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WO2002022162A1 - Remede contre la sclerose laterale amyotrophique - Google Patents

Remede contre la sclerose laterale amyotrophique Download PDF

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Publication number
WO2002022162A1
WO2002022162A1 PCT/JP2001/001539 JP0101539W WO0222162A1 WO 2002022162 A1 WO2002022162 A1 WO 2002022162A1 JP 0101539 W JP0101539 W JP 0101539W WO 0222162 A1 WO0222162 A1 WO 0222162A1
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Prior art keywords
hgf
als
mice
gene
transgenic mice
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Japanese (ja)
Inventor
Toshikazu Nakamura
Hiroshi Funakoshi
Woong Sun
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Sumitomo Pharma Co Ltd
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Sumitomo Pharmaceuticals Co Ltd
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Priority to CA2422774A priority Critical patent/CA2422774C/fr
Publication of WO2002022162A1 publication Critical patent/WO2002022162A1/fr
Anticipated expiration legal-status Critical
Priority to US15/399,211 priority patent/US10058590B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a therapeutic agent for amyotrophic lateral sclerosis (hereinafter, referred to as ALS). More specifically, the present invention relates to an ALS therapeutic agent that makes HGF (hepatocyte growth factor) and / or HGF gene effective.
  • HGF hepatocyte growth factor
  • ALS Amyotrophic lateral sclerosis
  • FALS familial ALS
  • S0D1 Cu 2+ / 3 ⁇ 4i 2+ superoxide dismutase
  • Hepatocyte growth factor was first identified as a potent mitogene for mature hepatocytes, and its gene was cloned in 1989 (Biochem. Biophys. Res. Commun •, 122, 1450 1459 ( 1984), Nature, 342, 440-443 (1989)). HGF was discovered as a hepatocyte growth factor.A number of recent studies on expression and functional keratoconi, including mosquito, knockout, and knock-in mouse techniques, have revealed that HGF is a novel neurotrophic factor. (Ciba. Found. Symp., 212, 198-211 (1997), Nat Neurosci., 2, 213-217 (1999)).
  • HGF exerts neurotrophic factor activity on hippocampus, cerebral cortex, dopaminergic midbrain, cerebellar granules, sensory, motor neurons, and sympathetic neuroblasts (Ciba.Found.Symp., 212, 198-211 (1 997), Brain Res. Mol. Brain Res., 32, 197-210 (1995)).
  • HGF de novo and trophic factor activity on mature motor neurons after developmental fetal movement motor neurons and axotomy of the hypoglossal nerve have been demonstrated in vivo (J. Neurosci., 20, 326-337). (2000), Eur. J. Neurosci., 11, 4139-4144 (1999)).
  • HGF de novo and trophic factor activity on mature motor neurons after developmental fetal movement motor neurons and axotomy of the hypoglossal nerve
  • the present inventors first examined the involvement of HGF in ALS using G93A transgenic mice, which are ALS model mice. The results showed that c-Met / HGF receptor homozygous immunoreactivity (c-Met-IR) was localized in G93A transgenic mice as well as in wild-type littermates. Was found. Furthermore, with the development of ALS in G93A transgenic mice, we found that c-me1: and TIGF mRNA expression levels in the circulatory horn (localization of motor neurons) increased with the growth of mice. Was. These results suggest that HGF plays a role in ALS motor exercise.
  • mice expressing rat HGF in a Ron-specific manner were prepared, and whether or not HGF actually had an effect on ALS was examined using the mice.
  • HGF neuron-specific enolase promoter-1
  • HGF reduces motor neuron death and suppresses motor neuron axon degeneration.
  • HGF exerts a neuroprotective effect not only on motor neurons but also on DRG sensory nerves related to ALS-related neurons.
  • HGF was shown to have a neuroprotective effect in delaying muscle loss in ALS.
  • HGF HGF could actually suppress (delay) the progress of ALS using the transgenic mice described above.
  • the results showed that the expression of HGF in very small amounts (about twice that of wild-type and G93A mice) surprisingly delayed the onset of paralysis, prolonged paralysis, and improved motor performance. It became.
  • HGF has an ALS improving effect.
  • caspase-1 In the metaphase of ALS, caspase-1 has been shown to be activated and / or induced by motor neurons in transgenic mice expressing the mutant S0D1; 13 ⁇ 4 (Proc. Natl. Acad. Sci. USA, 95, 15763-15768 (1998), Science, 288, 335-339 (2000), and the introduction of a dominant negative inhibitor of caspase-1 into G93A mice successfully delayed mortality for about two weeks. (Nature, 388, 31 (1997), J. Exp. Ed., 185, 933-940 (1997)), it is thought that caspase-1 plays an important role in the progression of this ⁇ . Thus, the present inventors examined whether HGF alters caspase-1 induction, and as a result, it has been clarified that HGF has an effect of suppressing caspase-1 induction in ALS motor neurons.
