALS include progressive spinal muscular atrophy, progressive bulbar paralysis, primary lateral sclerosis, pediatric and juvenile muscular atrophy, Fogio-Ronde syndrome, Charcot-Marie Examples include, but are not limited to, tooth disease.

  The therapeutic agent of the present invention can take various preparation forms (for example, liquids, solids, etc.), but is generally an injection with only the active ingredient HGF or a conventional carrier. The injection can be prepared by a conventional method. For example, HGF is dissolved in an appropriate solvent (for example, sterilized water, buffer solution, physiological saline, etc.), then sterilized by filtration with a filter or the like, and then sterile. It can be prepared by filling a container. The content of HGF in the injection is usually adjusted to about 0.0002 to 0.2 (W / V%), preferably about 0.001 to 0.1 (W / V%).

  In formulating, a stabilizer is preferably added, and examples of the stabilizer include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol and the like. Furthermore, the preparation of the present invention may contain additives necessary for formulation, for example, excipients, solubilizers, antioxidants, soothing agents, tonicity agents and the like. In the case of a liquid preparation, it is desirable to store it after removing moisture by freeze storage or freeze drying. The freeze-dried preparation is used by re-dissolving and adding distilled water for injection at the time of use.

  The preparation of the present invention is administered by an appropriate administration route according to the form of the preparation. The administration route may be any administration route as long as HGF exerts its action and can suppress the progression of ALS. In general, subcutaneous administration, intradermal administration, intravenous administration, intraarterial administration, intramuscular administration, local injection, intraventricular administration, intraspinal administration and the like can be mentioned. Furthermore, the administration route through a device implanted in the body is also mentioned. Specifically, a method of gradually and gradually administering to the affected area using an osmotic pump or the like, or a sustained-release preparation (for example, a mini-pellet preparation) ) Is embedded near the affected area. Among these, intrathecal administration is a preferable administration route because the preparation of the present invention can directly reach motor neurons in the spinal cord.

  The dosage is appropriately adjusted according to the patient's symptoms, age, weight, etc., but is usually 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg as HGF, and is administered once to several times a day. It is appropriate to do.

  Moreover, as described in Examples below, it was shown that ALS is improved by the expression of a very small amount of HGF. That is, the HGF expression level approximately twice the HGF expression level in the spinal cord of wild-type mice and G93A transgenic mice sufficiently improved the motor function at 6 months, when ALS symptoms begin to appear, It was shown to prolong the life span of 1 month in mice. Therefore, the HGF preparation of the present invention is preferably administered to ALS patients in such an amount that the HGF expression level in the spinal cord is at least twice that before HGF administration. The present invention also provides a therapeutic agent for ALS comprising administering such an amount that the expression level of HGF in the spinal cord is twice or more that before administration of HGF.

  In recent years, reports on gene therapy using the HGF gene have been made (Circulation, 96, No 3459 (1997), Nature Medicine, 5, 226-230 (1999), Circulation, 100, No. 1672 (1999), Gene Therapy, 7,417-427 (2000), etc.), and is also a technically established technology. The present invention includes not only the administration of the HGF protein as described above but also an ALS gene therapy agent consisting of introducing an HGF gene. The following describes HGF gene therapy.

  The “HGF gene” used in the present invention refers to a gene capable of expressing HGF (HGF protein). Specifically, HGF described in Nature, 342, 440 (1989), Patent No. 2777678, Biochem. Biophys. Res. Commun., 163, 967 (1989), Biochem. Biophys. Res. Commun., 172, 321 (1990), etc. In which the cDNA is incorporated into an appropriate expression vector (non-viral vector, viral vector) as described below. Here, the base sequence of cDNA encoding HGF is described in the above-mentioned document, and is also registered in databases such as Genbank. Therefore, HGF cDNA can be cloned by using an appropriate DNA portion as a PCR primer based on these sequence information and performing, for example, RT-PCR reaction on mRNA derived from liver or leukocytes. These clonings can be easily carried out by those skilled in the art according to basic books such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989).

  Furthermore, the HGF gene of the present invention is not limited to those described above, and can be used as the HGF gene of the present invention as long as the expressed protein is a gene having substantially the same action as HGF. That is, 1) DNA that hybridizes with the cDNA under stringent conditions, or 2) one or more (preferably several) amino acids are substituted or deleted from the amino acid sequence of the protein encoded by the cDNA. In addition, DNA encoding a protein having an added amino acid sequence, and the like encoding a protein having an action as HGF are included in the category of the HGF gene of the present invention. Here, the DNAs 1) and 2) can be easily obtained by, for example, site-directed mutagenesis, PCR, or ordinary hybridization, and specifically, the basic documents such as Molecular Cloning. Can be done with reference to.

