Classifications

• • 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
  • A61K38/22—Hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/425—Thiazoles
• • 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
• • 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
  • A61K38/22—Hormones
  • A61K38/25—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

• the present invention relates to a therapeutic agent for amyotrophic lateral sclerosis containing a growth hormone secretion promoting factor receptor agonist as an active ingredient.
• ALS Amyotrophic Lateral Sclerosis
• ALS is the most common motor neuron disease in adults, and is a neurodegenerative disease in which upper and lower motor neurons are selectively and systematically killed.
• motor neuron death muscle atrophy and muscle weakness of the upper and lower limbs progress, and in many cases, bulbous symptoms such as speech disorder and difficulty in swallowing and respiratory muscle paralysis are often added.
• ALS develops mainly after middle age and is a serious disease that causes many patients to die due to respiratory failure 2 to 3 years after onset without ventilator management.
• SOD1 Cu / Zn superoxide dismutase
• transgenic mice that express the Ala (A) substitution protein at position 93 Gly (G) of human SOD1 that is, studies using mutant SOD1 gene (G93A) -introduced mice (hereinafter referred to as SOD1 G93A mice) are the most advanced. Evaluation using this animal is recommended by the European ALS / MND Society (Non-patent Document 2).
• Non-patent Document 1 Currently, the only ALS treatment approved worldwide is riluzole. This drug antagonizes glutamate toxicity, which is one of the causes of ALS, but its life-prolonging effect is only a few months, and its effect is limited (Non-patent Document 1).
• Ghrelin is a peptide hormone discovered as an endogenous ligand of growth hormone secretagogue receptor (GHS-R) (Non-patent Document 3). Ghrelin enhances growth hormone (GH) secretion in humans and animals (Non-Patent Document 4, etc.). GH is known to promote insulin-like growth factor-1 (hereinafter, IGF-1) production in the liver and skeletal muscle, and IGF-1 is one of the trophic factors of motor neurons. When a recombinant AAV4 viral vector containing the IGF-1 gene was injected into the lateral ventricle of SOD1 G93A mice, the survival period was significantly prolonged, and the reduction of motor function and muscle strength was significantly suppressed (Patent Document 1).
• IGF-1 insulin-like growth factor-1
• Non-Patent Document 5 the effects of forced expression of IGF-1 in the brain have been reported in this way, overexpression of human IGF-1 in the skeletal muscle of SOD1 G93A mice also affects motor neuron death and survival.
• GH or IGF-1 when GH or IGF-1 is clinically administered to ALS patients, GH therapy is ineffective (Non-Patent Document 6), and IGF-1 is also not affected by any of the muscle strength, functional outcome, and survival time of ALS patients. The effect was not shown (nonpatent literature 7). Therefore, activation of peripheral GH and IGF-1 systems cannot be said to be effective for ALS.
• Ghrelin also promotes GH secretion from the pituitary gland and increases blood GH concentration (eg, Non-Patent Documents 3 and 4), but it is not known to increase IGF-1 in the brain or spinal cord. Whether ghrelin is effective against ALS pathology is unknown. Ghrelin is the only food-enhancing substance present in the periphery and has been reported to increase food intake in humans and animals (eg, Non-Patent Document 4). However, ALS is a disease that causes skeletal muscles to atrophy due to the death of motor neurons, resulting in a decrease in muscle strength and death, so ghrelin administration only increases food intake and maintains body weight and skeletal muscle mass. The relationship with the suppression of motor neuron death is also unclear, and it is unclear whether ghrelin is effective against ALS pathology.
• Non-patent Document 8 Similarly, it has been reported that ALS patients with high blood cholesterol and triglyceride concentrations have a good prognosis (Non-patent Document 9). Although administration of ghrelin to mice did not affect blood triglyceride levels, it has been reported that blood total cholesterol levels increased (Non-patent Document 10), but ghrelin is used to treat hyperlipidemia. There is also a report that it is useful (Patent Document 2).
• Non-patent Document 11 It has also been reported that when ghrelin was repeatedly administered to patients with chronic respiratory inflammation for 3 weeks, weight and nutritional status were improved, but blood cholesterol levels were not affected. As described above, certain knowledge about the effect of ghrelin on blood cholesterol and neutral fat has not been obtained.
• ALS is an example of a non-ischemic neurodegenerative disease in which the compound group is effective.
• ALS is a disease in which motor neurons selectively die after adulthood, and hippocampal cells are not damaged. Therefore, even if GHRP-2 or ghrelin suppresses cell death of fetal rat hippocampal neurons induced by an increase in Ca concentration in vitro, it is unclear whether these compounds are effective for ALS.
• Non-Patent Documents 12 and 13 In in vitro rat spinal cord organ culture systems, ghrelin has been reported to inhibit glutamate-induced motor neuron death (Non-Patent Documents 12 and 13). However, the in ⁇ ⁇ vitro spinal organ culture system does not fully reflect the pathology of complex diseases such as ALS.
• Plarmorelin increases the proportion of cells with dendrites and axons more than 1.5 times the cell body in rat adrenal pheochromocytoma-derived PC-12 cells by about 1.2 times compared to the control group
• GHRP-2 is useful for ALS.
• GHRP-6 growth-Hormone Releasing Peptide-6
• a cell growth factor a single EGF alone only improves gait pattern disorders, but there are reports that administration of both simultaneously behaviorally improves gait in mice and significantly improves skeletal muscle action potentials.
• the animal model used differs from the clinical pathology of ALS, it cannot be determined whether GHRP-6 is useful for ALS.
• GHS-R antagonists that are ghrelin receptors are also useful for the treatment of ALS (Patent Literature 5, Patent Literature 6), so GHS-R agonists containing ghrelin are ALS It is not necessarily presumed to be useful for treatment.
• the present inventors have conducted intensive studies on a therapeutic agent useful for ALS for which there is no currently effective therapeutic agent, particularly a drug that can be treated more effectively than riluzole, which is the only existing drug.
• the present inventors have found that an R agonist exhibits a more effective motor neuron protective effect than the existing drugs. That is, the present invention includes the following inventions.