  • Akt phosphorylation is involved in HGF survival-promoting activity in cerebral cortical neurons and kidney epithelial cells HGF induces BoL-xL expression in the liver of a fulminant hepatitis model, and has been shown to block large amounts of apoptosis (Biochein. Biophys. Res. Commun. , 244, 683-690 (1998), Hepatology, 30, 15 159 (1999)).
  • HGF induces BoL-xL expression in the liver of a fulminant hepatitis model, and has been shown to block large amounts of apoptosis (Biochein. Biophys. Res. Commun. , 244, 683-690 (1998), Hepatology, 30, 15 159 (1999)).
  • the present inventors examined whether Akt phosphorylation occurs due to HGF expression. As a result, it was found that Akt phosphorylation was conspicuous in ALS.
  • HGF is responsible for at least three mechanisms: by preventing caspase-1 induction in motor neurons, by phosphorylating Akt, and by glial cell-specific glutamate transporters in reactive astrocytes ( It has been shown to improve ALS by preventing down-regulation of EAAT2 / GLT1).
  • HGF exerts both direct neurotrophic factor activity on motoneurons and indirect improvement of dalminic acid cytotoxicity on motoneurons by maintaining glutamate transporter levels in stellate cells.
  • ALS motor function and ⁇ It has the effect of improving.
  • bifunctional growth factors have not been previously known, and HGF is the first example. These functions of HGF indicate that HGF (gene or protein) can be used therapeutically in ALS and related motoneuron diseases. Disclosure of the invention
  • ALS therapeutic agent containing HGF and / or HGF gene as active ingredient
  • ALS progress inhibitor containing HGF and Z or HGF gene as active ingredient
  • HGF and / or an HGF gene-containing active ingredient an inhibitor of induction of cas or o-ze1 in motor neurons
  • an Akt phosphorylation promoter in the spinal cord comprising an HGF and HGF gene or an HGF gene as an active ingredient
  • Figure 1 shows immunohistological analysis of c-Met expression (c-Met-IR) in motoneurons of G93A transgenic mice (G93A in the figure) and wild-type littermates (wild dtype in the figure).
  • 6 is a micrograph showing the result of further examination. In the figure, 3 ⁇ 4 ⁇ is 2 months old, and 8mo is 8 months old.
  • FIG. 2 is a graph showing the results obtained by performing competitive RT-PCR on HGF and ciet A in the spinal cord in the progression of disease in G98A mice, and comparing the results with those 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).
  • the arrowhead indicates the c_met or HGF band, and the bar indicates the competition. This shows the band of the Italian.
  • FIG. 3 is an electrophoresis photograph showing the results of analyzing the expression of exogenous HGF in each tissue of an HGF transgenic mouse prepared using a neuron-specific enolase promoter using an RNase protection assay.
  • the arrow indicates a band corresponding to exogenous HGF
  • the bar indicates a band corresponding to endogenous HGF.
  • Br is the brain
  • Lu is the lung
  • Ht is the heart
  • Li is the liver
  • Kd is the kidney
  • Sp is the spleen
  • St is the stomach
  • Sc is the spinal cord
  • Ts is the testis
  • In indicates intestine
  • Sk indicates skin.
  • FIG. 5 is a graph showing the results of analysis by ELISA of HGF content in whole litters of wild-type and transgenic mice at the postnatal development stage. In the figure, P2 indicates the second day after birth, P14 indicates the 14th day after birth, and lmo indicates 1 month old and 6mo indicates 6 months old. 4 is a photomicrograph showing the results of histological analysis of the number and morphology of.
  • wildtype shows the results of wild-type mice
  • HGF shows the results of HGF-only transgenic mice
  • G93A shows the results of G93A transgenic mice (ALS terminal stage)
  • G93A / HGF shows the results of double transgenic mice (ALS terminal stage).
  • FIG. 7 is a graph showing the results of quantitatively comparing the number of motoneurons in the lumbar spinal cord in each transgenic mouse.
  • ⁇ indicates wild-type
  • Hata indicates HGF-only transgenic mouse
  • A3 ⁇ 4G93A transgenic mouse indicates double transgenic mouse.
  • FIG. 8 is a graph showing the wet weight of the gastrocnemius muscle in each transgenic mouse.
  • ⁇ indicates wild-type
  • HGF indicates transgenic mice with HGF alone
  • ⁇ indicates double transgenic mice.
  • FIG. 9 is a photomicrograph showing the morphology of a section (lm) of the anterior root (V, upper panel) and dorsal root (D, lower panel) of each 8-month-old transgenic mouse.
  • FIG. 10 is a graph showing the results obtained by ELISA for circulating HGF levels of 2, 6 and 8 months old in each transgenic mouse.
  • Figure 15 shows transgenic mice with wild-type ((, ri-14) and HGF alone ( ⁇ ).
  • FIG. 16 is a micrograph showing the results of color-free analysis of the aggregation of mutant S0D1 in the lumbar spinal cord of G93A and G93A / HGF littermates during disease progression.