  The HGF gene of the present invention can be applied to the same diseases as the aforementioned HGF protein. Further, the HGF gene and the HGF protein can be used independently or both can be used in combination.

  Next, the gene introduction method, the introduction form, the introduction amount, etc. used in the gene therapy of the present invention will be described.

  When administering a gene therapy agent containing the above gene as an active ingredient to a patient, the administration form is roughly divided into two cases, using a non-viral vector and using a viral vector, and is described in an experiment manual, etc. The preparation method and administration method are explained in detail (separate volume experimental medicine, basic technology of gene therapy, Yodosha, 1996, separate volume experimental medicine, gene transfer & expression analysis experiment method, Yodosha, 1997, Japanese gene therapy Gene Therapy Development Research Handbook, NTS, 1999). This will be specifically described below.

A. When a non-viral vector is used, a target gene can be introduced into a cell or tissue by the following method using a recombinant expression vector in which the target gene is incorporated into a conventional gene expression vector. Examples of the method for introducing a gene into a cell include a lipofection method, a phosphate-calcium coprecipitation method, a DEAE-dextran method, and a direct DNA injection method using a micro glass tube. In addition, gene transfer methods to tissues include gene transfer methods using internal type liposomes, gene transfer methods using electrostatic type liposomes, HVJ-liposome method, and improved HVJ-liposome method (HVJ). -AVE liposome method), receptor-mediated gene introduction method, method of transferring DNA molecules with cells (metal particles) with a particle gun, direct introduction of naked-DNA, introduction method using positively charged polymer, etc. By using the method, it is possible to incorporate the recombinant expression vector into cells.

  Among them, HVJ-liposomes are obtained by encapsulating DNA in liposomes made of a lipid bilayer, and further fusing the liposomes with inactivated Sendai virus (Hemagglutinating virus of Japan: HVJ). The HVJ-liposome method is characterized by a very high fusion activity with the cell membrane as compared with the conventional liposome method, and is a preferable introduction form. For the preparation method of HVJ-liposomes, refer to the literature (Experimental Medicine separate volume, Basic technology of gene therapy, Yodosha, 1996, Gene transfer & expression analysis experiment method, Yodosha, 1997, J.Clin.Invest.93,1458- 1464 (1994), Am. J. Physiol. 271, R1212-1220 (1996)) and the like. The HVJ is preferably a Z strain (available from ATCC), but basically other HVJ strains (eg, ATCC VR-907 and ATCC VR-105) can also be used. Furthermore, the direct introduction method of naked-DNA is the simplest method among the above methods, and is a preferable introduction method from this viewpoint.

  The expression vector used here may be any expression vector as long as it can express the target gene in vivo. For example, pCAGGS (Gene 108,193-200 (1991)) or pBK-CMV , PcDNA3.1, pZeoSV (Invitrogen, Stratagene) and the like.

B. When using a viral vector, a typical method using a viral vector such as a recombinant adenovirus or a retrovirus is used as the viral vector. More specifically, for example, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, shinbis virus, Sendai virus, SV40, immunodeficiency virus (HIV), etc. It is possible to introduce a gene into a cell by introducing the gene of interest into a DNA virus or RNA virus of the above and infecting the cell with a recombinant virus. Among the viral vectors, it is known that adenovirus infection efficiency is much higher than when other viral vectors are used. From this viewpoint, it is preferable to use an adenoviral vector system.

  As a method for introducing the gene therapy agent of the present invention into a patient, an in vivo method in which the gene therapy agent is directly introduced into the body, or a certain cell is taken out from a human and introduced into the cell outside the body. There is an ex vivo method for returning the cells to the body (Nikkei Science, April 1994, 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Extra Number, 12 (15) (1994), Gene Therapy Society of Japan, Gene Therapy Development Research Handbook, NTS, 1999). In the present invention, the in vivo method is preferable.

  The administration site to the patient may be any administration route as long as HGF expressed in vivo exerts an action and can suppress the progression of ALS. In general, subcutaneous administration, intradermal administration, intravenous administration, intraarterial administration, intramuscular administration, local injection, intraventricular administration, intraspinal administration and the like can be mentioned. Furthermore, the administration route through a device implanted in the body is also mentioned. Specifically, a method of gradually and gradually administering to the affected area using an osmotic pump or the like, or a sustained-release preparation (for example, a mini-pellet preparation) ) Is embedded near the affected area. Among these, intrathecal administration is a preferable administration route because the preparation of the present invention can directly reach motor neurons in the spinal cord.