• a growth hormone secretion promoting factor receptor agonist or a pharmaceutically acceptable salt thereof as an active ingredient and administered to an individual suffering from amyotrophic lateral sclerosis with no severe dysphagia
• a therapeutic agent for amyotrophic lateral sclerosis A growth hormone secretion promoting factor receptor agonist or a pharmaceutically acceptable salt thereof as an active ingredient, and administered to an individual suffering from amyotrophic lateral sclerosis with no severe dysphagia.
• the above growth hormone secretagogue receptor agonist is ghrelin, pralmorelin, GHRP-6, hexarelin, ipamorelin, ibutamorene methanesulfonate, urimorelin, anamorelin, masmorelin, capromorelin or SM-130686 (1)
• the therapeutic agent according to any one of (5) to (5).
• Peptide compound wherein ghrelin has the amino acid sequence set forth in SEQ ID NO: 1 and the third amino acid residue from the amino terminus is a modified amino acid residue in which a fatty acid is introduced into the side chain of the amino acid residue Or in the amino acid sequence shown in SEQ ID NO: 1 having an amino acid sequence in which one to several amino acids are deleted, substituted and / or added in the amino acid sequence from the amino terminus to the fifth to 28th amino acids, and 3 from the amino terminus.
• the intracellular amino acid residue is a peptide compound in which the amino acid residue is a modified amino acid residue in which a fatty acid is introduced into the side chain of the amino acid residue, and the intracellular calcium ion concentration by binding to the growth hormone secretagogue receptor
• the therapeutic agent according to the above (6) which is a peptide-based compound having an activity of increasing the level.
• a peptide in which ghrelin has the amino acid sequence set forth in SEQ ID NO: 1 and the third amino acid residue from the amino terminus is a modified amino acid residue in which a fatty acid is introduced into the side chain hydroxyl group of the amino acid residue The therapeutic agent according to the above (7), which is a system compound.
• the above ghrelin is a peptide compound having the amino acid sequence set forth in SEQ ID NO: 1, wherein the side chain hydroxyl group of the third amino acid residue from the amino terminus is acylated with an n-octanoyl group ( The therapeutic agent as described in 8).
• a method for treating amyotrophic lateral sclerosis comprising administering to an individual suffering from the disease.
• the above-mentioned growth hormone secretion promoting factor receptor agonist is ghrelin, pralmorelin, GHRP-6, hexarelin, ipamorelin, ibutamorene methanesulfonate, urimorelin, anamorelin, masmorelin, capromorelin or SM-130686 (10) The treatment method of any one of (14) thru
• a peptide compound in which ghrelin has the amino acid sequence set forth in SEQ ID NO: 1 and the third amino acid residue from the amino terminus is a modified amino acid residue in which a fatty acid is introduced into the side chain of the amino acid residue
• an amino acid sequence represented by SEQ ID NO: 1 having an amino acid sequence in which 1 to several amino acids have been deleted, substituted and / or added in the amino acid sequence from the 5th to the 28th amino acid from the amino terminus
• the intracellular amino acid residue is a peptide compound in which the amino acid residue is a modified amino acid residue in which a fatty acid is introduced into the side chain of the amino acid residue, and the intracellular calcium ion concentration by binding to the growth hormone secretagogue receptor
• a peptide in which ghrelin has the amino acid sequence set forth in SEQ ID NO: 1 and the third amino acid residue from the amino terminus is a modified amino acid residue in which a fatty acid is introduced into the side chain hydroxyl group of the amino acid residue The treatment method according to the above (16), which is a system compound.
• the above ghrelin is a peptide compound having the amino acid sequence set forth in SEQ ID NO: 1, wherein the side chain hydroxyl group of the third amino acid residue from the amino terminus is acylated with an n-octanoyl group ( The therapeutic method as described in 17).
• the growth hormone secretion promoting factor receptor agonist or the pharmaceutically acceptable salt thereof according to the above (20) or (21), wherein the existing therapeutic agent for amyotrophic lateral sclerosis is riluzole.
• the growth hormone secretion promoting factor receptor agonist or the pharmaceutically acceptable salt thereof is administered as a subcutaneous injection, as described in any one of (19) to (22) above A growth hormone secretagogue receptor agonist or a pharmaceutically acceptable salt thereof.
• the above-mentioned growth hormone secretion promoting factor receptor agonist is ghrelin, pralmorelin, GHRP-6, hexarelin, ipamorelin, ibutamorene methanesulfonate, urimorelin, anamorelin, masmorelin, capromorelin or SM-130686 (19)
• the growth hormone secretion promoting factor receptor agonist or the pharmaceutically acceptable salt thereof according to any one of (1) to (23).
• the intracellular amino acid residue is a peptide compound in which the amino acid residue is a modified amino acid residue in which a fatty acid is introduced into the side chain of the amino acid residue, and the intracellular calcium ion concentration by binding to the growth hormone secretagogue receptor A growth hormone secretagogue receptor agonist or a drug thereof according to the above (24), which is a peptide-based compound having an activity to increase the activity Acceptable salts.
• the above ghrelin is a peptide compound having the amino acid sequence set forth in SEQ ID NO: 1, wherein the side chain hydroxyl group of the third amino acid residue from the amino terminus is acylated with an n-octanoyl group
• a growth hormone secretagogue receptor agonist or a pharmaceutically acceptable salt thereof A growth hormone secretagogue receptor agonist or a pharmaceutically acceptable salt thereof.
• the GHS-R agonist of the present invention has the effect of prolonging the survival period by suppressing the progression of the disease state in ALS, and is therefore very useful for the treatment of ALS without currently effective therapeutic agents.
• it is further useful because it has the effect of suppressing muscle weakness by suppressing the decrease in the number of motor neurons, which is a feature of the onset and progression of ALS, and significantly suppressing the death of the cells.
• This effect is superior to riluzole because it extends the survival time even when GHS-R agonists are administered from the time when riluzole, an existing ALS treatment, is ineffective. Satisfies fulfillment needs.
• the GHS-R agonist shows a protective effect on motor neurons, it is useful for motor neuron diseases other than ALS such as spinal muscular atrophy.
