WO2015147244A1 - Cachexia treatment or preventative agent - Google Patents

Cachexia treatment or preventative agent Download PDF

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WO2015147244A1
WO2015147244A1 PCT/JP2015/059561 JP2015059561W WO2015147244A1 WO 2015147244 A1 WO2015147244 A1 WO 2015147244A1 JP 2015059561 W JP2015059561 W JP 2015059561W WO 2015147244 A1 WO2015147244 A1 WO 2015147244A1
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interferon
cachexia
peg
administration
treatment
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PCT/JP2015/059561
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French (fr)
Japanese (ja)
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智勝 岩村
成見　英樹
明子 曽根田
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東レ株式会社
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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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/215—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a cachexia treatment or prevention agent.
  • Cachexia is a syndrome of complex metabolic abnormalities that occurs in connection with the underlying disease and is defined as characterized by a loss of muscle mass with or without fat loss ( Non-patent document 1).
  • Base diseases that cause cachexia include chronic diseases such as malignant tumors, tuberculosis, diabetes, blood diseases, endocrine diseases, infectious diseases, or acquired immune deficiency syndromes.
  • Significant weight loss, anemia, edema, anorexia In addition, it is known that the main symptoms are generalized weakness or fatigue (Non-patent Documents 2 and 3).
  • cachexia having a malignant tumor as a basic disease is called cancer cachexia and is said to account for about 20% of the causes of malignant tumor death (Non-patent Document 4).
  • Non-patent Document 5 Non-patent Document 5
  • IL-1 interleukin-1
  • IL-6 interleukin-6
  • TNF- tumor necrosis factor
  • Non-patent Document 8 For the treatment of cachexia, appetite stimulants such as megesterol acetate and medroxyprogesterone (Non-patent Document 8) are mainly used. Besides these, steroidal anti-inflammatory drugs 1, 2 such as dexamethasone and prednisolone are used. -Diphenylpyrrole derivatives (Patent Literature 1), carboxylic acid amide derivatives (Patent Literature 2), parathyroid hormone related peptide antibodies (Patent Literature 3), ghrelin-like low molecular weight compounds (Patent Literature 4) and androgen receptor modulators (Patent Literature) Drugs such as 5) are considered effective. In addition, thalidomide is sometimes used to improve chronic inflammation. Recently, anti-TNF- ⁇ antibody, anti-IL-6 antibody and anti-IL-6 receptor antibody, which are in clinical trials, are not effective. It is considered effective for treatment of liquid quality (Non-patent Documents 8 and 9).
  • interferon- ⁇ is widely used as a therapeutic agent for cancer, a therapeutic agent for multiple sclerosis, a therapeutic agent for chronic hepatitis B, and a therapeutic agent for chronic hepatitis C, and ischemic reperfusion injury or treatment for multiple organ failure
  • a medicine a medicine for cancerous ascites and cancerous pleural effusion
  • a therapeutic agent for cancerous ascites and cancerous pleural effusion are also disclosed.
  • appetite stimulants used for the treatment of cachexia have a limited therapeutic effect because no improvement effect on metabolic abnormalities is observed, and steroidal anti-inflammatory drugs have long-term side effects due to strong side effects. There is a problem that it is difficult to use.
  • interferon- ⁇ there is no report on the therapeutic effect or preventive effect of cachexia. The creation of effective medicine for the treatment of cachexia is expected.
  • an object of the present invention is to provide a cachexia treatment or prevention agent.
  • interferon- ⁇ has an excellent therapeutic effect and preventive effect on cachexia, and have completed the present invention.
  • the present invention provides a cachexia treatment or prevention agent containing interferon- ⁇ as an active ingredient.
  • interferon- ⁇ is preferably covalently bonded to polyalkylene glycol, and more preferably covalently bonded to polyethylene glycol.
  • a covalent conjugate of interferon- ⁇ and polyalkylene glycol When administered, a covalent conjugate of interferon- ⁇ and polyalkylene glycol has a long blood half-life and acts continuously on the living body, and thus has an excellent therapeutic or preventive effect on cachexia.
  • the cachexia treatment or prevention agent is preferably a cancer cachexia treatment or prevention agent.
  • the agent for treating or preventing cachexia of the present invention has a therapeutic or preventive effect on cachexia in chronic diseases.
  • the cachexia treatment or prevention agent of the present invention is characterized by containing interferon- ⁇ as an active ingredient.
  • interferon- ⁇ is not only one having the same amino acid sequence as the naturally occurring interferon- ⁇ (hereinafter referred to as natural interferon- ⁇ ), but also one or more in the amino acid sequence of natural interferon- ⁇ .
  • Individual, for example, 1 to 20, typically 1 or several amino acids deleted, substituted or added, and having biological activity as interferon (hereinafter referred to as interferon activity) Includes variants of interferon- ⁇ .
  • Natural interferon- ⁇ may be derived from any organism, but is preferably derived from the organism to which the cachexia therapeutic or preventive agent of the present invention is administered.
  • Such organisms may be mammals, for example, rodents such as mice, rats, hamsters, or primates such as humans, gorillas, chimpanzees, and cows, horses, pigs, goats. , Sheep, dogs, cats, rabbits and the like.
  • the natural interferon- ⁇ is derived from a human.
  • Natural human interferon- ⁇ may include, for example, the amino acid sequence represented by SEQ ID NO: 1.
  • the above-mentioned interferon- ⁇ also includes interferon- ⁇ in which the sugar chain portion of natural interferon- ⁇ is modified and interferon- ⁇ having no sugar chain portion.
  • the above-described interferon- ⁇ is an amino acid having a sequence identity of 70% or more, 80% or more, preferably 90% or more, more preferably 95% or more, such as 98% or more with the amino acid sequence represented by SEQ ID NO: 1. It may be a polypeptide consisting of a sequence and having interferon activity.
  • “having X% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1” means that the total length of the sequence to be compared is most identical to the total length of the sequence represented by SEQ ID NO: 1. This means that the sequence identity (%) is X% when aligned so as to increase the property.
  • the above-mentioned interferon- ⁇ also includes recombinant interferon- ⁇ produced by gene recombination technology based on the amino acid sequence of interferon- ⁇ including natural type interferon- ⁇ or the base sequence encoding it.
  • the nucleotide sequence encoding the amino acid sequence of interferon- ⁇ is, for example, 70% or more, 80% or more, preferably 90% or more, more preferably 95% or more, such as 98% or more of the amino acid sequence represented by SEQ ID NO: 1. It may be a base sequence consisting of an amino acid sequence having sequence identity and encoding a polypeptide having interferon activity.
  • interferon- ⁇ is obtained by using a known method such as tissue extraction, protein synthesis using gene recombination technology, biological production using natural cells or recombinant cells that express interferon- ⁇ . be able to.
  • tissue extraction protein synthesis using gene recombination technology
  • biological production using natural cells or recombinant cells that express interferon- ⁇ .
  • interferon- ⁇ commercially available interferon- ⁇ can also be used.
  • interferon- ⁇ belongs to type I interferon.
  • other type I interferons interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , and interferon- ⁇ , can also be used as active ingredients in cachexia treatment or prevention agents. The same effect can be expected with interferon- ⁇ .
  • these interferons like interferon- ⁇ , these interferons, such as type I interferon, also lack one or more, for example 1 to 20, typically one or several amino acids in the amino acid sequence of natural interferon. It may be an interferon mutant having a lost, substituted or added amino acid sequence and having biological activity as an interferon (hereinafter referred to as interferon activity).
  • interferons also consist of amino acid sequences having a sequence identity of 70% or more, 80% or more, preferably 90% or more, more preferably 95% or more, such as 98% or more, with the amino acid sequence of natural interferon, and It may be a polypeptide having interferon activity.
  • interferon- ⁇ chemically modified as an active ingredient of the cachexia treatment or prevention agent of the present invention.
  • This chemical modification may be any as long as interferon- ⁇ can have interferon activity.
  • chemically modified interferon-beta includes covalent conjugates of interferon-beta and polymers, covalent conjugates of interferon-beta and lipids, and fusion proteins of interferon-beta and other proteins. To do.
  • other interferons such as type I interferon are chemically modified can be suitably used in the present invention.
  • the covalent conjugate of interferon- ⁇ and polymer is preferably a covalent conjugate of interferon- ⁇ and polyalkylene glycol.
  • one molecule or two or more molecules can be bonded to one molecule of interferon- ⁇ .
  • the total molecular weight of the polyalkylene glycol bonded to one molecule of interferon- ⁇ is preferably 5,000 to 240,000, more preferably 10,000 to 80,000, and more preferably 39,000 to 45,000. 000 or less is more preferable.
  • polyalkylene glycol having a molecular weight of 40,000 is covalently bonded to one molecule of interferon- ⁇ .
  • one molecule or two or more molecules of interferon- ⁇ can be bonded to one molecule of the above polyalkylene glycol.
  • the molecular weight of one molecule of the polyalkylene glycol is preferably 5,000 or more and 520,000 or less, more preferably 10,000 or more and 200,000 or less, and further preferably 39,000 or more and 45,000 or less. preferable.
  • polyalkylene glycol is composed of a large number of repeating units in one molecule, and the molecular weight of polyalkylene glycol is generally different depending on each molecule, and thus is represented by an average molecular weight. Therefore, the “molecular weight” of the polyalkylene glycol in the present specification means an average molecular weight.
  • polyalkylene glycol a polyalkylene glycol which is a polymer compound acceptable as a drug carrier, inactive or extremely low in the living body, and non-toxic or extremely low in toxicity can be preferably used.
  • Covalent conjugate of interferon- ⁇ and polyalkylene glycol is one molecule of polyalkylene glycol covalently bonded to one molecule of interferon- ⁇ and has interferon activity. And those having one or more molecules of interferon- ⁇ covalently bonded to one molecule of polyalkylene glycol and having interferon activity.
  • a covalent conjugate of interferon- ⁇ and polyalkylene glycol one molecule or two or more molecules of polyalkylene glycol is covalently bonded to one molecule of interferon- ⁇ and has interferon activity. It is more preferable.
  • the covalent conjugate of interferon- ⁇ and polyalkylene glycol may be either a direct covalent bond of interferon- ⁇ and polyalkylene glycol, or a bond obtained through a linker or the like.