  As a preparation form, various preparation forms (for example, a liquid agent etc.) suitable for each of the above administration forms can be taken. For example, when an injection containing a gene which is an active ingredient is used, the injection can be prepared by a conventional method, for example, dissolved in an appropriate solvent (buffer solution such as PBS, physiological saline, sterilized water, etc.). Then, if necessary, it can be prepared by sterilizing by filtration with a filter or the like and then filling into an aseptic container. A conventional carrier or the like may be added to the injection as necessary. Liposomes such as HVJ-liposomes can be in the form of liposome preparations such as a suspension, a freezing agent, and a centrifugal concentrated freezing agent.

  The DNA content in the preparation can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient, but is usually 0.0001-100 mg, preferably 0.001-10 mg as the DNA of the present invention. This is preferably administered once every several days to several months. Further, as in the case of the aforementioned HGF protein, in the present invention, an ALS therapeutic agent comprising administering an amount such that the expression level of HGF in the spinal cord is twice or more that before administration of the HGF gene. Is also provided.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples.

Materials and methods
(1) Transgenic mice Transgenic G93A mice ((SOD1-G93A) 1Gurdl) were purchased from Jackson Laboratory (Science, 264, 1772-1775 (1994)). For maintenance, male G93A mice were mated with female C57B6 mice and genotyped by PCR and dot-blot hybridization.

  A full-length rat HGF cDNA (Proc. Natl. Acad. Sci. USA 87, 3200-3204 (1990)) tagged with the KT3 epitope is amplified by PCR and is neuron specific in the pNSE-Ex vector (provided by Dr. Doherty) It was inserted downstream of the enolase promoter (Neuron, 18, 231-241 (1997)). For subcloning, a Not I site was introduced into the pNSE-Ex vector by addition of a linker. Thereafter, the entire nucleotide sequence of the plasmid was confirmed. HGF-KT3 showed similar activity to human recombinant HGF in MDCK cell migration assay and in vitro survival assay of primary culture of hippocampal neurons. Transgenic mice can be obtained by making slight improvements to the methods already described (J. Cell. Biol., 128, 185-199 (1995), Proc. Natl. Acad. Sci., USA, 95, 5269-5274 (1998)). Were prepared and analyzed for transgene incorporation. Briefly, the transgenic cassette was excised from the vector by Sal I restriction enzyme treatment and the DNA was injected into C57B6 embryos whose genetic background was consistent with G93A transgenic mice. Transgene integration was confirmed by PCR and dot-blot using SV40 poly (A) sequences in the transgene. The expression of exogenous HGF was examined by RNase protection assay using a probe covering the 3 'end of HGF-KT3 cDNA and the 5' region of SV40 poly (A) signal sequence.

  Offspring of NSE-HGF transgenic mice were crossed with G93A transgenic mice. Children from one parent were kept in the same cage until the phenotype appeared. After the first sign of phenotype, the animals were kept isolated. Food and water were placed at the bottom of the cage for easy consumption. This time was used as death time when the animal could not stand its body within 30 seconds.

  (2) Quantification of HGF and c-met RNA in the anterior horn Quantitative competitive RT-PCR for the anterior horn of the spinal cord was performed using a previously reported method (Brain Res. Brain Res. Protoc., 5, 190-197 (2000) ).

(3) RNase protection assay RNase protection assay was performed as described previously (J. Cell. Biol., 123, 455-465 (1993), Science, 268, 1495-1499 (1995)), RPAII ribonuclease protection assay kit (Ambion , Austin, TX). In order to prepare an antisense cRNA probe specific for HGF mRNA, a 326 bp fragment containing a portion of the polyA sequence from the HGF 3 ′ end of HGF-KT-3-polyA was inserted into the pGEM-T vector. The plasmid was linearized and transcribed in the presence of α- [ ³² P] CTP, and the prepared cRNA probe was used for hybridization. Exogenous HGF RNA gives a 326 bp protection band and endogenous HGF RNA gives a shorter protection band (251 bp) because it lacks the KT-3 and poly (A) sequences.

  (4) Behavioral testing Behavioral testing was performed on 15 animals of each phenotype from 6 families. Extension of the hind limb is usually observed when the mouse is hung in the air with its tail. Motor neuron disease mice commonly show regression of the hind limbs. The score corresponds to the number of stretched hind limbs. For the rotor rod test, the mouse was placed on a rotating rod at 20 rpm. The duration of each mouse remaining on the rod was measured. If the animal could remain on the rod for 4 minutes, the study was terminated in 4 minutes and recorded as 4 minutes. Footprints were collected by allowing the mouse to walk on a straight path after the foot was then dipped in black ink. The stride was measured in the area showing normal walking.