• GHS-R agonist of the present invention known peptide compounds and non-peptide compounds can be used.
• ghrelin which is an endogenous ligand
• examples of the peptide compound include pralmorelin (GHRP-2), GHRP-6, hexarelin, ipamorelin, and the like.
• Acid salt ibutamoren mesilate; MK-0677
• urimorelin ulimorelin
• TZP-101 urimorelin
• anamorelin anamorelin; RC-1291
• masmorelin macimorelin, EAZS-130
• capromorelin CP-424391
• SM-130686 Etc SM-130686 Etc.
• ghrelin is desirable for peptide compounds
• anamorelin is desirable for non-peptide compounds.
• ghrelin examples include ghrelin derived from humans, ghrelin derived from other animals such as rats, mice, pigs, cows, and the like (for example, see International Publication WO01 / 07475).
• Human-derived ghrelin consists of 28 amino acids (SEQ ID NO: 1), and is a peptide compound in which the side chain hydroxyl group of the third serine residue from the amino terminus is acylated with a fatty acid (n-octanoyl group).
• Ghrelin which is an endogenous ligand, is a hormone present in the living body, and since it has already been administered to humans and its safety has been confirmed, it can be a particularly safe ALS therapeutic agent.
• the ghrelin derivative includes an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the 5th to 28th amino acid residues from the amino terminus in the amino acid sequence described in SEQ ID NO: 1.
• a peptide in which the third amino acid (serine) residue from the amino terminus is a modified amino acid residue in which a fatty acid is introduced into the side chain (hydroxyl group) of the amino acid residue and binds to GHS-R Can be used as long as the peptide has an activity to increase the intracellular calcium ion concentration (see International Publication WO01 / 07475).
• “several” means 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 means.
• the amino acid sequence of the ghrelin derivative desirably has 70%, preferably 80%, more preferably 90%, particularly preferably 95%, and most preferably 97% homology compared to the natural amino acid sequence.
• agonist activity against GHS-R and physiological actions described in the above publications can be used as indicators, and the target ghrelin derivatives can be selected based on the indicators.
• the agonist activity against GHS-R can be examined using the intracellular calcium ion concentration as an index, and a known method can be used as a measurement method according to the index, for example, Fluo-4 due to a change in calcium ion concentration.
• FLIPR® Fluorometric Imaging Plate Reader, Molecular Devices Co., Ltd.
• AM® Molecular® Probe
• a well-known method can be utilized. For example, in order to confirm the feeding-enhancing effect in healthy mice, a peptide compound or a non-peptide compound having a calcium-elevating activity is administered subcutaneously or intraperitoneally to an animal, and the amount of food consumed for 1 hour after administration is determined. What is necessary is just to compare with a solvent administration group.
• GHRP-2 D-alanyl-D- (2-naphthyl) alanyl-L-alanyl-L-tryptophyll-D-phenylalanyl-L-lysine amide
• US Pat. No. 5,663,146 D-alanyl-D- (2-naphthyl) alanyl-L-alanyl-L-tryptophyll-D-phenylalanyl-L-lysine amide
• L-histidyl-D-tryptophyll-L-alanyl-L-tryptophyll-D-phenylalanyl-L-lysine amide (Endocrinology (1984) 114 (5) 1537-1545) is used. be able to.
• L-histidyl-2-methyl-D-tryptophyll-L-alanyl-L-tryptophyll-D-phenylalanyl-L-lysine amide US Pat. No. 5,646,301
• US Pat. No. 5,646,301 L-histidyl-2-methyl-D-tryptophyll-L-alanyl-L-tryptophyll-D-phenylalanyl-L-lysine amide
• Ipamorelin includes ⁇ -methylalanyl-L-histidyl-D- ⁇ - (2-naphthyl) -L-alanyl-D-phenylalanyl-L-lysine amide (Journal of Medicinal Chemistry (1998) 41 3699-704 ) Can be used.
• Ibutamolen methanesulfonate includes 2-amino-2-methyl-N-[(1R) -2- (1-methanesulfonylspiro [indoline-3,4'-piperidine] -1 '-Yl) -2-oxo-1- (phenylmethoxymethyl) ethyl] propanamide methanesulfonate (US Pat. No. 5,536,716) can be used.
• Anamorelin includes 2-amino-N- ⁇ (1R) -2- [3-benzyl-3- (N, N ', N'-trimethylhydrazinocarbonyl) piperidin-1-yl] -1-((1H -Indol-3-yl) -2-oxoethyl) -2-methylpropionamide ⁇ (US Pat. No. 6,576,648) can be used.
• Masmorelin includes 2-methylalanyl-N- [1 (R) -formamido-2- (1H-indol-3-yl) ethyl] -D-tryptophanamide (US Pat. No. 6,861,409). Can be used.
• Capromorelin includes 2-amino-N- [2- [3a (R) -benzyl-2-methyl-3-oxo-3,3a, 4,5,6,7-hexahydro-2H- Pyrazolo [4,3-c] pyridin-5-yl] -1 (R)-(benzyloxymethyl) -2-oxoethyl] isobutyramide (European Patent 0869968) can be used.
• SM-130686 includes (+)-3 (S)-(2-chlorophenyl) -1- [2- (diethylamino) ethyl] -3-hydroxy-2-oxo-4- (trifluoromethyl) -2, 3-Dehydro-1H-indole-6-carboxamide hydrochloride (US Pat. No. 6,576,656) can be used.
• the salt relating to the GHR-S agonist that can be used in the present invention is preferably a pharmaceutically acceptable salt, such as a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, or a salt with an organic acid. And salts with basic or acidic amino acids.
• the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; and aluminum salt and ammonium salt.
• salt with an organic base examples include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N′-dibenzylethylenediamine and the like.
• salt with inorganic acid examples include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
• salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p -Salts with toluenesulfonic acid and the like.
• salts with basic amino acids include salts with arginine, lysine, ornithine and the like
• salts with acidic amino acids include salts with aspartic acid and glutamic acid, for example. It is done. Of these salts, sodium salt and potassium salt are most preferable.