  • the dosage of a covalent conjugate of interferon-beta, preferably chemically modified interferon-beta, such as interferon-beta and polyalkylene glycol, to provide a therapeutic or prophylactic effect on cachexia is determined by the interferon titer. (Unit; hereinafter, U) or mass (g).
  • the covalent conjugate of interferon- ⁇ and polyalkylene glycol maintains a specific activity of 10% or more compared to the specific activity of interferon- ⁇ before the polyalkylene glycol is covalently bound (interferon activity per weight). It is preferable.
  • the binding of the polymer compound to the protein may be referred to as chemical modification or modification of the protein with the polymer compound, and the covalent conjugate of interferon- ⁇ and polyalkylene glycol was chemically modified with polyalkylene glycol.
  • interferon- ⁇ or simply modified interferon- ⁇ .
  • a covalent conjugate of interferon- ⁇ and polyethylene glycol hereinafter PEG is also referred to as PEG-modified interferon- ⁇ , PEGylated interferon- ⁇ , or PEG-conjugated interferon- ⁇ .
  • Examples of the covalent bond site between interferon- ⁇ and polyalkylene glycol include the amino group, thiol group, N-terminus, C-terminus or sugar chain of interferon- ⁇ .
  • a non-natural (artificial) amino acid introduced into interferon- ⁇ using a known gene recombination technique can also be used as a covalent bond site with polyalkylene glycol.
  • the kind of non-natural amino acid and a method for introducing a non-natural amino acid into a protein are described in International Publication No. 2011/158895.
  • the interferon activity of interferon- ⁇ can be measured by a known biological measurement method. Specifically, after a sample containing interferon- ⁇ is allowed to act on interferon-sensitive cells (for example, FL cells), Sindbis virus or blister that has the ability to infect these cells. Interferon was measured by adding a certain amount of stomatitis virus (abbreviated as VSV), etc., and infecting it, and measuring the degree of virus resistance induced by interferon- ⁇ (hereinafter referred to as antiviral activity). Activity can be measured.
  • VSV stomatitis virus
  • Antiviral activity is measured using suppression of virus growth as an indicator, and dilution of a specimen that inhibits cytopathic effect (abbreviated as Cytopathic Effect; CPE) that destroys cells as a result of virus growth by 50%.
  • CPE Cytopathic Effect
  • a covalent conjugate of interferon- ⁇ and polyalkylene glycol can be prepared by a known method. For example, methods for covalently attaching a polyalkylene glycol to the amino group of interferon are described in US Pat. No. 4,917,888 and WO 87/00056. A method for covalently bonding a polyalkylene glycol to the thiol group of interferon is described in WO 99/55377. Methods for covalently bonding a polymer to the N-terminus of interferon are described in International Publication No. 00/23114 and JP-A-9-25298.
  • a method for covalently bonding a polyalkylene glycol to the sugar chain of interferon is described in 1978 Makromolecular Chemistry (Vol. 179, p. 301), US Pat. No. 4,101,380 or US Pat. No. 4,179,337.
  • a method for covalently bonding a polyalkylene glycol to an unnatural (artificial) amino acid introduced into interferon is described in 2004 Bioorganic & Medicinal Chemistry Letters (Vol. 14, p. 5743-5745).
  • polyalkylene glycol is covalently bonded to the C-terminus of interferon- ⁇ , it can be achieved by introducing cysteine or other non-natural (artificial) amino acid at or near the C-terminus of interferon- ⁇ .
  • polyalkylene glycol derivatives whose ends are activated with hydroxysuccinimide ester, nitrobenzene sulfonate ester, maleimide, orthopyridyl disulfide, vinyl sulfone, maleimide, iodoacetamide, carboxylic acid, azide, phosphine or amine structure are interferon- ⁇ and poly It can be used to form a covalent bond with alkylene glycol, and these polyalkylene glycol derivatives can be synthesized by known methods.
  • the covalent conjugate of interferon- ⁇ and polyalkylene glycol is preferably a covalent conjugate of interferon- ⁇ and PEG.
  • a covalent conjugate of interferon- ⁇ and PEG has a molecular weight of 5,000 to 240,000 (preferably a molecular weight of 10,000 to 80,000, more preferably a molecular weight of 39,000 or more) to one molecule of interferon- ⁇ . 45,000 or less) is preferably a covalent conjugate in which one molecule of PEG is covalently bonded.
  • the covalent conjugate of interferon- ⁇ and PEG include a covalent conjugate of human interferon- ⁇ and PEG.
  • the covalent conjugate of interferon- ⁇ and PEG can be prepared by the method described in International Publication No. 2005/019260. it can.
  • the binding site of PEG in human interferon- ⁇ the amino group of the 19th or 134th lysine in the amino acid sequence of human interferon- ⁇ is preferable, and a covalent conjugate in which one molecule of PEG is covalently bonded to this site is more preferable. preferable.
  • a covalent conjugate in which one molecule of PEG having a molecular weight of 40,000 or more (preferably a molecular weight of 42,000) is covalently bonded to the amino group of the 134th lysine of the amino acid sequence of human interferon- ⁇ can be exemplified.
  • 134th lysine of the amino acid sequence of human interferon- ⁇ means that the N-terminal amino acid (methionine) of the amino acid sequence of human interferon- ⁇ consisting of 166 amino acids (SEQ ID NO: 1) is the first.
  • the position of the amino acid residue of interferon- ⁇ is represented with the N-terminal amino acid of interferon- ⁇ being the first.
  • the covalent conjugate of interferon- ⁇ and polyalkylene glycol may also be one in which polyalkylene glycol is bonded to the amino group of lysine in the amino acid sequence of interferon- ⁇ .
  • the lysine of the amino acid sequence of interferon- ⁇ that serves as a polyalkylene glycol binding site may be at a position corresponding to the 19th or 134th lysine of the amino acid sequence represented by SEQ ID NO: 1 in the alignment of the amino acid sequence, It does not have to be in the corresponding position.
  • Cachexia refers to significant weight loss, anemia, edema, loss of appetite, generalized weakness or malaise in chronic diseases such as malignant tumor, tuberculosis, diabetes, blood disease, endocrine disease, infection or acquired immune deficiency syndrome. Includes systemic syndromes with feelings as main symptoms. Examples include cancer cachexia, tuberculosis cachexia, diabetic cachexia, blood disease cachexia, endocrine disease cachexia, infectious cachexia, or acquired immunodeficiency syndrome.
  • the cachexia treatment or prevention agent of the present invention can be used for any cachexia, but can be particularly preferably used for cancer cachexia caused by malignant tumors.
  • malignant tumor (also referred to as cancer or malignant neoplasm) includes cancer derived from epithelial tissue (also referred to as carcinoma), sarcoma derived from non-epithelial tissue, and malignant tumor derived from hematopoietic organs.
  • the type of malignant tumor is not particularly limited.
  • malignant melanoma malignant bone tumor, gastric cancer, hepatocellular carcinoma, acute myeloid leukemia, acute lymphocytic leukemia, cervical cancer, endometrial cancer, esophageal cancer Pancreatic cancer, prostate cancer, colon cancer, breast cancer, lung cancer, bladder cancer or ovarian cancer.
  • the agent for treating or preventing cachexia of the present invention contains interferon, for example, interferon- ⁇ as an active ingredient, and improves cachexia, that is, malignant tumor, tuberculosis, diabetes, blood disease, endocrine disease, infection or It has the effect of improving systemic syndromes such as marked weight loss, anemia, edema, loss of appetite, generalized weakness or malaise that occur in chronic diseases such as acquired immune deficiency syndrome. Hypoalbuminemia, high IL-6emia, weight loss, and worsening prognosis (shortening survival) are typical symptoms of cachexia.
  • interferon for example, interferon- ⁇ as an active ingredient
  • the cachexia therapeutic or preventive agent of the present invention includes, as examples of cachexia improving action, hypoalbuminemia improving action (blood albumin concentration lowering suppressing effect), high IL-6 blood serum improving action (blood IL-6 concentration increase inhibitory effect), weight loss improving effect (weight loss inhibitory effect), and / or life prolonging effect (survival effect of prolonging survival).
  • the cachexia treatment or prevention agent of the present invention may be a medicament containing an interferon, for example, interferon- ⁇ as an active ingredient.
  • the cachexia improving action of the cachexia treatment or prevention agent of the present invention is based on a mechanism independent of the antitumor action (tumor growth inhibitory effect).
  • treatment of cachexia refers to symptoms of cachexia (such as weight loss, anemia, edema, loss of appetite, general weakness or malaise) and pathological indicators (decrease in albumin concentration, IL-6 concentration). Improvement of at least one of increase, shortening of survival period, etc.).
  • prevention of cachexia refers to symptoms of cachexia (such as weight loss, anemia, edema, loss of appetite, general weakness or malaise) and pathological indicators (decrease in albumin concentration, IL-6 concentration). Or the like, or to reduce the degree of inhibition or delay of at least one expression.
  • interferon for example, interferon- ⁇
  • interferon- ⁇ which is an active ingredient of the above-mentioned cachexia treatment or prevention agent
  • the cachexia treatment or prevention agent of the present invention is an arbitrary administration subject, preferably a mammal (for example, human, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, etc.), particularly preferably It can be used as a cachexia treatment or prevention agent for humans.
  • the subject of administration of the cachexia treatment or prevention agent of the present invention is a subject in which cachexia (eg, cancer cachexia) is observed, or a chronic disease that is likely to cause cachexia (eg, malignant tumor, tuberculosis, diabetes) , Blood diseases, endocrine diseases, infectious diseases, acquired immune deficiency syndrome, etc.).
  • the administration target of the cachexia treatment or prevention agent of the present invention may or may not have pleural effusion or ascites.
  • the active ingredient is interferon such as interferon- ⁇ as it is or as a composition containing it and an additive such as a pharmaceutically acceptable carrier. Or it can be administered parenterally.
  • the administration route of the cachexia treatment or prevention agent of the present invention is not particularly limited, and is subcutaneous, intradermal, transdermal, transmucosal, transpulmonary, nasal, enteral, intravenous, intraarterial, intramuscular, abdominal cavity. It may be a parenteral route such as internal, rectal and intravaginal administration, or oral administration.