  • 4mo is 4 months old
  • 8mo is 8 months old.
  • Figure 17 shows the amount of mutant S0D1 in the lumbar spinal cord of wild-type (W), HGF alone (H), G93ACG), and double transgenic (G / H) mice.
  • 9 is an electrophoresis photograph showing the result of squaring by winging. In the figure, 2mo indicates 2 months old, 6mo indicates 6 months old, and 8mo indicates 8 months old.
  • FIG. 18 shows the phosphorylated Akt (8-month-old lumbar spinal cord) in wild-type (W), HGF alone (H), G93A (G), and double transgenic (G / H) mice.
  • 9 is an electrophoretic photograph showing the results of immunostaining of (p-Akt), Akt, Bcl-xL, and ⁇ cl-2.
  • Figure 19 shows the immunohistochemical analysis of the lumbar anterior horn at 8 months of age of the four mice using GFAP. It is a microscope picture which shows a result.
  • FIG. 20 is a graph showing the immunohistochemical analysis using the GFAP shown in FIG.
  • ⁇ indicates wild type
  • Hata indicates HGF-only transgenic
  • FIG. 21 is an electrophoretic photograph showing the results of immunoblotting of EAAT2, GFAP and c-Met in the spinal cord of the four mice at 2, 6 and 8 months.
  • W indicates wild-type
  • H indicates HGF-only transgenic
  • G indicates G93A transgenic
  • G / H indicates double transgenic.
  • FIG. 23 is an electrophotograph showing EAAT2 and c-Met immunoblotting in primary cultured astrocytes after HGF treatment.
  • H GF used in the present invention is a substance, and its base sequence and amino acid sequence are described in Nature, 342, 440 (1989), Patent No. 2777678, Biochera. Biophys. Res. Comraun.,
  • HGF human growth factor
  • those purified by various methods can be used as long as they are purified to the extent that they can be used as pharmaceuticals.
  • commercially available products for example, Toyobo Code No. HGF-101, etc. can be used.
  • the HGF can be obtained by culturing primary cultured cells or cell lines that produce HGF, and separating and purifying it from a culture supernatant or the like.
  • a gene encoding HGF is inserted into an appropriate vector by a genetic genetic technique, inserted into an appropriate host, and transformed, and the recombinant HGF of interest is obtained from the culture supernatant of the transformant.
  • the host cell is not particularly limited, Various host cells conventionally used in genetic engineering techniques, such as Escherichia coli, yeast, or animal cells, can be used.
  • animal cells include CH0 cells, COS cells', and mouse C127 cells (all available from ATCC and the like).
  • the HGF thus obtained may have a structure similar to the natural HGF as long as it has substantially the same action as the natural HGF. That is, 1) a protein encoded by DNA that hybridizes with the HGF cDM under stringent conditions, and 2)
  • a protein comprising an amino acid sequence in which one or more (preferably several) amino acids have been substituted, deleted and / or added to the amino acid sequence of GF; For example, it is included in the category of the HGF of the present invention.
  • code the evening protein of 1) and 2)! ) NA can be easily obtained by, for example, site-directed mutagenesis, PCR, or ordinary hybridization, and specifically, Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989) ) Etc. can be used as a reference.
  • HGF is a therapeutic agent for ALS, specifically, an ALS progress inhibitor.
  • HGF is a direct neurotrophic factor activity on motor neurons and an indirect effect of glutamate cytotoxicity on motor neurons by maintaining dalminic acid transporter levels in star cells. It has the effect of improving the motor function and lifespan of ALS through the two functions of the effective improvement.
  • Such bifunctional growth factors are not known before and are the first example of HGF. By exerting these two effects, HGF can slow or inhibit the progression of ALS and related motor neuron disease.
  • HGF is considered to exhibit ALS-improving effects by exerting at least the following three actions.
  • HGF HGF Is the first growth factor to be shown.
  • motor neuron diseases related to ALS include progressive spinal muscular atrophy, advanced '1 live bulb paralysis, primary lateral sclerosis, pediatric and juvenile muscular atrophy, fogio-Ronde's disease, and Charcoal's disease. 'Mary' disease is not limited to these.
  • the therapeutic agent of the present invention may be injectable with various forms of preparation (eg, liquid, solid, etc.), generally with only effective HGF alone or with a conventional carrier.
  • the injection can be prepared by a conventional method. For example, after dissolving HGF in an appropriate solvent (eg, sterilized water, buffer solution, physiological saline, etc.), sterilize by filtration with a filter or the like, and then sterilely. It can be prepared by filling into a container.
  • the content of HGF in the preparation is usually adjusted to about 0.0002 to 0.2 (f / V%), preferably to about 0.001 to 0.1 (ff / V%).
  • a stabilizer is preferably added, and examples of the stabilizer include albumin, globulin, gelatin, mannitol, glucose, dextran, and ethylene glycol.