  (5) Histological analysis In order to count motor neurons, the spinal cord was fixed with a continuous concentration of ethanol, embedded in paraffin, and then continuously cut from L5 to L4 (14 μm). The number of motor neurons (within L4-L5) in the anterior horn was measured for a total of 20 sections every 7 sections. Neurons with distinct nucleoli stained strongly with crystal violet in the defined area of the anterior horn were counted.

  c-Met specific antibody (Santa Cruz, 1: 100), HGF specific antibody (Tokushu Meneki, 1: 1000), human SOD specific antibody (Sigma, 1: 200), GFAP specific antibody (Sigma, 1: 1000), and caspase-1 specific antibody (Santa Cruz, 1: 500) with 1% at room temperature after blocking with 5% goat serum and mouse IgG blocking agent for 1 hour (MOM kit, Vector science) Time-4 hours, or added to sections at 4 ° C overnight. After washing, a biotinylated or fluorescently labeled secondary antibody was added and incubated for 15 minutes. For fluorescent immunostaining, sections were now observed under a fluorescence microscope after counterstaining with Hoechst33342. Fluorescence images were captured with a CCD camera (Hamamatsu), digitized, and fluorescence levels were measured using Adobe PhotoShop. In order to visualize the signal recognized by the biotinylated secondary antibody, ABC solution was added for 10 minutes and developed in DAB solution. The specificity of each staining was tested by the previously reported technique (Sun et al., 1999).

  The L5 root was dissociated and fixed overnight with 4% paraformaldehyde / 0.25% glutaraldehyde, post-fixed for 2 hours on ice with osmium tetroxide, the root was dehydrated and embedded in Epon812. The embedded route was cut (1 μm) and stained with toluidine blue. The sample morphology was examined under an optical microscope.

  (6) Cell culture Primary astrocytes were cultured with a slight modification to the method already described (Brain Res. Mol. Brain Res., 41, 259-268 (1996)). Two-day-old G93A mice or wild-type littermates of the cerebral cortex were dissected into small pieces. Tissue blocks were degraded by incubating with 0.25% trypsin and 100 μg / ml DNase I for 12 minutes while shaking in a 37 ° C. water bath. Tissue was pulverized by aspiration with a pipette, and the separated cells were DF (high glucose DMEM / HamF12, 50:50 containing 10% fetal bovine serum on poly-L-ornithine coated dishes in the indicated number of cells. ) Plated in medium (GibCO BRL). When the cells became confluent 7 days after plating, the cells were washed twice with PBS and treated with the indicated concentrations of recombinant HGF. The astrocyte purity was usually over 95% after 7 days of culture and was examined by morphology or NSE / GFAP double staining.

  (7) Western blotting lumbar spinal extract was prepared in RIPA buffer. 50 μg of lysate was electrophoresed on a 10% SDS-polyacrylamide gel (SDS-PAGE) and transferred to a PVDF membrane. Membranes were incubated overnight at 4 ° C. with 5% non-fat skim milk in PBS and anti-EAAT2 antibody (1: 2,000, Chemicon) was added for 2 hours. After washing, the membrane was incubated with HRP-labeled anti-guinea pig IgG antibody (1: 3,000, Chemicon) and subjected to ECL chemifluorescence reaction (Amersham, Buckinghamshire, UK). The antibody was removed from the membrane and reprobed with anti-GFAP (1: 3000, Sigma), anti-human SOD (1: 500, Sigma), and anti-c-Met (1: 400, Santa Curz) antibodies. 50 μg of lysate was subjected to 12% SDS-PAGE and phosphorylated Akt (Cell Signaling Tech.), Akt (Cell Signaling Tech.), Bcl-xL / S (Santa Cruz) and Bcl-2 as described above. (Santa Cruz) antibody was added. Band intensity was measured with a Fluorchem image analyzer (IS-8000).

  (8) Detection of HGF concentration HGF in tissue or plasma was measured by enzyme-linked immunoassay (ELISA) using anti-rat HGF polyclonal antibody (Tokushu Meneki, Tokyo, Japan). The rat HGF ELISA system specifically detects rat and mouse HGF with similar affinity.

  (9) Statistical analysis Statistical comparison was performed by Student's t-test. For the survival test, statistical significance (p <0.05) was assessed by log rank test.