• the GHS-R agonist of the present invention can be obtained by a conventional method. For example, it can be isolated from natural sources or produced by recombinant DNA techniques and / or chemical synthesis techniques.
• examples of vectors incorporating the target gene encoding the peptide-based compound according to the present invention include E. coli vectors (pBR322, pUC18, pUC19, etc.), hay Examples include fungal vectors (pUB110, pTP5, pC194, etc.), yeast vectors (YEp type, YRp type, YIp type), and animal cell vectors (retrovirus, vaccinia virus, etc.). However, any can be used as long as it can stably hold the target gene in the host cell.
• the vector is introduced into a suitable host cell.
• a method for incorporating a target gene into a plasmid or a method for introducing it into a host cell for example, the method described in Molecular®Cloning: A • Laboratory • Manual (Cold • Spring • Harbor • Laboratory • Press, 1989) can be used.
• a promoter is connected upstream of the gene so as to function.
• the promoter used in the present invention may be any promoter as long as it is appropriate for the host cell used for expression of the target gene.
• the host cell to be transformed is Escherichia genus
• lac promoter, trp promoter, lpp promoter, ⁇ PL promoter, recA promoter, etc. can be used
• Bacillus genus is used
• SPO1 promoter, SPO2 promoter, etc. can be used.
• GAP promoter, PHO5 promoter, ADH promoter and the like can be used, and in the case of animal cells, SV40-derived promoter, retrovirus-derived promoter and the like can be used.
• the host cell is transformed with the vector containing the target gene obtained as described above.
• bacteria eg, Escherichia genus, Bacillus genus, etc.
• yeast Sacharomyces genus, Pichia genus, Candida genus, etc.
• animal cells CHO cells, COS cells etc.
• a liquid medium is suitable as a medium for culturing, and it is particularly preferable that the medium contains a carbon source, a nitrogen source and the like necessary for the growth of the transformed cells to be cultured. If desired, vitamins, growth promoting factors, serum and the like can be added.
• the peptide compound according to the present invention is separated and purified from the culture by a conventional method.
• the cells or cells are collected after culturing, suspended in a buffer solution containing a protein denaturant (guanidine hydrochloride, etc.), and the cells are obtained by ultrasound or the like.
• the cells are disrupted and then centrifuged.
• gel filtration, ultrafiltration, dialysis, SDS-PAGE various chromatographies are performed in consideration of the molecular weight, solubility, charge (isoelectric point), affinity, etc. of the target substance. Separation and purification methods such as chromatography can be appropriately combined.
• the peptide-based compound of the present invention is produced using a chemical synthesis technique, for example, an amino acid having a protecting group is condensed by a liquid phase method and / or a solid phase method, the peptide chain is extended, and the whole protecting group is added with an acid. And the resulting crude product is purified by a known purification method.
• Ghrelin and its derivatives according to the present invention can also be obtained by conventional methods, for example, can be obtained by isolation and purification from natural raw materials, or produced by recombinant DNA technology and / or chemical synthesis technology. can do.
• ghrelin contains amino acid residues whose side chains are modified (acylated), when the amino acid residues are modified (acylated), the modification reaction can be performed according to known means.
• a host cell transformed with an expression vector having DNA encoding ghrelin or a derivative thereof is cultured, and the target peptide is collected from the culture, and the present invention is applied.
• Such ghrelin or a derivative thereof can also be obtained.
• a compound in which the target peptide is modified (acylated) in the cell can be obtained.
• a processing / protease activity capable of cleaving the precursor polypeptide of the peptide at an appropriate position and can acylate a serine residue in the peptide. It is desirable to use cells having activity.
• a host cell having such processing protease activity and serine acylation activity is transformed with an expression vector containing cDNA encoding the precursor polypeptide, and the transformed cell has calcium-elevating activity or growth hormone secretion. Selection can be made by confirming that a fatty acid-modified peptide compound having an accelerating activity is produced.
• an amino acid having a protecting group is condensed by a liquid phase method and / or a solid phase method, the peptide chain is extended, and all the protecting groups are removed with an acid.
• the product can be obtained by purification by the above purification method.
• the side chain of the amino acid at the target position can be selectively acylated with an acylating enzyme or an acyltransferase.
• a method using a combination of recombinant DNA technology and chemical synthesis technology may be used. Fragments containing modified amino acid residues are produced by chemical synthesis, and other fragments not containing modified amino acid residues are produced using recombinant DNA technology. Can also be produced by fusing each fragment and then fusing each fragment (see International Publication WO01 / 07475, WO03 / 084983).
• pralmorelin GHRP-2
• GHRP-6 GHRP-6
• hexarelin ipamorelin
• MK-0677 ibutamorene methanesulfonate
• urimorelin anamorelin
• masmorelin capromorelin
• SM -130686 SM -130686 and the like
• GHS-R agonists As an administration subject of the present invention, among individuals suffering from ALS, as individuals that can increase food intake by administration of GHS-R agonists, in other words, individuals who can orally ingest the food intake desired by individuals Of "individuals who are not seriously swallowed.” Furthermore, since the GHS-R agonist has a therapeutic effect even when administered from the time when riluzole is ineffective, the individual may be an “individual refractory to or insufficiently responsive to riluzole”.
• ALSFRS-R ALS Functional Rating Scale
• ghrelin As a GHS-R agonist, as detailed in the Examples, SOD1 G93A mice are also fed to the floor after 18 weeks of age when feeding from the food box becomes difficult. Even under conditions that helped to eat food, ghrelin exhibits an effect of suppressing muscle weakness and / or prolonging survival. That is, when ghrelin is administered under conditions that can achieve its feeding-enhancing effect, it is possible to suppress a decrease in muscle strength of the ALS model animal and prolong the survival period.
• ALS therapeutic agents Individuals that are refractory to existing amyotrophic lateral sclerosis therapeutic agents or who are not responsive to existing amyotrophic lateral sclerosis therapeutic agents (ALS therapeutic agents) It shows a state where no therapeutic effect is observed or is not maintained by an existing ALS therapeutic agent at present, that is, a state where progression of a disease state is not suppressed even when an existing ALS therapeutic agent is taken. Refractory to treatment with existing ALS therapeutic agents is evaluated by examining one or more clinical evaluation indices in the above-mentioned ALSFRS-R. Therefore, it can be judged by a clinician skilled in the treatment of ALS that it is refractory to existing ALS therapeutic agents.