  • the cachexia treatment or prevention agent of the present invention may be a pharmaceutical composition containing any pharmaceutically acceptable additive.
  • additives include carriers, excipients, stabilizers, suspending agents, emulsifiers, thickeners, dispersants, absorption enhancers, binders, lubricants, disintegrants, wetting agents, buffering agents, Examples include, but are not limited to, flavoring agents, preservatives, and coloring agents.
  • Examples of the dosage form for oral administration of the cachexia treatment or prevention agent of the present invention include tablets, pills, capsules, granules, syrups, emulsions or suspensions, It can be produced by a known method. These dosage forms can contain additives such as carriers or excipients usually used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, maltose, saccharose, starch, and magnesium stearate.
  • Examples of the dosage form for parenteral administration of the cachexia treatment or prevention agent include injections, suppositories, nasal absorption agents, pulmonary absorption agents, transdermal absorption agents, and topical sustained release agents. These can be produced by known methods.
  • the solution preparation can be prepared, for example, by interferon such as interferon- ⁇ dissolved in a sterile aqueous solution used for injection or suspended and emulsified in an extract and embedded in liposomes.
  • a solid preparation can be prepared, for example, as a lyophilized product by adding mannitol, trehalose, sorbitol, lactose, glucose or the like as an excipient to an interferon such as interferon- ⁇ .
  • the gel can be prepared by, for example, dissolving interferon such as interferon- ⁇ in a thickener or polysaccharide such as glycerin, PEG, methylcellulose, carboxymethylcellulose, hyaluronic acid or chondroitin sulfate.
  • any dosage form may contain human serum albumin, human immunoglobulin, ⁇ 2 macroglobulin, amino acid or the like as a stabilizer, and alcohol as a dispersant or absorption enhancer as long as it does not impair interferon activity.
  • Alcohol as a dispersant or absorption enhancer as long as it does not impair interferon activity.
  • Sugar alcohol, ionic surfactant or nonionic surfactant may be included.
  • you may contain trace metal or organic acid salt as needed.
  • the cachexia treatment or prevention agent of the present invention is administered at daily doses of once or twice a day at a dose of 10,000 U to 18 million U / dose of the active ingredient interferon- ⁇ . can do.
  • the cachexia treatment or prevention agent of the present invention may also be administered at a single administration interval with a period of 2 days or more, and further, intermittent administration at an administration interval of once every 2 days to 1 month. May be.
  • the cachexia treatment or prevention agent of the present invention comprising a chemically modified interferon (for example, a covalent conjugate of interferon- ⁇ and polyalkylene glycol) as an active ingredient is once or twice a week. It is preferable to administer intermittently at an administration interval of once a month, particularly preferably intermittently at an interval of once or twice a week.
  • the cachexia treatment or prevention agent of the present invention may be administered alone, or one or more other arbitrary agents used for treatment or prevention of diseases, or reduction or suppression of symptoms. May be administered in combination.
  • the drug to be combined may be a low molecular compound, or may be a high molecular protein, polypeptide, antibody, vaccine or the like.
  • the cachexia treatment or prevention agent of the present invention can be administered simultaneously or with a time difference from the combined agents.
  • medical agent may be administered together and can also be administered as a mixture.
  • the dose of the drug to be combined can be appropriately selected based on the clinically used dose.
  • the combination ratio of the cachexia treatment or prevention agent of the present invention when combined with the drug combined with the drug is the subject of administration, age of administration subject, body weight, symptom, administration time, dosage form, administration method, or combination of drugs. It can be selected as appropriate.
  • the agent for treating or preventing cachexia of the present invention can also be used in combination with drugs such as chemotherapeutic agents, immunotherapeutic agents or diuretics.
  • chemotherapeutic agents include cyclophosphamide, ifosfamide, melphalan, busulfan, nimustine, ranimustine, temozolomide and other alkylating agents, methotrexate, fluorouracil, tegafur, carmofur, doxyfluridine, capecitabine, cytarabine, ancitabine, phositabine, cytarabine, cytarabine Nucleic acid metabolism antagonists such as phart, gemcitabine, mercaptopurine or fludarabine, doxorubicin, daunorubicin, pirarubicin, epirubicin, idarubicin, mitoxantrone, mitomycin C, bleomycin or peplomycin, antitumor antibiotics, vincristine, vinblastine, vindesine, vinorelbine , Microtubule inhibitors such as paclitaxel or docetaxel, syl
  • immunotherapeutic agent examples include muramyl dipeptide derivatives, lentinan, schizophyllan, ubenimex, picibanil, krestin, interleukin, granulocyte colony stimulating factor or erythropoietin.
  • diuretics examples include xanthine derivative preparations such as sodium salicylate theobromine or calcium salicylate theobromine, ethiazide, cyclopenthiazide, trichloromethiazide, hydrochlorothiazide, hydroflumethiazide, benchylhydrochlorothiazide, penflutide, polythiazide, or methycrothiazide.
  • xanthine derivative preparations such as sodium salicylate theobromine or calcium salicylate theobromine, ethiazide, cyclopenthiazide, trichloromethiazide, hydrochlorothiazide, hydroflumethiazide, benchylhydrochlorothiazide, penflutide, polythiazide, or methycrothiazide.
  • Antialdosterone preparations such as spironolactone or triamterene, carbonic acid dehydrogenase inhibitors such as acetazolamide, chlorbenzenesulfonamide preparations such as chlorthalidone, mefluside or indapamide, azosemide, isosorbide, ethacrynic acid, piretanide, bumetanide or furosemide.
  • the cachexia treatment or prevention agent of the present invention may also contain other above-mentioned agents used in combination.
  • the present invention also provides a method for treating or preventing cachexia, comprising administering the agent for treating or preventing cachexia of the present invention to the administration subject.
  • Example 1 Preparation of Covalent Conjugate of Interferon- ⁇ and PEG Recombinant human interferon ⁇ (SEQ ID NO: 1) was prepared according to Goeddel et. al. Nucleic Acid Research (1980), Vol. 8, p.
  • Recombinant mouse interferon- ⁇ (NCBI Protein database accession number 1IFA_A) was prepared according to Matsuda et. al. , Journal of Interferon Research (1986), Vol. 6, p. It was prepared according to the method described in 519-526.
  • PEG having a molecular weight of 42,000 is added to the amino group of the 134th lysine of the amino acid sequence of recombinant human interferon ⁇ (SEQ ID NO: 1).
  • SEQ ID NO: 1 “Covalent conjugate of interferon- ⁇ and PEG” in which one molecule is covalently bonded
  • Molecular interferon- ⁇ and PEG in which one molecule of PEG having a molecular weight of 42,000 is covalently bonded to recombinant mouse interferon ⁇ .
  • a “covalent conjugate” was prepared.
  • ethylene glycol was added to recombinant human interferon- ⁇ (final concentration 200 ⁇ g / mL) dissolved in 100 mmol / L acetate buffer (pH 5.0) containing 0.5 mol / L sodium chloride. After addition (at a final concentration of 20%), the pH was adjusted to 7.6 using a 1 mol / L disodium hydrogen phosphate solution. To this, hydroxysuccinimide ester activated PEG (molecular weight 42,000, NOF Corporation; product number 61G99122B01) was added and mixed, and a binding reaction was carried out at 4 ° C. overnight.
  • a cation exchange column (TOYOPEARL CM 650 (S)) was added to this binding reaction solution by adding 5 volumes of 10 mmol / L acetate buffer (pH 4.5) and equilibrated with 10 mmol / L acetate buffer (pH 4.5). ; Tosoh Corporation). The protein was eluted and fractionated with the following developing solvent.
  • Solvent A 10 mmol / L acetate buffer (pH 4.5)
  • Solvent B 10 mmol / L acetate buffer (pH 4.5) containing 1 mol / L sodium chloride
  • the mixed solution of the solvents A and B is passed through 40 times the resin amount of the cation exchange column, and the solvent B feed rate is continuously increased from 0% to 65% during the passage. Elution.
  • the eluted fraction was further subjected to a cation exchange column SP-5PW (Tosoh Corporation), and the amino group of the 134th lysine of the amino acid sequence of recombinant human interferon- ⁇ (SEQ ID NO: 1) was used.
  • a “covalent conjugate of human interferon- ⁇ and PEG” (hereinafter, “PEG-IFN- ⁇ ”) in which one molecule of PEG having a molecular weight of 42,000 was covalently bonded was isolated.
  • Example 2 Hypoalbuminemia-improving effect and hyper-IL-6-emia-improving effect of interferon- ⁇ in a cachexia model The method of Huynh et al. (Molecular cancer therapeutics, 2007, Vol. 6, P. 2959-2966) ), A cachexia model mouse was prepared by peritoneal seeding of ovarian cancer cells, and the effect of interferon- ⁇ on hypoalbuminemia and hyper-IL-6 in the cachexia was examined. As interferon- ⁇ , PEG-mIFN- ⁇ prepared in Example 1 was used.
  • Human ovarian cancer cell line OV-90 (CRL-11732; American type culture collection) was cultured and maintained in a MEM medium containing 15% fetal calf serum at 37 ° C. in a 5% CO 2 incubator.
  • OV-90 cells were collected with trypsin / EDTA and then washed with phosphate buffer (PBS) to prepare an OV-90 cell suspension.
  • An OV-90 cell suspension (5 ⁇ 10 6 cells / mouse) was transplanted into the abdominal cavity of female SCID mice (CB-17 / lcr-scid / scid-Jcl; Japan Marie).
  • the collected blood sample was euthanized under anesthesia, and after laparotomy, the tumor mass in the abdominal cavity was removed using tweezers and scissors, and its weight was measured.
  • the albumin concentration in plasma was measured using a mouse Albumin ELISA Quantitation Set (BETHYL Lab) according to the instructions attached to the kit.
  • the plasma was diluted 1 million times with a diluent (Sample Diluent: 50 mmol / L Tris, 0.14% NaCl, 1% BSA, 0.5% Tween 20) and used for measurement.