Example 1
Expression and regulation of HGF and c-Met in G93A transgenic mice
Whether or not c-Met / HGF receptor-like immunoreactivity (c-Met-IR) was shown in motor neurons of G93A transgenic mice was examined by immunohistological analysis. As a result, c-Met-IR was found to be localized in the motor neurons of 2-month-old G93A mice, similar to those of wild-type littermates (Fig. 1). In addition, c-Met-IR was observed in many cells in the anterior horn at 8 months when the G93A mouse reached the end stage, while the wild-type littermates of the same age were c-Met- IR was shown (Figure 1).

  Next, the quantification of HGF and c-met mRNA within the anterior horn of the spinal cord during disease progression of G93A transgenic mice was performed by the above-mentioned competitive RT-PCR and compared with that of wild-type mice of the same age. The results revealed that c-met and HGF mRNA expression levels within the anterior horn of the spinal cord, where motor neurons are localized, progressively increased during the development of ALS in G93A transgenic mice. (FIG. 2). These results suggest the role of HGF in ALS motoneurons. c-Met-IR was expressed not only in the remaining motor neurons but also in the surrounding astroid cells at the end stage, suggesting a role for HGF in cells other than these motor neurons. These results raised the possibility that HGF may be one of the intrinsic factors that slows disease progression.

Example 2
Generation and characterization of transgenic mice that overexpress HGF specifically in neurons
In order to examine the effect of HGF on ALS, transgenic mice expressing rat HGF in a neuron-specific manner were generated using a neuron-specific enolase promoter (NSE). From eight independent HGF transgenic mouse strains, one strain was selected that specifically expressed foreign HGF specifically at the nervous system but at a relatively low level. Figures 3, 4 and 5 show the characteristics of the HGF transgenic mouse strain. The RNase protection assay showed that exogenous HGF was expressed exclusively in the brain and spinal cord among the 12 tissues tested (arrows in FIG. 3). In tissues of HGF transgenic mice, plasma endogenous and total HGF levels were not different from those of wild-type littermates, respectively (FIGS. 3 and 4). On the other hand, the level of HGF protein increased with the growth of mice in the spinal cord only after the major differentiation of postnatal spinal nerves was completed (FIG. 5).

  HGF transgenic mice could not be distinguished from wild type transgenic mice unless genotyping was performed. That is, no differences were found in size, weight, morphology, or behavior or changes in the motor nervous system containing many motor neurons and astrocytes, and muscle weight at any developmental stage examined. This indicates that the introduction of HGF has been successful in a nervous system-specific manner without affecting development (see FIGS. 6 to 10, and FIGS. 19 and 20).

Example 3
Effect of HGF on ALS (1)
In order to investigate the role of HGF in ALS, the heterozygote (+/-) of HGF transgenic mice was crossed with the heterozygote (+/-) of G93A mice, thereby directly transferring HGF to neurons in ALS. An introduced transgenic mouse was prepared. Note that the crossing resulted in four different groups of mice: wild type (W), transgenic for HGF only (HGF), transgenic for G93A only (G93A), and double transgenic for both G93A and HGF (G93A / HGF) is generated.

  First, we examined whether HGF expression exerts a neuroprotective effect on motor neurons by histological analysis. The results are shown in FIGS. A paraffin section (14μm) in the anterior horn of the lumbar spinal cord stained with crystal violet clearly shows a decrease in the number of motor neurons in G93A mice (G93A), and the remaining motor neurons are also atrophied. (Figure 6). On the other hand, double transgenic littermates (G93A / HGF) retained spinal motor neurons with clearly more healthy morphology than that of G93A mice (Figure 6). Wild-type and HGF-only transgenic mice showed a similar number of healthy motor neurons (FIG. 6).

  Next, the number of motor neurons in the lumbar spinal cord was quantitatively compared using 6 independent animals per group for each mouse. As a result, G93A mice began to lose lumbar motoneurons at 6 months of age, and at 8 months of age, G93A mice motoneurons compared to wild-type or HGF-only transgenic littermates. Only 40% remained (Figure 7). In contrast, double transgenic littermates showed a marked improvement in the number of motor neurons compared to G93A transgenic mice (FIG. 7).

  Since it has been shown that the pro-survival activity of HGF on motor neurons differs between lumbar and cervical levels in chick embryos, the introduction of HGF can then reduce motor neuron death at the cervical level Whether it was also tested. As a result, in G93A mice, 55% of cervical motor neurons remained at 8 months of age. The death of motor neurons at a lower number of cervical levels than at the lumbar level is characteristic of this model mouse. The double transgenic mice still have a greater number (87.8 ± 2.4%) of motor neurons at the cervical level, indicating that HGF is effective for both lumbar and cervical motor neurons. It is shown that.