• a clinician evaluates an individual's condition at the time of medical examination every 3 months using ALSFRS-R, and the degree of change (decrease in the evaluation scale for a certain period (eg 3 months) after the start of existing ALS treatment If the score / period) is equal to or greater than the degree of change in the evaluation scale during a certain period (for example, 6 months) before the start of treatment with an existing ALS treatment, the existing ALS treatment for the individual Is not effective and the individual is judged to be refractory to existing ALS treatments.
• individuals who are refractory to treatment with existing ALS treatment agents include individuals who have previously obtained a therapeutic effect in response to the treatment, but are no longer able to obtain the same effect by the treatment at this time. It is.
• “An inadequate response” to an existing ALS therapeutic agent indicates that the therapeutic effect of the existing ALS therapeutic agent is insufficient, and the progression of the disease state is not sufficiently suppressed even by the existing ALS therapeutic agent treatment.
• Insufficient response to treatment with existing ALS therapeutic agents can be assessed by examining one or more clinical evaluation indices in the above-mentioned ALSFRS-R IV. Therefore, it can be judged by clinicians skilled in the treatment of ALS that the response to existing ALS therapeutic agents is insufficient.
• the clinician evaluates the patient's condition at the time of medical care every 3 months using ALSFRS-R, and the degree of change (decrease in the evaluation scale for a certain period (eg 3 months) after the start of treatment with existing ALS treatment If the reduction tendency is less than 30% when compared with the degree of change in the evaluation scale for a certain period (for example, 6 months) before the start of treatment with the existing ALS treatment agent, The effectiveness of the ALS treatment is insufficient and the individual is considered to be “insufficient” to the existing ALS treatment.
• treatment refers to the suppression, reduction or elimination of the progression of ALS pathology, which includes progressive degeneration of motor neurons, denervation of muscle fibers, muscle atrophy, muscle weakness, spasticity. Or the suppression, reduction or elimination of paralysis, and prolongation of survival.
• the “therapeutic effect” used herein can be determined by a known method. For example, judgment can be made by evaluating functional status based on ALSFRS-R, respiratory function based on forced vital capacity (FVC), muscle strength based on MRC scale (Medical Research Council Scale), etc. it can. In addition, any method can be used as long as it can determine the degree of ALS symptoms. For example, grip strength, back muscle strength, availability of independent walking, presence / absence of tube feeding, period until tube feeding (the starting date of the period can be arbitrarily set.
• the ALS therapeutic agent of the present invention or the existing ALS It may be the start date of administration of the therapeutic agent, or when ALS symptoms such as muscle weakness are observed), presence / absence of tracheostomy, presence / absence of ventilator, intubation or tracheostomy for ventilator (The start date of the period can be arbitrarily set.
• the administration start date of the ALS therapeutic agent of the present invention or the existing ALS therapeutic agent may be used, or when ALS symptoms such as muscle weakness are observed.
• the survival date (starting date of the period) can be arbitrarily set.
• the administration start date of the ALS therapeutic agent of the present invention or the existing ALS therapeutic agent may be used, or the symptoms of ALS such as muscle weakness may be present.
• the timing for determining the “therapeutic effect” of a drug using these methods may be after the dosing period of the ALS therapeutic agent of the present invention or the existing ALS therapeutic agent or during the dosing period.
• the ALS therapeutic agent containing the GHS-R agonist in the present invention as an active ingredient may be administered alone to an individual to be administered, or may be administered in combination with other drugs.
• “concomitant” and “combined” refers to administering two or more drugs to the same individual. These agents may be administered simultaneously or nearly simultaneously (eg, within 1 hour) or may be administered after several hours.
• the second drug is administered immediately after the first drug is administered every day.
• both the first and second drugs are administered at a time when they properly exert their effects.
• an ALS treatment containing ghrelin as an active ingredient takes the existing ALS treatment before every breakfast and dinner and immediately after that Both drugs can be used in combination by administering an ALS therapeutic agent containing as an active ingredient (for example, subcutaneous administration) or by administering in the reverse order.
• an ALS therapeutic agent containing as an active ingredient for example, subcutaneous administration
• Ghrelin exhibits muscle weakness-inhibiting action and / or prolongation of survival under conditions where existing ALS treatments are ineffective, effective for individuals with ALS who are refractory or unresponsive to existing ALS treatments.
• a combined effect with the existing ALS therapeutic agent is also expected.
• riluzole can be mentioned.
• GHS-R agonists or pharmacologically acceptable salts thereof that can be used in the present invention are blended with a pharmaceutically acceptable carrier, tablets, capsules, granules, fine granules, powders, etc. It can be administered orally or parenterally as a solid formulation or a liquid formulation such as a syrup or injection.
• various organic or inorganic carrier substances commonly used as pharmaceutical materials are used as the pharmaceutically acceptable carrier. Excipients, lubricants, binders, disintegrants in solid preparations; solvents, dissolution aids in liquid preparations , Suspending agents, isotonic agents, pH adjusting agents, buffers, soothing agents and the like. Further, if necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used.
• Preferable examples of the excipient include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid and the like.
• Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
• binder examples include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like.
• disintegrant examples include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like.
• the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.
• solubilizer examples include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.
• Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate;
• surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate
• hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.
• the isotonic agent include sodium chloride, glycerin, D-mannitol and the like.
• buffer solutions such as phosphate, acetate, carbonate and citrate.
• Preferable examples of the soothing agent include benzyl alcohol.
• Preferable examples of the preservative include paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
• antioxidant examples include sulfite and ascorbic acid.
• Examples of the dosage form suitable for parenteral administration include, for example, injections for intravenous administration, intradermal administration, subcutaneous administration, intramuscular administration, drops, suppositories, transdermal absorption agents, transmucosal absorption agents, or Examples of the dosage form suitable for oral administration include capsules, tablets, syrups and the like.