  • the concentration of mouse IL-6 in plasma was measured using Quantikine mouse IL-6 kit (R & D Systems), and the concentration of human IL-6 in plasma was measured using Quantikine human IL-6 kit (R & D Systems). Used according to the instructions attached to the kit.
  • the plasma was diluted 5-fold with physiological saline (Otsuka Pharmaceutical Factory) and used for measurement.
  • “control” on the horizontal axis indicates the control group
  • solvent indicates the solvent administration group
  • PEG-mIFN- ⁇ indicates the PEG-mIFN- ⁇ administration group.
  • the symbol * (asterisk) in these figures indicates that it is statistically significant (P ⁇ 0.05) compared to the solvent group.
  • Interferon- ⁇ is known to have an action of suppressing the growth of cancer cells (hereinafter referred to as an antitumor action).
  • administration of PEG-mIFN- ⁇ did not change plasma human IL-6 concentration and intraperitoneal tumor weight. That is, the cachexia ameliorating effect observed with the administration of PEG-mIFN- ⁇ was observed when the antitumor effect on human ovarian cancer cells was not sufficiently exhibited. As a result of this, it was shown that the action was not a secondary action but an action independent of the antitumor action.
  • interferon- ⁇ has a cachexia improving action independent of the antitumor action.
  • Example 3 Weight loss improving effect and life prolonging effect of interferon- ⁇ in cachexia model mice The effect of interferon- ⁇ on weight loss and days of survival in cachexic subjects was examined. As interferon- ⁇ , PEG-IFN- ⁇ and PEG-mIFN- ⁇ were used.
  • a cell suspension of human ovarian cancer cell line OV-90 (5 ⁇ 10 6 cells / mouse) was treated with female SCID mice (CB-17 / lcr-scid / scid-jcl) in the same manner as in Example 2. Transplanted intraperitoneally; In addition, the female SCID mouse
  • mouth of the same week age which did not transplant a human ovarian cancer cell was made into the control group (n 4).
  • test substance a mixed solution prepared by mixing PEG-IFN- ⁇ prepared in Example 1 and PEG-mIFN- ⁇ (hereinafter referred to as PEG-IFN- ⁇ s) was obtained by combining PEG-IFN- ⁇ and PEG-mIFN.
  • PEG-IFN- ⁇ s a mixed solution prepared by mixing PEG-IFN- ⁇ prepared in Example 1 and PEG-mIFN- ⁇
  • Subcutaneous administration was performed so that each of - ⁇ was a dose of 50000 U / animal / dose.
  • the test substance and the solvent were administered 6 times in 12 days.
  • the body weight of each individual in the control group, the solvent administration group, and the PEG-IFN- ⁇ s administration group was measured.
  • the survival rate of the control group, the solvent administration group, and the PEG-IFN- ⁇ s administration group (number of surviving individuals 12 days after the administration start date / number of surviving individuals on the administration start date) was evaluated 12 days after the administration start date.
  • the results of weight measurement are shown in FIG.
  • the symbol * (asterisk) in the figure indicates that it is statistically significant (P ⁇ 0.05) compared to the solvent group.
  • Table 1 shows the evaluation results of the survival rate 12 days after the administration start date.
  • the survival rate in Table 1 is shown as “the number of surviving individuals 12 days after the administration start date / the number of surviving individuals on the administration start date”.
  • interferon- ⁇ has a cachexia improving action independent of the antitumor action and is effective in the treatment and prevention of cachexia.
  • the cachexia treatment or prevention agent of the present invention can be used for the treatment or prevention of cachexia in patients with chronic diseases. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
  • SEQ ID NO: 1 amino acid sequence of human interferon- ⁇

Abstract

The present invention provides a cachexia treatment or preventative agent which includes interferon-β as an active ingredient.

Description

悪液質の治療又は予防剤Cachexia treatment or prevention agent
　本発明は、悪液質の治療又は予防剤に関する。 The present invention relates to a cachexia treatment or prevention agent.
　悪液質とは、基礎疾患に関連して生ずる複合的代謝異常の症候群のことであり、脂肪量の減少の有無に関わらず筋肉量の減少を特徴とするものであると定義されている（非特許文献１）。悪液質が発症する基礎疾患としては、悪性腫瘍、結核、糖尿病、血液疾患、内分泌疾患、感染症又は後天性免疫不全症候群等の慢性疾患が挙げられ、著しい体重減少、貧血、浮腫、食欲不振、全身衰弱又は倦怠感等を主症状とすることが知られている（非特許文献２及び３）。中でも悪性腫瘍を基礎疾患とする悪液質は、がん悪液質と呼ばれ、悪性腫瘍死因の約２０％を占めると言われている（非特許文献４）。 Cachexia is a syndrome of complex metabolic abnormalities that occurs in connection with the underlying disease and is defined as characterized by a loss of muscle mass with or without fat loss ( Non-patent document 1). Base diseases that cause cachexia include chronic diseases such as malignant tumors, tuberculosis, diabetes, blood diseases, endocrine diseases, infectious diseases, or acquired immune deficiency syndromes. Significant weight loss, anemia, edema, anorexia In addition, it is known that the main symptoms are generalized weakness or fatigue (Non-patent Documents 2 and 3). Among them, cachexia having a malignant tumor as a basic disease is called cancer cachexia and is said to account for about 20% of the causes of malignant tumor death (Non-patent Document 4).
　がん悪液質では、症状の進行に伴って患者の体力が著しく減弱するため、抗腫瘍剤の投与ができなくなり、悪液質の症状を改善するために栄養補給を行うと、かえって悪性腫瘍の増悪を加速させることになるため、がん悪液質の発症は、悪性腫瘍の治療に重大な支障を来たしている。仮に、患者に体力があり、抗腫瘍剤の投与が可能であったとしても、骨髄毒性等の副作用が生じ、悪液質は改善しないことが報告されている（非特許文献５）。 In cancer cachexia, as the symptoms progress, the patient's physical strength is significantly weakened, so it becomes impossible to administer anti-tumor agents, and if nutrition is supplied to improve cachexia symptoms, malignant tumors The development of cancer cachexia has seriously hindered the treatment of malignant tumors. Even if the patient has strength and can administer an antitumor agent, side effects such as bone marrow toxicity occur and cachexia is not improved (Non-patent Document 5).
　悪液質では、活性化された免疫システムや腫瘍細胞から産生されるインターロイキン－１（以下、ＩＬ－１）、インターロイキン－６（以下、ＩＬ－６）及び腫瘍壊死因子（以下、ＴＮＦ－α）等の炎症性サイトカインが病態形成に深く関わっていることが知られており（非特許文献６）、悪液質患者の血中のＩＬ－６濃度は、体重減少の程度及び生存率と関連しているため、ＩＬ－６で誘発される血中Ｃ反応性蛋白（ＣＲＰ）濃度は、血中アルブミン濃度と共に悪液質の診断マーカーとされている（非特許文献７）。 In cachexia, interleukin-1 (hereinafter referred to as IL-1), interleukin-6 (hereinafter referred to as IL-6) and tumor necrosis factor (hereinafter referred to as TNF-) produced from the activated immune system and tumor cells. It is known that inflammatory cytokines such as α) are deeply involved in pathogenesis (Non-patent Document 6), and the concentration of IL-6 in the blood of cachexia patients depends on the degree of weight loss and the survival rate. Since it is related, the blood C-reactive protein (CRP) concentration induced by IL-6 is regarded as a diagnostic marker for cachexia together with the blood albumin concentration (Non-patent Document 7).
　悪液質の治療には、酢酸メゲステロールやメドロキシプロゲステロン等の食欲刺激剤（非特許文献８）が主に使用され、これら以外にも、デキサメサゾンやプレドニゾロン等のステロイド系抗炎症薬１，２－ジフェニルピロール誘導体（特許文献１）、カルボン酸アミド誘導体（特許文献２）、副甲状腺ホルモン関連ペプチド抗体（特許文献３）、グレリン様低分子化合物（特許文献４）及びアンドロゲン受容体モジュレーター（特許文献５）等の薬剤が有効とされている。さらに、慢性炎症の改善をする場合には、サリドマイドが使用されるケースもあり、最近では、臨床試験段階にある抗ＴＮＦ－α抗体、抗ＩＬ－６抗体及び抗ＩＬ－６受容体抗体が悪液質の治療に有効であると考えられている（非特許文献８及び９）。 For the treatment of cachexia, appetite stimulants such as megesterol acetate and medroxyprogesterone (Non-patent Document 8) are mainly used. Besides these, steroidal anti-inflammatory drugs 1, 2 such as dexamethasone and prednisolone are used. -Diphenylpyrrole derivatives (Patent Literature 1), carboxylic acid amide derivatives (Patent Literature 2), parathyroid hormone related peptide antibodies (Patent Literature 3), ghrelin-like low molecular weight compounds (Patent Literature 4) and androgen receptor modulators (Patent Literature) Drugs such as 5) are considered effective. In addition, thalidomide is sometimes used to improve chronic inflammation. Recently, anti-TNF-α antibody, anti-IL-6 antibody and anti-IL-6 receptor antibody, which are in clinical trials, are not effective. It is considered effective for treatment of liquid quality (Non-patent Documents 8 and 9).
　一方、インターフェロン－βについては、癌治療薬、多発性硬化症治療薬、慢性Ｂ型肝炎治療薬及び慢性Ｃ型肝炎治療薬としての用途が広く知られ、虚血性再灌流傷害又は多臓器不全治療薬としての用途（特許文献６）、癌性腹水及び癌性胸水の治療薬としての用途（特許文献７）も開示されている。 On the other hand, interferon-β is widely used as a therapeutic agent for cancer, a therapeutic agent for multiple sclerosis, a therapeutic agent for chronic hepatitis B, and a therapeutic agent for chronic hepatitis C, and ischemic reperfusion injury or treatment for multiple organ failure The use as a medicine (patent document 6) and the use as a therapeutic agent for cancerous ascites and cancerous pleural effusion (patent document 7) are also disclosed.