  • the preparation of the present invention may contain additives necessary for preparation, for example, excipients, solubilizing agents, antioxidants, soothing agents, isotonic agents and the like.
  • excipients for example, excipients, solubilizing agents, antioxidants, soothing agents, isotonic agents and the like.
  • a liquid preparation it is desirable to store it after freezing or freezing it by freeze-drying. The freeze-dried preparation is used after reconstitution by adding distilled water for injection at the time of use.
  • the preparation of the present invention is administered by an appropriate administration route depending on the form of the preparation.
  • the administration route may be any administration route as long as HGF exerts an action and the progress of ALS can be suppressed.
  • examples include subcutaneous administration, intradermal administration, intravenous administration, intraarterial administration, intramuscular administration, local injection, intraventricular administration, and intraspinal administration.
  • there is also a route of administration via a device implanted in the body Specifically, a method of continuously and gradually administering to the affected area using an osmotic pump or the like, or a sustained-release preparation (for example, a mini pellet) Formulation) near the affected area.
  • intraspinal administration is a preferred route of administration because the preparation of the present invention can directly reach motor neurons in the spinal cord.
  • the dosage is appropriately adjusted depending on the patient's condition, age, body 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 administer it separately.
  • the HGF preparation of the present invention is preferably administered to an ALS patient in such an amount that the expression level of HGF in the spinal cord is at least twice as high as before HGF administration.
  • an ALS Jito U comprising administering such an amount that the expression level of HGF in the # 1 way becomes twice or more that before administration of HGF. .
  • the present invention includes not only the administration of the HGF protein as described above, but also an ALS gene therapy agent comprising the introduction of the HGF gene. The following describes gene therapy for HGF.
  • the “HGF gene” used in the present invention refers to a gene capable of expressing HGF (HGF protein). Specifically, Nature, 342, 440 (1989), Patent No. 2777678, Biochem. Biophys. Res. Commun., 163, 967 (1989), Biochem. Biophys. Res. Coramun., 172, 321 ( 1990), etc., into a suitable expression vector (non-viral vector, viral vector) as described below.
  • a suitable expression vector non-viral vector, viral vector
  • the nucleotide sequence of the cDNA encoding HGF is described in the Kasumi literature and also registered in databases such as Genbank.
  • an appropriate DNA portion is used as a primer for PCR, for example, by performing an RT-PCR reaction on mRNA derived from the liver or leukocytes, and the like. can do.
  • clonings can be performed by those skilled in the art, for example, according to a basic book such as "Molecular Cloning 2nd Edt, Cold Spring Harbor Laboratory Press (1989)". It can be done easily.
  • 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 has a gene substantially the same action as HGF. That is, 1) a DNA that hybridizes with the cDNA under stringent conditions, or 2) one or more (preferably several) amino acids substituted with the amino acid sequence of the protein encoded by the cDNA, Among the DNAs encoding a protein consisting of a deleted and / or added amino acid sequence, and the like, as long as they encode a protein having an action as an HGF, they are included in the category of the HGF gene of the present invention.
  • the DNAs of 1) and 2) can be easily obtained by, for example, site-directed mutagenesis, PCR, or ordinary hybridization, and specifically, This can be done with reference to basic books such as.
  • the HGF gene of the present invention can be applied to the same diseases as the aforementioned HGF protein.
  • the HGF gene and the HGF protein can be used independently, or both can be used in combination.
  • the dosage form can be broadly divided into two types: using a non-viral vector and using a viral vector.
  • the methods of preparation and administration are described in detail in the experimental manuals. (Separate volume experimental medicine, Basic technology of gene therapy, Yodosha, 1996, Separate volume experimental medicine, Gene transfer & expression analysis experiment method, Co., 1997, Gene Therapy Development Research Handbook, edited by The Japan Society for Gene Therapy, N.T.S., 1999). Hereinafter, a specific description will be given.
  • the target gene can be introduced into cells or tissues by the following method. Examples include lipofection method, monocalcium phosphate coprecipitation method, DEAE-dextran method, and direct DNA injection method using a micro glass tube.
  • lipofection method monocalcium phosphate coprecipitation method
  • DEAE-dextran method direct DNA injection method using a micro glass tube.
  • gene transfer into tissues includes gene transfer using internal liposomes, gene transfer using electrostatic liposomes, HVJ-liposome method, and improved HV.
  • J-liposome method HVJ-AVE liposome method
  • receptor-mediated gene transfer method method of transferring DNA molecules into cells with carrier (metal particles) using protein, naked-DNA
  • the recombinant expression vector can be taken into cells by subjecting it to any of the methods such as direct introduction method and introduction method using positively charged polymer.
  • HVJ-ribosome is a fusion of ribosome and inactivated Sendai virus (Hemagglutinating virus of Japan: HVJ) that encapsulates DNA in ribosome made of lipid bilayer. It is.
  • the HVJ-liposome method is characterized by having a very high fusion activity with the cell membrane as compared with the conventional liposome method, and is a preferred introduction form.