• a dosage form suitable for parenteral administration is preferable, and for example, a dosage form such as injection, infusion, or inhalation is preferred.
• a medicine in the form of an injection or infusion comprises one or more pharmaceutical additives such as an isotonic agent, pH adjuster, soothing agent, preservative, etc., together with an active ingredient GHS-R agonist. It can be prepared by dissolving in distilled water for injection and sterilizing.
• a medicine in the form of injection or infusion can be provided as a medicine in lyophilized form. Such a preparation can be used as an injection or an instillation by adding and dissolving distilled water for injection or physiological saline at the time of use.
• ghrelin Since human ghrelin is subcutaneously administered to suppress muscle weakness and prolong survival in ALS model animals, ghrelin, its derivatives or their pharmacologically acceptable salts, or peptide or non-peptide GHS-R
• the agonist can be administered as an injection, such as a subcutaneous injection.
• the active ingredient when it is a peptide-based compound, it can be orally administered as a microcapsule in which a preparation that is not easily degraded in the digestive tract, for example, a peptide that is an active ingredient is encapsulated in a ribosome.
• the administration method which absorbs from mucous membranes other than digestive tracts, such as a rectum, a nose, and a sublingual, is also possible. In this case, it can be administered to an individual in the form of a suppository, a nasal spray, an inhalant, or a sublingual tablet.
• the active ingredient is a non-peptidic compound and is used for oral administration, it will be tableted with solid carriers such as excipients, lubricants, binders, disintegrants, filled into hard capsules in powder or small pill form Or may be in the form of a troche.
• solid carriers such as excipients, lubricants, binders, disintegrants, filled into hard capsules in powder or small pill form Or may be in the form of a troche.
• the amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g.
• a liquid carrier is used, the preparation comprising the active ingredient and the liquid carrier can be administered in the form of a syrup, emulsifier, soft capsule, aqueous or non-aqueous liquid suspension or solution.
• the dose of the GHS-R agonist that can be used as an active ingredient of the ALS therapeutic agent according to the present invention can be appropriately selected depending on the age, weight, degree of symptoms, and administration route of the individual (patient).
• the upper limit of the daily dose for an individual human adult is about 100 mg / kg or less, preferably about 10 mg / kg or less, more preferably about 10 mg / kg or less as the weight of the substance. Is 1 mg / kg or less.
• the lower limit of the daily dose is, for example, about 0.1 ⁇ g / kg or more, preferably 1 ⁇ g / kg or more, more preferably 10 ⁇ g / kg or more.
• the substance that can be used as an active ingredient of the pharmaceutical composition according to the present invention suppresses the progression of the pathology of ALS under the condition that realizes its increased feeding, so that at the end of the administration period, dysphagia of ALS-affected individuals becomes prominent, Until dysphagia is no longer a serious condition and means of involuntary oral intake such as tube feeding is required, it is repeated once or twice a day for several months to several years. Administration can be continuous or continuous. In addition, when the substance is repeatedly administered, it is desirable to administer it before breakfast and / or evening meals.
• SOD1 G93A mice were used as ALS model animals.
• B6SJL-Tg (SOD1-G93A) 1Gur / J mice (SOD1 G93A mice) and wild-type mice of the same strain were purchased from The Jackson Laboratory (ME, USA) and bred to obtain offspring.
• the obtained SOD1 G93A mouse and wild-type littermates (WT mice) were further mated and bred, and the born SOD1 G93A mouse was used in the experiment. In some examples, WT mice were also used.
• SOD1 G93A mice and WT mice were bred by free consumption of tap water and rodent standard feed pellets (CRF-1, 13.6% of total calories derived from fat, 3570 kcal / kg, Oriental Yeast Co., Ltd.).
• rodent standard feed pellets CRF-1, 13.6% of total calories derived from fat, 3570 kcal / kg, Oriental Yeast Co., Ltd.
• food was sprinkled on the floor to assist the mice to eat.
• Example 1 Comparison of body weight and forelimb muscle strength between 10-week-old SOD1 G93A mice and WT mice SOD1 G93A mice, like human ALS, began to selectively kill motor neurons during maturation. It will eventually die, with skeletal muscle atrophy and muscle weakness. Therefore, the body weight and forelimb muscle strength of 10-week-old SOD1 G93A mice were compared with those of WT mice, and the onset of ALS at this age was confirmed.
• Table 1 shows the weight and forelimb muscle strength of each group.
• the average body weight was about 1 g less than in WT mice, and forelimb muscle strength was about 0.1 N lower on average, which was significantly lower. From this, it was confirmed that SOD1 G93A mice had already weakened muscular strength at 10 weeks of age and developed ALS.
• Example 2 Effect of riluzole on SOD1 G93A mice Materials and Methods Riluzole, an existing ALS treatment, has been reported to show a life-prolonging effect when administered from the 4th and 7th week before the onset of SOD1 G93A mice (Amyotrophic Lateral Sclerosis (2009) ) 10, 85-94, Annals of Neurollogy (1996) 39, 147-157). In Example 1, it was confirmed that SOD1 G93A mice exhibited muscle weakness that is an ALS symptom at 10 weeks of age.
• SOD1 G93A mice were divided into two groups, a solvent (saline solution) group and a riluzole group, at the time of 10 weeks of age, and saline or riluzole (Sigma-Aldrich, 16 mg / kg) died. Until intraperitoneal administration was performed once a day, and the survival period of each individual was analyzed. The dose of riluzole was set according to the previous report (Amyotrophic Lateral Sclerosis (2009), Vol. 10, pp. 85-94).
• Results Table 2 shows the average survival time of each group. The median survival time of the solvent group and the riluzole group was comparable, and no life-prolonging effect of riluzole was observed under the administration conditions from 10 weeks of age.
• Example 3 Action of human-derived ghrelin (hereinafter human ghrelin) on SOD1 G93A mice (1): Effect on skeletal muscle mass, muscle strength and survival time after continuous subcutaneous administration In Example 2, from the age of 10 weeks of SOD1 G93A mice It was confirmed that riluzole administration did not show any prolongation of survival. Thus, the action of human ghrelin (SEQ ID NO: 1) was examined under the conditions in which SOD1 G93A mice already showed muscle weakness, an ALS symptom, and riluzole did not show an effect on survival time.