  Next, the effect of HGF on axonal degeneration was examined by histological analysis using at least 3 independent mice for each group. Degeneration of the large root axon of the anterior root was seen in 8-month-old G93A mice (Fig. 9), whereas in the double transgenic littermates, the anterior degeneration of the anterior root was mild and most of the anterior root deteriorated The form which has not been shown. The dorsal root showed moderate degeneration in G93A mice, whereas the morphology in double transgenic littermates was visually normal (FIG. 9). These indicate that HGF effectively prevents both anterior and dorsal root degeneration. Furthermore, the terminal double transgenic mice showed similar healthy morphology of the dorsal roots even when the anterior roots were poor. Therefore, it was shown that HGF exerts a neuroprotective effect not only on motor neurons but also on DRG sensory nerves related to ALS-related neurotoxicity. Furthermore, the neuroprotective effect of HGF was also shown from the delayed effect of reducing gastrocnemius muscle weight (FIG. 8).

Example 4
Effect of HGF on ALS (2)
Whether the introduction of HGF can improve the onset of paralysis, longevity and ALS motor ability was tested using the above four mice. The onset of paralysis was observed in heterozygote (+/-) G93A mice at an average age of 243.8 ± 4.7 days (mean ± standard deviation, median = 242 ± 14 days), whereas double transgenic mice In (G93A / HGF), this onset was apparently late and occurred at an age of 271.9 ± 5.6 days (median 282.5 ± 9.5 days, p = 0.004) (FIG. 11). As for the start of death, the average survival time of G93A mice was 259.5 ± 5.0 days (median = 259 ± 11 days). On the other hand, double transgenic mice were prolonged to 286.8 ± 6.5 days, and the life expectancy was 27.3 days (median = 294 ± 14.5 days, p = 0.003). , Figure 12).

  In addition, exercise ability was tested by measuring the hindlimb stretch reflex, holding time on the rotor rod, and stride length. HGF-only transgenic mice showed no obvious difference in motor function from wild-type littermates. G93A mice, on the other hand, showed a progressive decrease in hindlimb stretch from 5 months of age, from 6 months of age to rotarod ability, and from 7 months of age to stride. On the other hand, double transgenic mice showed a much slower decrease in all functional parameters tested than G93A (FIGS. 13-15).

  FIG. 10 shows spinal HGF protein levels in four groups during ALS progression. HGF levels were approximately twice as high at 2 months and 6 months in HGF transgenic mice and G93A / HGF littermates, indicating that the addition of a small amount of HGF sufficiently improved motor function at 6 months and a one month life span Indicates that the However, inadequate production of HGF in the G93A / HGF double transgenic at 8 months, that is, the level of HGF in HGF, G93A and G93A / HGF is the same at the end stage, indicating that the subsequent improvement of ALS is incomplete HGF expression at a higher level is desired. As described above, it was shown that the expression of HGF gene in ALS neurons clearly improved the life span and motor function in ALS.

Example 5
Study of action mechanism of HGF on ALS (1)
To investigate possible mechanisms for decreased motor neuron death, decreased motor and sensory axon degeneration, and slow decrease in motor function in double transgenic mice: One of the earliest signs of illness is to consider whether to change the first event, later events, or improve the speed of disease progression without affecting specific events. Was tested. Mutant SOD1 aggregation has been reported to be the earliest event in animal models, and SOD1 aggregation has been observed in familial ALS patients.

  The total amount of mutant SOD1 in the spinal cord extract was examined by immunoblotting using an antibody specific for human SOD1 antibody and thus recognizing only mutant human SOD1 and not endogenous mouse SOD1. Three independent animals were tested for each group. Mutant SOD1 was detected from 2 months of age in both G93A and double transgenic mice. And the total amount of mutant SOD1 increased in both G93A and double transgenic mice over the same time course. The total amount of mutant SOD1 in G93A mice was not different from that of HGF littermates (FIG. 17).

  Next, SOD1 aggregation was examined. Aggregation of mutant SOD1 in the spinal cord was preferentially detected in the anterior horn at an early stage of about 4 months of age in G93A mice, and the amount of aggregation was clearly increased at 8 months of age when the animals were at the end ( Figure 16). Aggregation of mutant SOD1 in double transgenic mice is comparable to that of G93A littermates at 4 months of age (Figure 16), and no specific immune response was detected in wild type or HGF-only transgenic littermates . Aggregation of mutant SOD1 in double transgenic mice clearly increased at 8 months (FIG. 16), and the amount appeared to be slightly less compared to G93A littermates, but the differences were very small. These results indicated that HGF did not improve the origin of neurotoxicity and the first event of disease in this model.