  • HV J_ ribosome refer to the literature (Experimental Medicine Supplement, Basic Technology of Gene Therapy, Yodosha, 1996, Gene Transfer & Expression Analysis Experimental Method, Yodosha, 1997, J. Clin. Invest. 93, 1458-1464 (1994), Am. J. Physiol. 271, R1212-1220C1996)), etc., please refer to them.
  • HV J strain Z (available from ATCC) is preferred, but basically other HV J strains (eg, ATCC VR-907 and ATCC VR-105) can also be used.
  • 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 is a vector capable of expressing the target gene in vivo.
  • pCAGGS Gene 108, 193-200 ( 1991)
  • pBK-CMV pcDNA3.1
  • pZeoSV Invitrogen, Stratagene
  • a typical example of a virus vector is a method using a vinoless vector such as a recombinant adenovirus or a retrovirus. More specifically, for example, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, Introducing a gene of interest into DNA or RNA viruses such as Nyavirus, Box virus, Poliovirus, Synvis virus, Sendai virus, SV40, and Immunodeficiency virus (HIV), and infecting cells with the recombinant virus. Thus, it is possible to introduce a gene into a cell.
  • a vinoless vector such as a recombinant adenovirus or a retrovirus. More specifically, for example, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, Introducing a gene of interest into DNA or RNA viruses such as Nyavirus, Box virus, Poliovirus, Synvis virus, Sendai virus, SV40, and Immunodeficiency virus
  • Adenovirus vector is used. I like that power.
  • Examples of the method for introducing the gene therapy agent of the present invention into a patient include an i ⁇ ⁇ i ⁇ ⁇ method in which the gene therapy agent is directly introduced into the body, and a method in which certain types of cells are removed from a human and the gene therapy agent is added outside the body.
  • There is an eXViV0 method that introduces the cells into the cells and returns the cells to the body (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994) ), Experimental Medicine Special Edition, 12 (15), (1994), Handbook of Gene Therapy Development Research, edited by the Japanese Society of Gene Therapy, N'TIS, 1999).
  • the invivo method is preferred.
  • the administration site to the patient may be any administration route as long as HGF expressed in the living body exerts an action and can suppress the progress of ALS.
  • examples include subcutaneous administration, intradermal administration, intravenous administration, intraarterial administration, intramuscular administration, local injection, intraventricular administration, and circulatory administration.
  • administration routes via devices implanted in the body include a method in which an osmotic pump or the like is used to continuously and gradually administer the disease to the affected area, and a sustained-release preparation (for example, a minipellet). Formulation) is implanted near the affected area.
  • intraspinal administration is a preferred route of administration because the preparation of the present invention can directly reach motor neurons in the spinal cord.
  • the formulation various formulations (for example, liquids, etc.) suitable for each of the above administration forms can be taken.
  • the drug in the case of a drug containing an effective ⁇ gene, the drug can be prepared by a conventional method.
  • it can be prepared in an appropriate solvent (buffer such as PBS, physiological food ⁇ ⁇ . Sterile water, etc.).
  • the mixture may be sterilized by filtration with a filter or the like, and then filled in a sterile container to prepare the composition.
  • Conventional carriers and the like may be added.
  • ribosomes such as HVJ-ribosomes, suspending agents,? It can be in the form of ribosome preparations such as east binding agent, centrifugal concentrated and jujube binding 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.
  • the DNA of the present invention is preferably 0.0001 to 100 mg. It is recommended that the dose be 0.0 0.01 to 10 mg, administered once every few days or months.
  • the present invention provides a therapeutic agent for ALS 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.
  • Transgenic G93A mice (SOD G93A) lGurdl) were purchased from Jackson Laboratory (Science, 264 1772-1775 (1994)). For maintenance, G93A mice were bred to female C57B6 mice and genotyped by PCR and dot-blot hybridization.
  • rat HGF cDNA (Proc. Natl. Acad. Sci. USA 87, 3200-3204 (1990)) tagged with KT3 epitope was amplified by PCR, and the pNSE-Ex vector (Dr. Doherty) The DNA was inserted downstream of the neuron-specific enolase promoter (Neuron, 18, 231-241 (1997) :). For subcloning, a Not I site was introduced into the pNSE-Ex vector by adding a linker. Thereafter, the entire nucleotide sequence of the plasmid was confirmed.
  • HGF-KT3 showed the same activity as human recombinant HGF in the migration assay of DCK cells and the in vitro survival assay of primary culture of hippocampal neurons.
  • the method described previously J. Cell. Biol., 128, 185-199 (1995), Proc. Natl. Acad. Sci., USA, 95, 5269-5274 (1998)) Transgenic mice were made with improvements and weighed for transgene incorporation. Briefly, the transgenic cassette was excised from the vector by treatment with Sal I restriction enzyme, and DNA was injected into C57B6 embryos whose genetic background was consistent with the G93A transgenic mouse. Transgene integration was confirmed by PCR and dot-blot using the SV40 poly (A) sequence in the transgene. Expression of exogenous HGF was determined by RNase protection assay using a probe covering the 3 'end of HGF-KT3 cDNA and the 5' region of the SV40 poly (A) signal sequence.