• human ghrelin SEQ ID NO: 1
• Body weight and food weight were measured before administration start and after administration for 8 weeks, and body weight change and food intake were calculated.
• Eight weeks after administration the skeletal muscle mass of the lower body of the mouse was measured using X-ray CT (Latheta LCT-200, Hitachi Aloka Medical Co., Ltd.). Plasma total cholesterol concentration was measured by cholesterol E-Test Wako (Wako Pure Chemical Industries, Ltd.) using plasma separated from blood collected from the tail vein after 8 weeks of administration.
• Forelimb muscle strength was measured at 16 weeks using a rat / mouse simple muscle strength meter; 200 g meter (Ohara Medical Industry Co., Ltd.).
• the survival time of each individual was analyzed.
• Results Table 3 shows the average body weight change, food intake, and lower body skeletal muscle mass after 8 weeks of administration in each group.
• changes in body weight, food intake, and lower body skeletal muscle mass after administration for 8 weeks were significantly increased compared to the solvent group.
• Table 4 shows the total plasma cholesterol concentration after 8 weeks of administration in each group. There was no difference between the solvent group and the human ghrelin group.
• Table 5 shows the average forelimb muscle strength after 6 weeks of administration in each group.
• the forelimb muscle strength was significantly stronger in the human ghrelin group than in the solvent group, indicating that the decline in muscle strength was suppressed.
• Table 6 shows the average survival time of each group.
• the survival time was significantly prolonged compared with the solvent group, and the average survival time was 13.8% longer in the human ghrelin group than in the solvent group.
• Example 4 Action of human ghrelin on SOD1 G93A mice (2): Protective effect on motor neurons during continuous subcutaneous administration
• human ghrelin was administered to SOD1 G93A mice from the age of 10 weeks to reduce forelimb muscle strength. It was confirmed that the survival time was prolonged. Since ALS is a disease in which muscles are atrophied by the death of motor neurons and eventually death, we investigated whether human ghrelin has a protective effect on motor neurons.
• SOD1 G93A mice and WT mice were used for the experiment.
• SOD1 G93A mice were divided into two groups, a solvent group and a human ghrelin group, and WT mice administered with the solvent were designated as the Control group.
• Human ghrelin 50 ⁇ g / day, about 2 mg / kg / day
• An osmotic pump (alzet (registered trademark) MINI-OSMOTIC PUMP MODEL1004, DURECT Corporation) filled with an administration solution or a physiological saline solution was implanted under the back of the skin and continuously administered subcutaneously.
• Administration was started from 10 weeks of age, and administration was continued after 4 weeks by replacing the osmotic pump. Seven weeks after the start of administration, dissection was performed, and the spinal cord in the T9 region was removed. Nissl-stained sections were prepared, and the number of motor neurons was identified histologically. Using three non-adjacent sections for each individual, the number of motor neurons present in the anterior horn was counted. The measured value was calculated as a relative value when the average value of the number of motor neurons in the Control group was 100%.
• Results Table 7 shows the relative number of motor neurons in each group when compared with the Control group.
• the number of motor neurons was about 1 ⁇ 2 compared with the Control group, which was significantly lower.
• the number of motor neurons in the human ghrelin group was significantly higher than that in the solvent group.
• Example 5 Effect of human ghrelin on SOD1 G93A mice (3): Effect on muscle strength and survival time after repeated subcutaneous administration
• human ghrelin was continuously administered subcutaneously to SOD1 G93A mice from the age of 10 weeks As a result, it was confirmed that the decrease in forelimb muscle strength was suppressed, the survival period was prolonged, and the motor neurons were protected.
• the effect of 10-week-old SOD1 G93A mice on the forelimb muscle strength and survival time after repeated subcutaneous administration of human ghrelin was examined.
• Results Table 8 shows the average body weight change and food intake after 8 weeks of administration in each group.
• the body weight change and food intake after administration for 8 weeks were significantly increased compared with the solvent group.
• Table 9 shows the average value of changes in forelimb muscle strength after administration for 8 weeks from 10 weeks of age in each group.
• the decrease in forelimb muscle strength was significantly suppressed in the human ghrelin group compared to the solvent group.
• Table 10 shows the average survival time of each group.
• the survival time was significantly prolonged compared to the solvent group, and the average survival time was 21.8% longer in the human ghrelin group than in the solvent group.
• Example 6 Effect of human ghrelin on SOD1 G93A mice (4): Effect on body weight, skeletal muscle mass, muscle strength and number of motor neurons under restricted feeding 10% on SOD1 G93A mice in Examples 3 and 5 It was shown that the decrease in forelimb muscle strength was suppressed and the survival period was prolonged by continuous subcutaneous administration or repeated administration of human ghrelin from the age of weeks. In Example 4, it was confirmed that motor neurons were protected when ghrelin was continuously administered subcutaneously to SOD1 G93A mice.
• SOD1 G93A mice restricted feeding under which was about 90% of the daily food intake in free-feeding conditions of SOD1 G93A mice, it can not be more food, i.e., hyperphagia ghrelin
• hyperphagia ghrelin For the expression of mRNA of Atrogin1 and Muscle RING-finger protein-1 (MuRF1) related to body weight, skeletal muscle mass, muscle strength, number of motor neurons, and skeletal muscle atrophy when human ghrelin is administered subcutaneously under conditions where no action is manifested The effect was examined.
• Human ghrelin (50 ⁇ g / day, about 2 mg / kg / day) was dissolved in a solvent (physiological saline) to obtain an administration solution.
• An osmotic pump (alzet (registered trademark) MINI-OSMOTIC PUMP MODEL1004, DURECT Corporation) filled with the administration solution or physiological saline was implanted under the back of the skin and continuously administered subcutaneously. Administration was started at 10 weeks of age, and the osmotic pump was replaced after 4 weeks. The body weight was measured on the administration start date and 6 weeks later, and the change in body weight was calculated. Forelimb muscle strength was measured 7 weeks after the start of administration using a rat / mouse simple muscle strength meter; 200 g meter (Ohara Medical Industry Co., Ltd.).