特開２０００－９５６８５号公報Japanese Patent Laid-Open No. 2000-95585 特開平５－４３４６６号公報JP-A-5-43466 国際公開第９８／０５１３２９号International Publication No. 98/051329 国際公開第０５／０９７２６１号International Publication No. 05/097261 国際公開第０２／０６６４７５号International Publication No. 02/066475 国際公開第０７／０４２６０２号International Publication No. 07/042602 国際公開第１３／１２９５４９号International Publication No. 13/129549
　しかしながら、悪液質の治療に使用される食欲刺激剤は、代謝異常に対する改善効果が認められないために治療効果が限定的であり、ステロイド系抗炎症薬は、副作用が強いために長期的な使用が困難であるといった問題点がある。また、インターフェロン－βについては、悪液質の治療効果又は予防効果については一切の報告がない。悪液質の治療に有効な医薬の創出が期待されている。 However, appetite stimulants used for the treatment of cachexia have a limited therapeutic effect because no improvement effect on metabolic abnormalities is observed, and steroidal anti-inflammatory drugs have long-term side effects due to strong side effects. There is a problem that it is difficult to use. In addition, regarding interferon-β, there is no report on the therapeutic effect or preventive effect of cachexia. The creation of effective medicine for the treatment of cachexia is expected.
　そこで本発明は、悪液質の治療又は予防剤を提供することを目的とする。 Therefore, an object of the present invention is to provide a cachexia treatment or prevention agent.
　本発明者らは、上記課題を解決すべく鋭意研究を重ねた結果、インターフェロン－βが悪液質に対する優れた治療効果及び予防効果を有することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that interferon-β has an excellent therapeutic effect and preventive effect on cachexia, and have completed the present invention.
　すなわち本発明は、インターフェロン－βを有効成分として含有する、悪液質の治療又は予防剤を提供する。 That is, the present invention provides a cachexia treatment or prevention agent containing interferon-β as an active ingredient.
　上記のインターフェロン－βは、ポリアルキレングリコールと共有結合していることが好ましく、ポリエチレングリコールと共有結合していることがより好ましい。 The above-mentioned interferon-β is preferably covalently bonded to polyalkylene glycol, and more preferably covalently bonded to polyethylene glycol.
　インターフェロン－βとポリアルキレングリコールとの共有結合体は、投与された場合、血中半減期が長く、生体に持続的に作用するため、悪液質に対する優れた治療効果又は予防効果を有する。 When administered, a covalent conjugate of interferon-β and polyalkylene glycol has a long blood half-life and acts continuously on the living body, and thus has an excellent therapeutic or preventive effect on cachexia.
　また上記の悪液質の治療又は予防剤は、がん悪液質の治療又は予防剤であることが好ましい。 The cachexia treatment or prevention agent is preferably a cancer cachexia treatment or prevention agent.
　本明細書は本願の優先権主張の基礎となる日本国特許出願２０１４－０６８９２７号の開示内容の全体を包含する。 This specification includes the entire disclosure of Japanese Patent Application No. 2014-068927, which is the basis for the priority claim of the present application.
　本発明の悪液質の治療又は予防剤は、慢性疾患における悪液質に対して治療又は予防効果を有する。 The agent for treating or preventing cachexia of the present invention has a therapeutic or preventive effect on cachexia in chronic diseases.
悪液質モデルにおけるマウス・インターフェロン－βとポリエチレングリコールとの共有結合体の血漿中アルブミン量に及ぼす作用を示す図である。It is a figure which shows the effect | action which acts on the albumin amount in plasma of the covalent conjugate of mouse | mouth interferon-beta and polyethyleneglycol in a cachexia model. 悪液質モデルにおけるマウス・インターフェロン－βとポリエチレングリコールとの共有結合体の血漿中マウスＩＬ－６量に及ぼす作用を示す図である。It is a figure which shows the effect | action which acts on the mouse | mouth IL-6 amount in plasma of the covalent conjugate of mouse | mouth interferon-beta and polyethyleneglycol in a cachexia model. 悪液質モデルにおけるマウス・インターフェロン－βとポリエチレングリコールとの共有結合体の血漿中ヒトＩＬ－６量に及ぼす作用を示す図である。It is a figure which shows the effect | action which acts on the amount of plasma human IL-6 of the covalent conjugate of mouse | mouth interferon-beta and polyethyleneglycol in a cachexia model. 悪液質モデルにおけるマウス・インターフェロン－βとポリエチレングリコールとの共有結合体の腫瘍重量に及ぼす作用を示す図である。It is a figure which shows the effect | action which acts on the tumor weight of the covalent conjugate of mouse | mouth interferon-beta and polyethyleneglycol in a cachexia model. 悪液質モデルにおけるマウス・インターフェロン－βとポリエチレングリコールとの共有結合体及びヒト・インターフェロン－βとポリエチレングリコールとの共有結合体の混合液を投与したときの体重低下改善作用を示す図である。It is a figure which shows the weight-loss reduction effect when the mixed liquid of the mouse | mouth interferon-beta and the polyethyleneglycol covalent conjugate and the human interferon-beta and the polyethyleneglycol covalent conjugate in the cachexia model is administered.
　本発明の悪液質の治療又は予防剤は、インターフェロン－βを有効成分として含有することを特徴とする。 The cachexia treatment or prevention agent of the present invention is characterized by containing interferon-β as an active ingredient.
　上記のインターフェロン－βは、天然に存在するインターフェロン－β（以下、天然型インターフェロン－β）のアミノ酸配列と同じアミノ酸配列を持つものだけでなく、天然型インターフェロン－βのアミノ酸配列に１個又は複数個、例えば１個～２０個、典型的には１個又は数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ、インターフェロンとしての生物活性（以下、インターフェロン活性）を有するインターフェロン－βの変異体を包含する。天然型インターフェロン－βは、任意の生物由来であってよいが、本発明の悪液質の治療又は予防剤の投与対象の生物由来であることが好ましい。そのような生物は、例えば哺乳動物であってよく、例えばマウス、ラット、ハムスターなどのげっ歯類、又はヒト、ゴリラ、チンパンジーなどの霊長類であってもよいし、ウシ、ウマ、ブタ、ヤギ、ヒツジ、イヌ、ネコ、ウサギなどであってもよい。好ましい実施形態では、天然型インターフェロン－βはヒト由来である。天然型ヒト・インターフェロン－βは例えば、配列番号１で示されるアミノ酸配列を含むものであってよい。上記のインターフェロン－βはまた、天然型インターフェロン－βの糖鎖部分が改変されたインターフェロン－β及び糖鎖部分を有しないインターフェロン－βをも包含する。 The above-mentioned interferon-β is not only one having the same amino acid sequence as the naturally occurring interferon-β (hereinafter referred to as natural interferon-β), but also one or more in the amino acid sequence of natural interferon-β. Individual, for example, 1 to 20, typically 1 or several amino acids deleted, substituted or added, and having biological activity as interferon (hereinafter referred to as interferon activity) Includes variants of interferon-β. Natural interferon-β may be derived from any organism, but is preferably derived from the organism to which the cachexia therapeutic or preventive agent of the present invention is administered. Such organisms may be mammals, for example, rodents such as mice, rats, hamsters, or primates such as humans, gorillas, chimpanzees, and cows, horses, pigs, goats. , Sheep, dogs, cats, rabbits and the like. In a preferred embodiment, the natural interferon-β is derived from a human. Natural human interferon-β may include, for example, the amino acid sequence represented by SEQ ID NO: 1. The above-mentioned interferon-β also includes interferon-β in which the sugar chain portion of natural interferon-β is modified and interferon-β having no sugar chain portion.
　例えば、上記のインターフェロン－βは、配列番号１で示されるアミノ酸配列と７０％以上、８０％以上、好ましくは９０％以上、より好ましくは９５％以上、例えば９８％以上の配列同一性を有するアミノ酸配列からなり、かつインターフェロン活性を有するポリペプチドであり得る。なお本発明に関して、「配列番号１で示されるアミノ酸配列とＸ％以上の配列同一性を有する」とは、比較対象の配列の全長を、配列番号１で示される配列の全長に対して最も同一性が高くなるようにアラインメントしたときの配列同一性（％）がＸ％であることを意味する。 For example, the above-described interferon-β is an amino acid having a sequence identity of 70% or more, 80% or more, preferably 90% or more, more preferably 95% or more, such as 98% or more with the amino acid sequence represented by SEQ ID NO: 1. It may be a polypeptide consisting of a sequence and having interferon activity. In the present invention, “having X% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1” means that the total length of the sequence to be compared is most identical to the total length of the sequence represented by SEQ ID NO: 1. This means that the sequence identity (%) is X% when aligned so as to increase the property.
　上記のインターフェロン－βは、天然型インターフェロン－βをはじめとするインターフェロン－βのアミノ酸配列又はそれをコードする塩基配列に基づいて、遺伝子組換え技術により作製された組換え型インターフェロン－βも包含する。インターフェロン－βのアミノ酸配列をコードする塩基配列は、例えば、配列番号１で示されるアミノ酸配列と７０％以上、８０％以上、好ましくは９０％以上、より好ましくは９５％以上、例えば９８％以上の配列同一性を有するアミノ酸配列からなり、かつインターフェロン活性を有するポリペプチドをコードする塩基配列であってよい。 The above-mentioned interferon-β also includes recombinant interferon-β produced by gene recombination technology based on the amino acid sequence of interferon-β including natural type interferon-β or the base sequence encoding it. . The nucleotide sequence encoding the amino acid sequence of interferon-β is, for example, 70% or more, 80% or more, preferably 90% or more, more preferably 95% or more, such as 98% or more of the amino acid sequence represented by SEQ ID NO: 1. It may be a base sequence consisting of an amino acid sequence having sequence identity and encoding a polypeptide having interferon activity.
　上記のインターフェロン－βは、組織からの抽出、遺伝子組換え技術を用いたタンパク質合成、インターフェロン－βを発現する天然細胞又は組換え細胞を用いた生物学的製造等の公知の方法を用いて得ることができる。また、インターフェロン－βとして、市販のインターフェロン－βも用いることができる。 The above-mentioned interferon-β is obtained by using a known method such as tissue extraction, protein synthesis using gene recombination technology, biological production using natural cells or recombinant cells that express interferon-β. be able to. As interferon-β, commercially available interferon-β can also be used.