  • NSE-HGF transgenic mice Progeny of NSE-HGF transgenic mice were crossed with G93A transgenic mice. Offspring from one parent were kept in the same cage until the phenotype was destroyed. After the first signs of the phenotype, the animals were isolated. Food and water were placed at the bottom of the cage for easy access. This time was used as the death time when the animal could not stand its body within 30 seconds.
  • the RNase protection assay was performed as previously described (J. Cell. Biol., 123, 455-465 (1993), Science, 268, 1495-1499 (1995)), using the RPAII ribonuclease protection assay kit ( Ambion, Austin, TX).
  • a 326 bp fragment containing the polyA sequence from the HGF 3 'end of HGF-KT-3-polyA was inserted into the pGEM-T vector. . Plasmids were linearized, transcribed in the presence of a- [ 32 P] CTP, and prepared cRNA probes were used for hybridization.
  • Exogenous HGF RNA gave protected band of 326 bp
  • endogenous HGF RNA is KT - gives 3 and poly (A) because it lacks a sequence shorter protection band (251b P) o
  • Hind limb extension is usually observed when the mouse is hung in the air with its tail. Exercise two euron chiji mouse Hind limb retraction is commonly shown. The score corresponds to the number of hind limbs extended.
  • the mouse was placed on a rod rotating at 202 °. The duration of each mouse remaining on the rod was measured. If the animal was able to remain on the rod for 4 minutes, the test was completed in 4 minutes and recorded as 4 minutes. »Was collected by dipping the feet in black ink and then walking the mouse along a straight path. The stride length was measured in the area showing normal walking.
  • the spinal cord was fixed by a double-armed concentration of ethanol to count two eurons of exercise. After embedding in raffin, it was cut continuously from L5 to L4 (14 m). The number of interlocking neurons (in L4-L5) in the anterior horn was counted for every 7 slices for a total of 20 slices. Neurons whose clear nucleoli were clearly stained by Chris Biorelet in defined areas 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) after blocking with 5% goat serum and mouse IgG blocking agent for 1 hour (M.O.M kit, Vector science).
  • the sections were added to the sections for 1 hour to 4 hours at room temperature or overnight at 4 ° C. After washing, a biotinylated or fluorescently labeled secondary antibody was added and incubated for 15 minutes. Sections were counterstained here with Hoechst33342 for fluorescence immunization and observed under a fluorescence microscope.
  • the L5 route was dissociated, fixed with 4% paraformaldehyde (0.25% dartaraldehyde) for 1 hour, fixed on ice with osmium tetroxide for 2 hours, dehydrated, and embedded in ⁇ 812.
  • the embedded root was cut (1 ⁇ ) and stained with Tolu Zimble I. The morphology of the sample was examined under an optical microscope.
  • the tissue is crushed by aspirating with a pipette and the separated cells are plated on a poly-L-ornithine-coated dish at the indicated cell number in DF containing 10% fetal bovine serum (DF MEM / HamF12, 50:50) Plated in medium (GibCO BRL). Seven days after plating When cells become confluent, cells were washed twice with PBS and treated with the indicated concentrations of recombinant HGF. The purity of the star cyst was usually 95% or higher after 7 days of culture, and was examined by morphology or NSE / GFAP double staining.
  • Lumbar spinal cord extract was prepared in RIPA buffer. 50 ⁇ g of the lysate was run on a 10% SDS-polyacrylamide gel (SDS-PAGE) in mm and transferred to a PVDF membrane. The membrane was incubated in PBS at 4 ° C. with 5% non-atmospheric skim milk 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 (Amersham, Buckinghamshire, UK).
  • SDS-PAGE 10% SDS-polyacrylamide gel
  • the membrane cap antibody was removed and reprobed with anti-GFAP (1: 3000, Sigma), anti-human SOD (1: 500, Sigma), and anti-c-Met (1: 400, Santa Curz) antibodies. 50 g of the lysate was subjected to 12% SDS-PAGE and phosphorylated Akt (Cell Signaling Tech.), Akt (Cell Signaling Tech.), Bcl-xL / S (Santa Cruz) and ⁇ cl-2 (Santa Cruz) antibody was added. Band intensities were measured on a Fluorchem imager instrument (IS-8 000).
  • HGF in plasma was measured using the anti-rat HGF polyclonal monoclonal antibody (Tokushu Meneki, Tokyo, Japan) by the ELISA method.
  • the rat HGF E LISA system specifically detects rat and mouse HGF with similar affinity.