  Caspase-1 is thought to play an important role in the progression of ALS (Nature, 388, 31 (1997), J. Exp. Med., 185, 933-940 (1997)). Therefore, whether HGF could improve the induction of caspase-1 was examined by double-label immunohistochemistry using anti-caspase-1 antibody and anti-tubulin III antibody staining mature neurons. Caspase-1-immunoresponsiveness (caspase-1-IR) was below the limit of detection in both wild type and HGF littermates at any time tested. In G93A mice, caspase-1-IR was specifically induced and colocalized in large tubulin III immunostained cells at 6 months. This indicates the induction of caspase-1 in motor neurons. The level of caspase-1 in G93A decreased at 8 months and only faint caspase-1-IR was detected. Double transgenic mice show very weak caspase-1-IR in large tubulin III immunoreactive cells at 6 months of age compared to G93A mice, indicating that HGF is a motor neuron in the middle phase of ALS. It has been shown to reduce the level of induction of caspase-1 at.

  Akt phosphorylation has been shown to be involved in HGF survival-promoting activity in cerebral cortical neurons and kidney epithelial cells, and HGF induces Bcl-xL expression in the liver of fulminant hepatitis models and blocks massive apoptosis (Biochem. Biophys. Res. Commun., 244, 683-690 (1998), Hepatology, 30, 151-159 (1999)). To further elucidate the mechanism of HGF that reduces apoptosis, we investigated phosphorylation of Akt and regulation of Bcl-2 family genes.

  Western blot shows that the amount of Akt itself does not change in any litter, in contrast, Akt is clearly clearly phosphorylated in the spinal cord of G93A / HGF mice at 8 months. (Figure 18). The rather low level of Akt phosphorylation in the spinal cord of HGF mice may reflect a lower level of c-Met in HGF mice than that in double transgenic mice (FIG. 21). These results indicate that HGF exhibits survival promoting activity partially through activation of Akt.

Bcl-xL and Bcl-2 proteins were not induced in the spinal cord at 8 months of age ( FIG. 18 ). These experiments indicate that HGF reduces the progression of ALS by preventing caspase-1 induction in motor neurons and by phosphorylating Akt in the spinal cord.

Example 6
Study of action mechanism of HGF on ALS (2)
It has been pointed out that glutamate-mediated excitotoxicity is involved in motor neuron degeneration of ALS by reducing glutamate clearance. Consistent with this hypothesis, glutamate transport activity was clearly reduced in the spinal cord and motor cortex of patients with sporadic ALS (N. Engl. J. Med., 326, 1464-1468 (1992)) and astrocytes The immune response to the glial cell-specific glutamate transporter (EAAT2 / GLT-1), which is thought to be a major transporter to suppress glutamatergic neurotoxicity localized in (Ann. Neurol., 38, 73-84 (1995)) has been reported. In addition, inactivation of the glial glutamate transporter associated with a decrease in EAAT2 in the ALS model G85R-type transgenic mice (Neuron, 18, 327-338 (1997), SOD1 mutations (A4V and I113T) in ALS ( Nat.Neurosci., 2,427-433 (1999)) As described above, it is thought that glutamatergic excitotoxicity plays a role in motor neuron degeneration of ALS. The involvement of was examined.

  First, we investigated whether c-Met-IR is localized in reactive astrocytes and whether HGF alters astrocyte pathogenesis and reactive astrocyte glutamate transporter levels It was. Double staining of the spinal cord with anti-GFAP and anti-c-Met antibodies revealed that c-Met-IR was localized in the remaining large neurons and GFAP-positive reactive astrocytes. Consistent with immunohistochemical results, c-Met levels increased in G93A and double transgenic mice, especially at 8 months (Figure 21, lower panel), not only motor neurons but also stars The cells were also ALS at 8 months (late stage), suggesting that they could be targets for HGF.

  In wild-type or HGF-only transgenic mice, astrocytes were predominantly white matter and localized near the central canal, but only slightly in the anterior horn (Figure 19). Reactive astrocytes in G93A mice progressively increased in the anterior horn at 6 months of age, when motoneuron death was not yet observed in G93A mice (Figure 19). In contrast, double transgenic littermates had significantly fewer reactive astrocytes in the anterior horn (FIG. 19). Quantification of the reactivity intensity of GFAP-IR showed about 40% and 60% less immune response activity at the anterior horn compared to that of 6 and 8 month old G93A mice, respectively (FIG. 20). ). Next, as a result of immunoblotting of EAAT2, GFAP and c-Met of each mouse spinal cord at 2, 6 and 8 months, induction of GFAP from 6 months in both G93A mice and G93A / HGF mice, Specific EAAT2 down-regulation in months G93A mice and specific c-Met up-regulation in 8 months G93A and G93A / HGF mice were shown. In G93A / HGF mice, the total level of EAAT2 was maintained at 8 months.