　上記のインターフェロン－βはＩ型インターフェロンに属する。さらに、他のＩ型インターフェロンであるインターフェロン－α、インターフェロン－ω、インターフェロン－ε及びインターフェロン－κも、悪液質の治療又は予防剤の有効成分として用いることができる。また、インターフェロン－λについても同様の効果が期待できる。インターフェロン－βと同様に、Ｉ型インターフェロン等のこれらのインターフェロンも、天然型インターフェロンのアミノ酸配列に１個又は複数個、例えば１個～２０個、典型的には１個又は数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ、インターフェロンとしての生物活性（以下、インターフェロン活性）を有するインターフェロン変異体であってもよい。これらのインターフェロンはまた、天然型インターフェロンのアミノ酸配列と７０％以上、８０％以上、好ましくは９０％以上、より好ましくは９５％以上、例えば９８％以上の配列同一性を有するアミノ酸配列からなり、かつインターフェロン活性を有するポリペプチドであってよい。 The above interferon-β belongs to type I interferon. Furthermore, other type I interferons, interferon-α, interferon-ω, interferon-ε, and interferon-κ, can also be used as active ingredients in cachexia treatment or prevention agents. The same effect can be expected with interferon-λ. Like interferon-β, these interferons, such as type I interferon, also lack one or more, for example 1 to 20, typically one or several amino acids in the amino acid sequence of natural interferon. It may be an interferon mutant having a lost, substituted or added amino acid sequence and having biological activity as an interferon (hereinafter referred to as interferon activity). These interferons also consist of amino acid sequences having a sequence identity of 70% or more, 80% or more, preferably 90% or more, more preferably 95% or more, such as 98% or more, with the amino acid sequence of natural interferon, and It may be a polypeptide having interferon activity.
　上記のインターフェロン－βが化学的に修飾されたものを本発明の悪液質治療又は予防剤の有効成分として用いることも好ましい。この化学的な修飾は、インターフェロン－βがインターフェロン活性を有することができる限り、任意のものであってよい。例えば、化学的に修飾されたインターフェロン－βは、インターフェロン－βとポリマーとの共有結合体、インターフェロン－βと脂質との共有結合体、及び、インターフェロン－βと他のタンパク質との融合タンパク質を包含する。なおＩ型インターフェロンをはじめとする他のインターフェロンが化学的に修飾されたものも、本発明において好適に用いることができる。 It is also preferred to use the above-mentioned interferon-β chemically modified as an active ingredient of the cachexia treatment or prevention agent of the present invention. This chemical modification may be any as long as interferon-β can have interferon activity. For example, chemically modified interferon-beta includes covalent conjugates of interferon-beta and polymers, covalent conjugates of interferon-beta and lipids, and fusion proteins of interferon-beta and other proteins. To do. In addition, those in which other interferons such as type I interferon are chemically modified can be suitably used in the present invention.
　インターフェロン－βとポリマーとの共有結合体は、インターフェロン－βとポリアルキレングリコールとの共有結合体であることが好ましい。 The covalent conjugate of interferon-β and polymer is preferably a covalent conjugate of interferon-β and polyalkylene glycol.
　上記のポリアルキレングリコールは、インターフェロン－βの１分子に対して、１分子又は２分子以上が結合することができる。この場合、インターフェロン－βの１分子に結合するポリアルキレングリコールの分子量の合計は、５，０００以上２４０，０００以下が好ましく、１０，０００以上８０，０００以下がより好ましく、３９，０００以上４５，０００以下がさらに好ましい。なお、インターフェロン－βの１分子に分子量２０，０００のポリアルキレングリコールの２分子が共有結合している場合は、インターフェロン－βの１分子に分子量４０，０００のポリアルキレングリコールが共有結合していると表現することがある。 In the above polyalkylene glycol, one molecule or two or more molecules can be bonded to one molecule of interferon-β. In this case, the total molecular weight of the polyalkylene glycol bonded to one molecule of interferon-β is preferably 5,000 to 240,000, more preferably 10,000 to 80,000, and more preferably 39,000 to 45,000. 000 or less is more preferable. When two molecules of polyalkylene glycol having a molecular weight of 20,000 are covalently bonded to one molecule of interferon-β, polyalkylene glycol having a molecular weight of 40,000 is covalently bonded to one molecule of interferon-β. Sometimes expressed.
　また、上記のポリアルキレングリコールの１分子に対して、インターフェロン－βの１分子又は２分子以上が結合することもできる。この場合であっても、ポリアルキレングリコールの１分子の分子量は、５，０００以上５２０，０００以下が好ましく、１０，０００以上２００，０００以下がより好ましく、３９，０００以上４５，０００以下がさらに好ましい。 In addition, one molecule or two or more molecules of interferon-β can be bonded to one molecule of the above polyalkylene glycol. Even in this case, the molecular weight of one molecule of the polyalkylene glycol is preferably 5,000 or more and 520,000 or less, more preferably 10,000 or more and 200,000 or less, and further preferably 39,000 or more and 45,000 or less. preferable.
　なお、ポリアルキレングリコールは、１個の分子が多数の繰り返しユニットからできており、ポリアルキレングリコールの分子量は個々の分子により異なるのが一般的であるため、平均分子量で表される。したがって、本明細書におけるポリアルキレングリコールの「分子量」とは、平均分子量の意味である。 Note that polyalkylene glycol is composed of a large number of repeating units in one molecule, and the molecular weight of polyalkylene glycol is generally different depending on each molecule, and thus is represented by an average molecular weight. Therefore, the “molecular weight” of the polyalkylene glycol in the present specification means an average molecular weight.
　ポリアルキレングリコールとしては、薬物キャリアとして許容される高分子化合物であり、生体に対して不活性若しくは極めて低活性であり、かつ、無毒若しくは極めて低毒性であるポリアルキレングリコールが好ましく使用できる。 As the polyalkylene glycol, a polyalkylene glycol which is a polymer compound acceptable as a drug carrier, inactive or extremely low in the living body, and non-toxic or extremely low in toxicity can be preferably used.
　「インターフェロン－βとポリアルキレングリコールとの共有結合体」は、インターフェロン－βの１分子に対してポリアルキレングリコールの１分子又は２分子以上が共有結合したものであり、かつ、インターフェロン活性を有しているもの、及び、ポリアルキレングリコールの１分子に対してインターフェロン－βの１分子又は２分子以上が共有結合したものであり、かつ、インターフェロン活性を有しているもの、を包含する。インターフェロン－βとポリアルキレングリコールとの共有結合体としては、インターフェロン－βの１分子に対してポリアルキレングリコールの１分子又は２分子以上が共有結合したものであり、かつ、インターフェロン活性を有しているものがより好ましい。なお、インターフェロン－βとポリアルキレングリコールとの共有結合体は、インターフェロン－βとポリアルキレングリコールとが直接共有結合したものであっても、リンカー等を介して結合したものであってもよい。 “Covalent conjugate of interferon-β and polyalkylene glycol” is one molecule of polyalkylene glycol covalently bonded to one molecule of interferon-β and has interferon activity. And those having one or more molecules of interferon-β covalently bonded to one molecule of polyalkylene glycol and having interferon activity. As a covalent conjugate of interferon-β and polyalkylene glycol, one molecule or two or more molecules of polyalkylene glycol is covalently bonded to one molecule of interferon-β and has interferon activity. It is more preferable. The covalent conjugate of interferon-β and polyalkylene glycol may be either a direct covalent bond of interferon-β and polyalkylene glycol, or a bond obtained through a linker or the like.
　悪液質に対する治療又は予防効果をもたらすための、インターフェロン－β、好ましくは化学的に修飾されたインターフェロン－β、例えばインターフェロン－βとポリアルキレングリコールとの共有結合体の投与量は、インターフェロン力価（ユニット；以下、Ｕ）又は質量（ｇ）で表すことができる。インターフェロン－βとポリアルキレングリコールとの共有結合体は、ポリアルキレングリコールを共有結合する前のインターフェロン－βの比活性（重量あたりのインターフェロン活性）と比較して、その比活性が１０％以上維持されていることが好ましい。 The dosage of a covalent conjugate of interferon-beta, preferably chemically modified interferon-beta, such as interferon-beta and polyalkylene glycol, to provide a therapeutic or prophylactic effect on cachexia is determined by the interferon titer. (Unit; hereinafter, U) or mass (g). The covalent conjugate of interferon-β and polyalkylene glycol maintains a specific activity of 10% or more compared to the specific activity of interferon-β before the polyalkylene glycol is covalently bound (interferon activity per weight). It is preferable.
　ここで、タンパク質に高分子化合物を結合させることを、タンパク質を高分子化合物で化学修飾する又は修飾するともいい、インターフェロン－βとポリアルキレングリコールとの共有結合体は、ポリアルキレングリコールで化学修飾したインターフェロン－β、又は、単に、修飾したインターフェロン－βともいう。特に、インターフェロン－βとポリエチレングリコール（以下、ＰＥＧ）との共有結合体は、ＰＥＧ修飾インターフェロン－β、ＰＥＧ化インターフェロン－β又はＰＥＧ結合インターフェロン－βとも呼ばれる。 Here, the binding of the polymer compound to the protein may be referred to as chemical modification or modification of the protein with the polymer compound, and the covalent conjugate of interferon-β and polyalkylene glycol was chemically modified with polyalkylene glycol. Also referred to as interferon-β or simply modified interferon-β. In particular, a covalent conjugate of interferon-β and polyethylene glycol (hereinafter PEG) is also referred to as PEG-modified interferon-β, PEGylated interferon-β, or PEG-conjugated interferon-β.
　インターフェロン－βとポリアルキレングリコールとの共有結合部位としては、例えば、インターフェロン－βのアミノ基、チオール基、Ｎ末端、Ｃ末端又は糖鎖が挙げられる。公知の遺伝子組換え技術を用いてインターフェロン－βに導入した非天然型（人工）アミノ酸もポリアルキレングリコールとの共有結合部位とすることができる。例えば、非天然型アミノ酸の種類及びタンパク質に非天然型アミノ酸を導入する方法は、国際公開第２０１１／１５８８９５号に記載されている。 Examples of the covalent bond site between interferon-β and polyalkylene glycol include the amino group, thiol group, N-terminus, C-terminus or sugar chain of interferon-β. A non-natural (artificial) amino acid introduced into interferon-β using a known gene recombination technique can also be used as a covalent bond site with polyalkylene glycol. For example, the kind of non-natural amino acid and a method for introducing a non-natural amino acid into a protein are described in International Publication No. 2011/158895.