  • c-Met-IR c-Met / HGF receptor uniform responsiveness
  • FIGS. 3, 4 and 5 show the characteristics of the HGF transgenic mouse strain. Exogenous HGF is restricted to the brain and spinal cord of the 12 lams tested by RNase Protection Technology It was shown that the gene was expressed (arrow in FIG. 3). During the organization of HGF transgenic mice, endogenous and total HGF levels in plasma did not differ from wild-type littermates, respectively (FIGS. 3 and 4). HGF protein levels, on the other hand, increased with mouse growth in the spinal cord only after the end of major postnatal 3 ⁇ 41 nerve differentiation (FIG. 5).
  • HGF transgenic mice could not be distinguished from wild-type transgenic mice without genotyping. That is, size, weight, morphology, is! /, Contains many motor neurons and astrocytes !; behaves in the motor nervous system, changes, examines, and muscles at a significant stage The difference in weight of t was not found. This suggests that HGF can be successfully introduced into the nervous system without the need to reach them (see FIGS. 6 to 10 and FIGS. 19 and 20). Difficult case 3
  • heterozygous (+/-) HGF transgenic mice were bred to heterozygous cells in G93A mice (//), thereby transducing ALS neurons.
  • Transgenic mice into which HGF was directly introduced were prepared. It should be noted that the cross resulted in four different groups of mice: wild type (ff), transgenic for only HGF (HGF), transgenic for G93A only (G93A), and double transgenic for both G93A and HGF.
  • Transgenic (G93A / HGF) power 'generated.
  • HGF transduction was demonstrated at the cervical level, as the survival-promoting activity of HGF on motoneurons has been shown to differ between lumbar and cervical levels in chick embryos. Was also tested for their ability to reduce their deaths.
  • G93A mice 55% of cervical motoneurons remained at 8 months of age. Less than at the waist level! The death of motorized mouths at the cervical level is characteristic of the present model mice.
  • a higher number (87.8 ⁇ 2.4%) of motoneurons remained at the cervical level in the double transgenic mice, indicating that HGF had lumbar and cervical motility. It is effective for both euros.
  • HGF was shown to exert neuroprotective effects not only on motor neurons but also on DRG sensory neurons for ALS-related neurotoxicity.
  • the protective effect of HGF on dementia was also demonstrated by a delayed effect on the reduction of gastrocnemius muscle weight (FIG. 8).
  • HGF-only transgenic mice did not show any significant differences in motor function from wild-type littermates.
  • G93A mice showed a progressive decrease in hind limb extension from 5 months of age, rotor-rod performance from 6 months of age, and stride length from 7 months of age.
  • the double transgenic mice showed much slower reductions than G93A in all functional parameters tested ( Figures 13-15).
  • FIG. 10 shows the HGF protein levels of 3 ⁇ 41 in four groups during ALS progression.
  • HGF levels were approximately twice as high in HGF transgenic mice and G93A / HGF littermates at 2 months and 6 months, indicating a significant improvement in motor function during the additional months of the addition of small amounts of HGF. Indicates that ⁇ is to be elongated.
  • the inadequate production of HGF in the G93A / HGF double transgenic at 8 months i.e., the HGF levels in HGF, G93A and G93A / HGF were terminally identical, suggesting that ALS Higher levels of HGF expression may be desirable as they may reflect incomplete improvements.
  • the total amount of mutant S0D1 in the circulating extract was determined by immunoblotting using an antibody specific for the human S0D1 antibody and thus recognizing only the mutant human S0D1, but not the endogenous mouse S0D1. Three independent animals from each group were tested. Mutation S0D1 was detected in both G93A and double transgenic mice from 2 months of age. And the total amount of mutant S0D1 increased in both G93A and double transgenic mice over a similar time course. The total amount of mutant S0D1 in G93A mice was not different from that of HGF littermates (FIG. 17). Next, SOW aggregation was examined.
  • Double transgenic mice at 6 months of age showed very weak caspase-11 IR in large tubulin III immunoreactive cells compared to G93A mice, indicating that HGF-induced It has been shown to reduce the level of caspase-1 induction in interlocking new mouths.
  • Akt Akt phosphorylation and regulation of the 3 ⁇ 4cl-2 family gene.
  • EAAT2 was reduced in G85R transgenic mice, an ALS model (Neuron, 18, 327-338 (1997)), and glial cell glutamate transporters associated with the S0D1 mutation (A4V and I113T) in ALS were also investigated. Glutamatergic excitotoxicity is thought to play a role in ALS motoneuron degeneration (Nat Neurosci., 2, 27-433 (1999)). Therefore, the involvement of HGF in ⁇ 2 was examined.
  • astrocytes were predominantly white matter and localized near the central canal, but slightly in the anterior horn (Figure 19). Reactive astrocytes in G93A mice progressively increased in the anterior horn at 6 months of age, when motor neuron death was not yet observed in G93A mice (FIG. 19). In contrast, double transgenic littermates had significantly lower numbers of reactive astrocytes in the anterior horn (FIG. 19). Quantification of the reactivity intensity of G FAP-IR showed that the immune response activity in the anterior horn was about 40% and 60% lower than that of 6 and 8 month old G93A mice, respectively ( (Fig.