  The level of EAAT2 in G93A mice at 8 months of age was clearly reduced by 40% compared to that of wild-type or HGF-only transgenic littermates (Figure 22). In contrast, double transgenic mice showed higher levels of EAAT2 (140%) in the lumbar spinal cord compared to littermates of wild type or HGF alone transgenic mice (FIG. 22). To assess the level of EAAT2 in individual astrocytes, the level of EAAT2 was divided by the level of GFAP. Only 11% of EAAT2 remained in G93A mice, while 63% of EAAT2 remained in double transgenic mice (Figure 22). It is a valuable record that astrocytosis begins in 6 months, but EAAT2 reduction and c-Met induction in astrocytes occurs only in parallel only at the end stage.

  Next, as a result of examining the effect of HGF on primary cultured astrocytes, it became clear that EAAT2 levels in primary cultured astrocytes from both G93A mice and wild-type littermates increased by HGF treatment (Fig. 23 and Fig. 24). This indicates that the prevention of EAAT2 depletion in astrocytes is directly due to HGF activity in astrocytes. As described above, it was shown that double transgenic mice retain more functional astrocytes at the end stage, while G93A mice overproduce nonfunctional astrocytes in terms of glutamate clearance.

Example 7
Inhibition of ALS progression by HGF administration or HGF gene introduction
In G93A mice, which are ALS model mice, from the age of 6 months when ALS symptoms began to be observed, the HGF protein preparation is administered daily to an appropriate site such as the spinal cord at an appropriate frequency. At that time, a group to which a preparation containing no HGF protein is administered is used as a control group. Further, when evaluating the dose, the number of administrations, etc., groups in which the dose and the number of administrations are appropriately changed are used. Thereafter, in each of the HGF administration group and the control group, the ALS progression inhibitory effect and the like as described in Examples 3 and 4 are examined. The presence of an ALS progression inhibitory effect in the HGF administration group confirms that HGF is a therapeutic agent for ALS. Furthermore, by conducting the same experiment using the HGF gene instead of HGF, it is confirmed that the HGF gene is a therapeutic agent for ALS.

Formulation Example 1
A solution containing 1 mg of HGF, 1 g of mannitol and 10 mg of polysorbate 80 in 100 ml of physiological saline was prepared aseptically, dispensed into vials in 1 ml portions, freeze-dried and sealed to obtain a freeze-dried preparation.

Formulation Example 2
An aqueous solution containing 1 mg of HGF and 100 mg of human serum albumin in 100 ml of 0.02 M phosphate buffer (containing 0.15 M NaCl and 0.01% polysorbate 80, pH 7.4) is aseptically prepared, and 1 ml is dispensed into vials. After that, freeze-dried and sealed to obtain a freeze-dried preparation.

  HGF and / or HGF gene is an unprecedented effective ALS therapeutic agent.

Claims (13)

  A therapeutic agent for amyotrophic lateral sclerosis (ALS) containing HGF as an active ingredient and administered into the spinal cord or ventricle.   The ALS therapeutic agent according to claim 1, wherein the ALS is familial ALS (FALS).   The ALS therapeutic agent according to claim 1 or 2, wherein HGF is administered in an amount of 0.01 to 100 mg per day.   An ALS progression inhibitor comprising HGF as an active ingredient and administered into the spinal cord or ventricle.   The ALS progression inhibitor according to claim 4, wherein ALS is FALS.   The ALS progression inhibitor according to claim 4 or 5, wherein HGF is administered in an amount of 0.01 to 100 mg per day.   A therapeutic agent for ALS comprising HGF as an active ingredient, wherein HGF is administered in an amount such that the HGF expression level in the spinal cord is twice or more that before administration.   The ALS therapeutic agent according to claim 7, wherein HGF is administered into the spinal cord or ventricle.   An agent for suppressing the progression of ALS, comprising HGF as an active ingredient, wherein HGF is administered in an amount such that the HGF expression level in the spinal cord is twice or more that before administration.   The ALS progression inhibitor according to claim 9, wherein HGF is administered into the spinal cord or ventricle.   A muscle weight loss inhibitor in ALS comprising HGF as an active ingredient.   The muscle weight loss inhibitor in ALS according to claim 11, wherein HGF is administered into the spinal cord or ventricle.   13. The ALS muscle weight loss inhibitor according to claim 11 or 12, wherein HGF is administered in an amount of 0.01 to 100 mg per day.

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