　インターフェロン－βのインターフェロン活性は、公知の生物学的測定法により測定できる。具体的には、インターフェロンに感受性の高い細胞（例えば、ＦＬ細胞）に対してインターフェロン－βを含む検体を作用させた後に、これら細胞に感染能を持つシンドビスウイルス（Ｓｉｎｄｂｉｓ　ｖｉｒｕｓ）又は水ほう性口内炎ウイルス（Ｖｅｓｉｃｕｌａｒ　ｓｔｏｍａｔｉｔｉｓ　ｖｉｒｕｓ；ＶＳＶと略される。）等を一定量添加して感染させ、インターフェロン－βにより誘導されたウイルス抵抗性の程度（以下、抗ウイルス活性）を測定することで、インターフェロン活性を測定できる。抗ウイルス活性は、ウイルス増殖の抑制を指標に測定されるものであり、ウイルス増殖の結果として細胞を破壊させる細胞変性効果（Ｃｙｔｏｐａｔｈｉｃ　Ｅｆｆｅｃｔ；ＣＰＥと略される。）を５０％阻止する検体の希釈倍率から、インターフェロン力価（Ｕ）として算出される（小林茂保ら著、「免疫生化学実験法（続生化学実験講座５）」、日本生化学会編、東京化学同人、１９８６年、ｐ．２４５）。 The interferon activity of interferon-β can be measured by a known biological measurement method. Specifically, after a sample containing interferon-β is allowed to act on interferon-sensitive cells (for example, FL cells), Sindbis virus or blister that has the ability to infect these cells. Interferon was measured by adding a certain amount of stomatitis virus (abbreviated as VSV), etc., and infecting it, and measuring the degree of virus resistance induced by interferon-β (hereinafter referred to as antiviral activity). Activity can be measured. Antiviral activity is measured using suppression of virus growth as an indicator, and dilution of a specimen that inhibits cytopathic effect (abbreviated as Cytopathic Effect; CPE) that destroys cells as a result of virus growth by 50%. Calculated as the interferon titer (U) from the magnification (Shimoho Kobayashi et al., “Immunobiochemistry Experimental Method (Second Life Chemistry Laboratory Lecture 5)”, edited by the Japanese Biochemical Society, Tokyo Chemical Dojin, 1986, p.245. ).
　インターフェロン－βとポリアルキレングリコールとの共有結合体は、公知の方法で作製することができる。例えば、インターフェロンのアミノ基にポリアルキレングリコールを共有結合する方法は、米国特許第４９１７８８８号及び国際公開第８７／０００５６号に記載されている。インターフェロンのチオール基にポリアルキレングリコールを共有結合する方法は、国際公開第９９／５５３７７号に記載されている。インターフェロンのＮ末端に高分子を共有結合する方法は、国際公開第００／２３１１４号及び特開平９－２５２９８号に記載されている。インターフェロンの糖鎖にポリアルキレングリコールを共有結合する方法は、１９７８年のＭａｋｒｏｍｏｌｅｃｕｌａｒ　Ｃｈｅｍｉｓｔｒｙ（第１７９巻、ｐ．３０１）、米国特許第４１０１３８０号又は米国特許第４１７９３３７号に記載されている。インターフェロンに導入した非天然型（人工）アミノ酸にポリアルキレングリコールを共有結合する方法は、２００４年のＢｉｏｏｒｇａｎｉｃ　＆　Ｍｅｄｉｃｉｎａｌ　Ｃｈｅｍｉｓｔｒｙ　Ｌｅｔｔｅｒｓ（第１４巻、ｐ．５７４３－５７４５）に記載されている。なお、インターフェロン－βのＣ末端にポリアルキレングリコールを共有結合する場合は、インターフェロン－βのＣ末端又はその近傍にシステインや他の非天然型（人工）アミノ酸を導入することで可能となる。 A covalent conjugate of interferon-β and polyalkylene glycol can be prepared by a known method. For example, methods for covalently attaching a polyalkylene glycol to the amino group of interferon are described in US Pat. No. 4,917,888 and WO 87/00056. A method for covalently bonding a polyalkylene glycol to the thiol group of interferon is described in WO 99/55377. Methods for covalently bonding a polymer to the N-terminus of interferon are described in International Publication No. 00/23114 and JP-A-9-25298. A method for covalently bonding a polyalkylene glycol to the sugar chain of interferon is described in 1978 Makromolecular Chemistry (Vol. 179, p. 301), US Pat. No. 4,101,380 or US Pat. No. 4,179,337. A method for covalently bonding a polyalkylene glycol to an unnatural (artificial) amino acid introduced into interferon is described in 2004 Bioorganic & Medicinal Chemistry Letters (Vol. 14, p. 5743-5745). When polyalkylene glycol is covalently bonded to the C-terminus of interferon-β, it can be achieved by introducing cysteine or other non-natural (artificial) amino acid at or near the C-terminus of interferon-β.
　インターフェロン－βとポリアルキレングリコールとの結合の方法に応じて、共有結合反応に使用するポリアルキレングリコールの末端を活性化する必要がある。例えば、末端がヒドロキシスクシンイミドエステル、ニトロベンゼンスルホネートエステル、マレイミド、オルトピリジルジスルフィド、ビニルスルフォン、マレイミド、ヨードアセトアミド、カルボン酸、アジド、ホスフィン又はアミン構造で活性化されたポリアルキレングリコール誘導体をインターフェロン－βとポリアルキレングリコールとの共有結合の形成に用いることができ、これらのポリアルキレングリコール誘導体は公知の方法により合成できる。 Depending on the method of binding between interferon-β and polyalkylene glycol, it is necessary to activate the end of the polyalkylene glycol used in the covalent bond reaction. For example, polyalkylene glycol derivatives whose ends are activated with hydroxysuccinimide ester, nitrobenzene sulfonate ester, maleimide, orthopyridyl disulfide, vinyl sulfone, maleimide, iodoacetamide, carboxylic acid, azide, phosphine or amine structure are interferon-β and poly It can be used to form a covalent bond with alkylene glycol, and these polyalkylene glycol derivatives can be synthesized by known methods.
　インターフェロン－βとポリアルキレングリコールとの共有結合体は、インターフェロン－βとＰＥＧとの共有結合体であることが好ましい。インターフェロン－βとＰＥＧとの共有結合体は、１分子のインターフェロン－βに、分子量５，０００以上２４０，０００以下（好ましくは分子量１０，０００以上８０，０００以下、より好ましくは分子量３９，０００以上４５，０００以下）のＰＥＧが１分子共有結合した共有結合体であることが好ましい。 The covalent conjugate of interferon-β and polyalkylene glycol is preferably a covalent conjugate of interferon-β and PEG. A covalent conjugate of interferon-β and PEG has a molecular weight of 5,000 to 240,000 (preferably a molecular weight of 10,000 to 80,000, more preferably a molecular weight of 39,000 or more) to one molecule of interferon-β. 45,000 or less) is preferably a covalent conjugate in which one molecule of PEG is covalently bonded.
　インターフェロン－βとＰＥＧとの共有結合体の好ましい例としては、ヒト・インターフェロン－βとＰＥＧとの共有結合体を挙げることができ、例えば、国際公開第２００５／０１９２６０号に記載された方法により作製できる。ヒト・インターフェロン－βにおけるＰＥＧの結合部位としては、ヒト・インターフェロン－βのアミノ酸配列の１９番目又は１３４番目のリジンのアミノ基が好ましく、この部位にＰＥＧが１分子共有結合した共有結合体がより好ましい。具体的には、ヒト・インターフェロン－βのアミノ酸配列の１３４番目のリジンのアミノ基に分子量４０，０００以上（好ましくは、分子量４２，０００）のＰＥＧが１分子共有結合した共有結合体を例示できる。 Preferable examples of the covalent conjugate of interferon-β and PEG include a covalent conjugate of human interferon-β and PEG. For example, the covalent conjugate of interferon-β and PEG can be prepared by the method described in International Publication No. 2005/019260. it can. As the binding site of PEG in human interferon-β, the amino group of the 19th or 134th lysine in the amino acid sequence of human interferon-β is preferable, and a covalent conjugate in which one molecule of PEG is covalently bonded to this site is more preferable. preferable. Specifically, a covalent conjugate in which one molecule of PEG having a molecular weight of 40,000 or more (preferably a molecular weight of 42,000) is covalently bonded to the amino group of the 134th lysine of the amino acid sequence of human interferon-β can be exemplified. .
　ここで、「ヒト・インターフェロン－βのアミノ酸配列の１３４番目のリジン」とは、１６６アミノ酸からなるヒト・インターフェロン－βのアミノ酸配列（配列番号１）のＮ末端のアミノ酸（メチオニン）を１番目としたときに、１３４番目に位置するアミノ酸であるリジンを意味する。本明細書では、インターフェロン－βのＮ末端のアミノ酸を１番目として、インターフェロン－βのアミノ酸残基の位置を表す。 Here, “134th lysine of the amino acid sequence of human interferon-β” means that the N-terminal amino acid (methionine) of the amino acid sequence of human interferon-β consisting of 166 amino acids (SEQ ID NO: 1) is the first. Means lysine, which is an amino acid located at position 134. In the present specification, the position of the amino acid residue of interferon-β is represented with the N-terminal amino acid of interferon-β being the first.
　インターフェロン－βとポリアルキレングリコールとの共有結合体もインターフェロン－βのアミノ酸配列のリジンのアミノ基にポリアルキレングリコールが結合したものであってよい。ポリアルキレングリコール結合部位となる、インターフェロン－βのアミノ酸配列のリジンは、アミノ酸配列のアラインメントにおいて配列番号１で示されるアミノ酸配列の１９番目又は１３４番目のリジンと対応する位置にあってもよいし、対応する位置になくてもよい。 The covalent conjugate of interferon-β and polyalkylene glycol may also be one in which polyalkylene glycol is bonded to the amino group of lysine in the amino acid sequence of interferon-β. The lysine of the amino acid sequence of interferon-β that serves as a polyalkylene glycol binding site may be at a position corresponding to the 19th or 134th lysine of the amino acid sequence represented by SEQ ID NO: 1 in the alignment of the amino acid sequence, It does not have to be in the corresponding position.