  • EAAT2 EAAT2
  • GFAP GFAP
  • c-Met immunoblotting of the spinal cord of each mouse at 2, 6 and 8 months were performed, and as a result, GFAP of both G93A mice and G93A / HGF mice from 6 months was Guidance And specific down-regulation of EAAT2 in 8-month G93A mice and specific c-Met up-regulation in 8-month G93A and G93A / HGF mice. In G93A / HGF mice, the total level of EAAT2 was maintained at 8 months.
  • EAAT2 levels in G93A mice at 8 months of age were reduced by 40%, compared to those of wild-type or HGF-only transgenic littermates (FIG. 22).
  • double transgenic mice were wild-type or showed higher levels of EAAT2 (140%) in the lumbar spinal cord compared to littermates of transgenic mice with HGF alone (FIG. 22).
  • EAAT2 levels in individual astrocytes were divided by GFAP levels. Only 11% of EAAT2 remained in mosquito G93A mice, while 63% remained in EAAT2 double transgenic mice (FIG. 22). Although astrocytosis begins at six months, it is a worthwhile record that EAAT2 depletion and c-Met induction in astrocytes only occur in late stages in parallel.
  • HGS administration or HGF gene transfer suppresses ALS progression
  • an HGF protein preparation is administered daily at an appropriate frequency and at an appropriate site, such as in the spinal cord, at the age of 6 months when ALS symptoms begin to be observed.
  • a group to which a preparation containing no HGF protein was administered is used as a control group.
  • the effect of suppressing the progression of ALS as described in Examples 3 and 4 is examined in each of the HGF administration group and the control group. The effect of inhibiting ALS progression in the HGF administration group This confirms that HGF can cure ALS.
  • a therapeutic agent (progression inhibitor) for ALS comprising HGF and / or an HGF gene as an active ingredient
  • HGF exerts direct neurotrophic factor activity on motoneurons and an indirect amelioration of glutamate cytotoxin I production on motoneurons by retaining the dalminic acid transporter level in astrocytes. It has the effect of improving the motor function and lifespan of ALS through two actions.
  • Such bifunctional growth factors have not been known so far, and therefore, HGF and / or the HGF gene can be effectively used as an unprecedented effective treatment; ⁇ IJ.

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Abstract

L'invention concerne des remèdes (inhibiteurs de progression) contre la sclérose latérale amyothrophique (SLA) contenant en tant que principe actif HGF et/ou un gène HGF. Le HGF (facteur de croissance des hétapocytes) présente deux fonctions, c'est-à-dire, une activité de facteur neurotrophique directe sur les motoneurones et un effet indirect de soulagement de la cytotoxicité de l'acide glutamique sur les motoneurones par maintien du niveau de transporteur d'acide glutamique dans les astrocytes, améliorant ainsi la fonction motrice et prolongeant la durée de vie de patients atteints de SLA. Ainsi, le HGF et/ou le gène du HGF peuvent être utilisés efficacement en tant que remèdes efficaces inconnus jusqu'à présent.
PCT/JP2001/001539 2000-09-14 2001-02-28 Remede contre la sclerose laterale amyotrophique Ceased WO2002022162A1 (fr)

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US7601365B2 (en) 2000-08-28 2009-10-13 Damavand Wound, AB Synergetic effects of HGF and antibacterial treatment
WO2004030702A1 (fr) * 2002-10-02 2004-04-15 Anges Mg, Inc. Médicament pour traiter des troubles auditifs
US7390482B2 (en) 2002-10-02 2008-06-24 Anges Mg, Inc. Drug for auditory dysfunction
AU2003268741B2 (en) * 2002-10-02 2009-06-11 Anges Mg, Inc. Drug for auditory dysfunction
CN102869376A (zh) * 2010-03-09 2013-01-09 葛兰素史密丝克莱恩生物有限公司 链球菌感染的治疗
EP3061457A4 (fr) * 2013-10-22 2017-06-07 Viromed Co., Ltd. Composition pour la prévention ou le traitement de la sclérose latérale amyotrophique au moyen de deux isoformes ou davantage du facteur de croissance des hépatocytes
US10639351B2 (en) 2013-10-22 2020-05-05 Helixmith Co., Ltd. Method for treating amyotrophic lateral sclerosis with a polynucleotide encoding two or more isoforms of hepatocyte growth factor
CN106714823A (zh) * 2014-09-10 2017-05-24 克霖固鲁制药股份有限公司 适合神经系统疾病的治疗的hgf制剂
US10213485B2 (en) 2014-09-10 2019-02-26 Kringle Pharma Inc. HGF preparation suitable for treatment of neurological disorders
US10702582B2 (en) 2014-09-10 2020-07-07 Kringle Pharma Inc. HGF preparation suitable for treatment of neurological disorders

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