• the skeletal muscle mass of the lower body of the mouse is measured using X-ray CT (Latheta LCT-200, Hitachi Aloka Medical Co., Ltd.) before the start of administration (10 weeks of age) and 7 weeks later to determine the amount of change in skeletal muscle mass. It was. Thereafter, the mouse was dissected and the spinal cord in the T9 region was removed. Nissl-stained sections were prepared, and the number of motor neurons was identified histologically. Using three non-adjacent sections for each individual, the number of motor neurons present in the anterior horn was measured. The measured value was calculated as a relative value when the average number of motor neurons in the WT-Control group was 100%.
• the gastrocnemius muscle was extracted, mRNA was extracted, and the expression levels of Atrogin1 and MuRF1 mRNA were measured by quantitative PCR. The measured value was calculated as a relative value when the mRNA expression level of the WT-Control group was 100%.
• the weight loss in the human ghrelin group was significantly smaller than when the solvent was administered to SOD1 G93A mice (G93A-solvent group).
• lower body skeletal muscle mass increased in freely fed WT mice but decreased in the restricted feeding group.
• skeletal muscle loss was significantly suppressed in the G93A-human ghrelin group.
• Table 12 shows the mRNA expression levels of Atrogin1 and MuRF1 in skeletal muscle 7 weeks after the start of administration.
• WT mice there was no significant change in the expression of these genes even if they were raised under restricted feeding.
• SOD1 G93A mice G93A- solvent group
• WT mice freely fed
• the expression of both genes was significantly increased compared to the control group.
• SOD1 G93A mice G93A- ghrelin group
• SOD1 G93A mice G93A- ghrelin group administered human ghrelin under restricted feeding
• the expression of Atrogin1 and MuRF1 mRNA was significantly lower than that in the solvent group, and skeletal muscle atrophy was suppressed in the human ghrelin group It has been suggested. This is consistent with Table 11 that the decrease in skeletal muscle mass was less in the human ghrelin group.
• ghrelin suppressed weight loss and skeletal muscle atrophy in SOD1 G93A mice by an action independent of its feeding enhancement effect even under restricted feeding.
• ghrelin achieves increased feeding, in other words, it needs to be administered to individuals whose food intake can be increased by ghrelin administration. It was done. That is, it was found that ghrelin indirectly suppresses motor neuron death by improving the energy state of the whole body through an effect of enhancing feeding, thereby suppressing the progression of ALS pathology.
• Example 7 Comparison of body weight and forelimb muscle strength of 16-week-old SOD1 G93A mice and WT mice
• human ghrelin was administered from 10-week-old when SOD1 G93A mice already showed decreased forelimb muscle strength It showed that food consumption and body weight were increased, motor nerve cell death and muscle strength were suppressed, and survival was prolonged.
• treatment begins only after a definitive diagnosis is made by the clinician. Since a definitive diagnosis of ALS takes from six months to one year or more (ALS Treatment Guidelines 2002, Japanese Neurological Society Treatment Guidelines), it is thought that symptoms will progress before the start of treatment.
• Body weight and muscle strength of forelimbs were measured using 16-week-old WT mice and SOD1 G93A mice for the experiments.
• Forelimb muscle strength was measured using a rat / mouse simple muscle strength meter; 200 g meter (Ohara Medical Sangyo Co., Ltd.).
• Table 14 shows the body weight and forelimb muscle strength of each group.
• SOD1 G93A mice have an average body weight of 2g or less compared to WT mice, and forelimb muscle strength is about 0.5 N lower on average. The difference with these WT mice is more than that at 10 weeks of age (Table 1). It was remarkable. Moreover, the forelimb muscle strength of 16-week-old SOD1 G93A mice was lower than that at 10-week-old (0.91 N) (Table 1).
• Example 8 Effect of human ghrelin on SOD1 G93A mice (5): Effect on survival time after continuous subcutaneous administration from 16 weeks of age In this example, 16-week-old mice with remarkable muscle weakness and more advanced ALS pathology We investigated the effect of human ghrelin on ALS disease progression in SOD1 G93A mice using survival time as an index.
• human ghrelin significantly prolonged the survival period compared to the solvent group even when administration was started from 16 weeks of age when SOD1 G93A mice had marked muscle weakness and ALS pathological condition further advanced.
• riluzole which is an existing ALS therapeutic agent, did not prolong survival time even when administered from 10 weeks of age. That is, it has been found that ghrelin exhibits a remarkable effect of significantly extending the survival time even when administered from the 16-week-old of SOD1 G93A mice, compared with existing ALS therapeutic agents. From this, it was shown that ghrelin remarkably suppresses the progression of ALS disease state and has a therapeutic effect.
• Example 9 Effects of GHRP-6 and anamorelin, which are growth hormone secretagogue receptor agonists, on SOD1 G93A mice: effects on survival time after repeated subcutaneous administration In Example 5, 10 weeks of age against SOD1 G93A mice It was confirmed that the survival time was prolonged by repeated subcutaneous administration of human ghrelin. In this example, the effect of 10-week-old SOD1 G93A mice on survival time after repeated subcutaneous administration of GHRP-6 and anamorelin, which are growth hormone secretagogue receptor agonists, was examined.
• Results Table 16 shows the average body weight change and food intake after 5 weeks of administration in each group.
• the change in body weight and food intake after 5 weeks of administration were significantly increased compared to the solvent group.
• the change in body weight after administration for 5 weeks was significantly increased and the amount of food intake tended to increase compared to the solvent group.
• Table 17 shows the average survival time of each group. Survival time was significantly prolonged in the GHRP-6 group and the anamorelin group compared to the solvent group.
• a pharmaceutical composition comprising a growth hormone secretagogue receptor agonist or a pharmaceutically acceptable salt thereof is amyotrophic for an individual suffering from amyotrophic lateral sclerosis in which dysphagia is not severe It can be a therapeutic agent for lateral sclerosis.

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