　「悪液質」とは、悪性腫瘍、結核、糖尿病、血液疾患、内分泌疾患、感染症又は後天性免疫不全症候群等の慢性疾患において、著しい体重減少、貧血、浮腫、食欲不振、全身衰弱又は倦怠感等を主症状とする全身性の症候群を包含する。例えば、がん悪液質、結核性悪液質、糖尿病性悪液質、血液疾患性悪液質、内分泌疾患性悪液質、感染症性悪液質又は後天性免疫不全症候群による悪液質が挙げられる。本発明の悪液質の治療又は予防剤は、任意の悪液質に対して用いることができるが、悪性腫瘍によって引き起こされる、がん悪液質に対して特に好ましく用いることができる。 “Cachexia” refers to significant weight loss, anemia, edema, loss of appetite, generalized weakness or malaise in chronic diseases such as malignant tumor, tuberculosis, diabetes, blood disease, endocrine disease, infection or acquired immune deficiency syndrome. Includes systemic syndromes with feelings as main symptoms. Examples include cancer cachexia, tuberculosis cachexia, diabetic cachexia, blood disease cachexia, endocrine disease cachexia, infectious cachexia, or acquired immunodeficiency syndrome. The cachexia treatment or prevention agent of the present invention can be used for any cachexia, but can be particularly preferably used for cancer cachexia caused by malignant tumors.
　本発明において「悪性腫瘍」（がん、悪性新生物とも呼ばれる）とは、上皮組織由来の癌（癌腫とも呼ぶ）、非上皮性組織由来の肉腫及び造血器由来の悪性腫瘍を包含する。悪性腫瘍の種類は特に限定されないが、例えば、悪性黒色腫、悪性骨腫瘍、胃がん、肝細胞がん、急性骨髄性白血病、急性リンパ性白血病、子宮頸がん、子宮体がん、食道がん、膵がん、前立腺がん、大腸がん、乳がん、肺がん、膀胱がん又は卵巣がんが挙げられる。 In the present invention, “malignant tumor” (also referred to as cancer or malignant neoplasm) includes cancer derived from epithelial tissue (also referred to as carcinoma), sarcoma derived from non-epithelial tissue, and malignant tumor derived from hematopoietic organs. The type of malignant tumor is not particularly limited. For example, malignant melanoma, malignant bone tumor, gastric cancer, hepatocellular carcinoma, acute myeloid leukemia, acute lymphocytic leukemia, cervical cancer, endometrial cancer, esophageal cancer Pancreatic cancer, prostate cancer, colon cancer, breast cancer, lung cancer, bladder cancer or ovarian cancer.
　本発明の悪液質の治療又は予防剤は、インターフェロン、例えばインターフェロン－βを有効成分として含有し、悪液質の改善作用、すなわち悪性腫瘍、結核、糖尿病、血液疾患、内分泌疾患、感染症又は後天性免疫不全症候群等の慢性疾患において発現する著しい体重低下、貧血、浮腫、食欲不振、全身衰弱又は倦怠感等を主症状とする全身性の症候群を改善する作用を有する。低アルブミン血症、高ＩＬ－６血症、体重低下、及び生命予後の悪化（生存期間の短縮化）は、悪液質の典型的症状である。本発明の悪液質の治療又は予防剤は、悪液質の改善作用の一例として、低アルブミン血症改善作用（血中アルブミン濃度低下抑制効果）、高ＩＬ－６血症改善作用（血中ＩＬ－６濃度上昇抑制効果）、体重低下改善作用（体重低下抑制効果）、及び／又は延命作用（生存期間の延長効果）を有する。本発明の悪液質の治療又は予防剤は、インターフェロン、例えばインターフェロン－βを有効成分として含有する医薬であってよい。本発明の悪液質の治療又は予防剤の悪液質改善作用は、抗腫瘍作用（腫瘍増殖抑制効果）とは独立したメカニズムに基づく。本発明において悪液質の「治療」とは、悪液質の症状（体重低下、貧血、浮腫、食欲不振、全身衰弱又は倦怠感等）及び病状指標（アルブミン濃度の低下、ＩＬ－６濃度の上昇、生存期間の短縮化等）の少なくとも１つを改善することをいう。本発明において悪液質の「予防」とは、悪液質の症状（体重低下、貧血、浮腫、食欲不振、全身衰弱又は倦怠感等）及び病状指標（アルブミン濃度の低下、ＩＬ－６濃度の上昇等）のうち少なくとも１つの発現の阻止若しくは遅延、又はその程度を軽減することをいう。 The agent for treating or preventing cachexia of the present invention contains interferon, for example, interferon-β as an active ingredient, and improves cachexia, that is, malignant tumor, tuberculosis, diabetes, blood disease, endocrine disease, infection or It has the effect of improving systemic syndromes such as marked weight loss, anemia, edema, loss of appetite, generalized weakness or malaise that occur in chronic diseases such as acquired immune deficiency syndrome. Hypoalbuminemia, high IL-6emia, weight loss, and worsening prognosis (shortening survival) are typical symptoms of cachexia. The cachexia therapeutic or preventive agent of the present invention includes, as examples of cachexia improving action, hypoalbuminemia improving action (blood albumin concentration lowering suppressing effect), high IL-6 blood serum improving action (blood IL-6 concentration increase inhibitory effect), weight loss improving effect (weight loss inhibitory effect), and / or life prolonging effect (survival effect of prolonging survival). The cachexia treatment or prevention agent of the present invention may be a medicament containing an interferon, for example, interferon-β as an active ingredient. The cachexia improving action of the cachexia treatment or prevention agent of the present invention is based on a mechanism independent of the antitumor action (tumor growth inhibitory effect). In the present invention, “treatment” of cachexia refers to symptoms of cachexia (such as weight loss, anemia, edema, loss of appetite, general weakness or malaise) and pathological indicators (decrease in albumin concentration, IL-6 concentration). Improvement of at least one of increase, shortening of survival period, etc.). In the present invention, “prevention” of cachexia refers to symptoms of cachexia (such as weight loss, anemia, edema, loss of appetite, general weakness or malaise) and pathological indicators (decrease in albumin concentration, IL-6 concentration). Or the like, or to reduce the degree of inhibition or delay of at least one expression.
　上記の悪液質の治療又は予防剤の有効成分であるインターフェロン、例えばインターフェロン－βが、悪液質の治療又は予防に有効であることは、例えば、実施例に記載の、悪液質モデル動物（坦がんによる悪液質を発症した動物）における、低アルブミン血症改善作用、高ＩＬ－６血症改善作用、体重低下改善作用又は延命作用や、骨格筋重量低下改善作用又は肝肥大抑制作用等により評価することができる。 The fact that interferon, for example, interferon-β, which is an active ingredient of the above-mentioned cachexia treatment or prevention agent, is effective for the treatment or prevention of cachexia is described in, for example, the cachexia model animal described in the Examples. Hypoalbuminemia-improving effect, high IL-6-emia-improving effect, weight-loss-improving effect or life-prolonging effect, skeletal muscle weight-loss-decreasing effect, or liver hypertrophy suppression It can be evaluated by action or the like.
　本発明の悪液質の治療又は予防剤は、任意の投与対象、好ましくは哺乳動物（例えば、ヒト、マウス、ラット、ウサギ、イヌ、ネコ、ウシ、ウマ、ブタ、サル等）、特に好ましくはヒトに対する悪液質の治療又は予防剤として用いることができる。本発明の悪液質の治療又は予防剤の投与対象は、悪液質（たとえばがん悪液質）が認められる対象、又は悪液質を生じやすい慢性疾患（例えば、悪性腫瘍、結核、糖尿病、血液疾患、内分泌疾患、感染症又は後天性免疫不全症候群等）を有する対象であってよい。本発明の悪液質の治療又は予防剤の投与対象は、胸水や腹水を有していてもよいが、有していなくてもよい。 The cachexia treatment or prevention agent of the present invention is an arbitrary administration subject, preferably a mammal (for example, human, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, etc.), particularly preferably It can be used as a cachexia treatment or prevention agent for humans. The subject of administration of the cachexia treatment or prevention agent of the present invention is a subject in which cachexia (eg, cancer cachexia) is observed, or a chronic disease that is likely to cause cachexia (eg, malignant tumor, tuberculosis, diabetes) , Blood diseases, endocrine diseases, infectious diseases, acquired immune deficiency syndrome, etc.). The administration target of the cachexia treatment or prevention agent of the present invention may or may not have pleural effusion or ascites.
　本発明の悪液質の治療又は予防剤の投与形態としては、有効成分であるインターフェロン－β等のインターフェロンをそのまま又はそれと医薬として許容される担体等の添加剤とを含む組成物として、経口的又は非経口的に投与することができる。本発明の悪液質の治療又は予防剤の投与経路は特に限定されず、皮下、皮内、経皮、経粘膜、経肺、経鼻、経腸、静脈内、動脈内、筋肉内、腹腔内、直腸内、及び腟内投与等の非経口経路であってもよいし、経口投与であってもよい。 As the administration form of the cachexia treatment or prevention agent of the present invention, the active ingredient is interferon such as interferon-β as it is or as a composition containing it and an additive such as a pharmaceutically acceptable carrier. Or it can be administered parenterally. The administration route of the cachexia treatment or prevention agent of the present invention is not particularly limited, and is subcutaneous, intradermal, transdermal, transmucosal, transpulmonary, nasal, enteral, intravenous, intraarterial, intramuscular, abdominal cavity. It may be a parenteral route such as internal, rectal and intravaginal administration, or oral administration.
　本発明の悪液質の治療又は予防剤は、医薬として許容される任意の添加剤を含有する医薬組成物であってもよい。添加剤としては、例えば担体、賦形剤、安定化剤、懸濁化剤、乳化剤、増粘剤、分散剤、吸収促進剤、結合剤、滑沢剤、崩壊剤、湿潤剤、緩衝剤、矯味剤、保存剤、着色剤などが挙げられるが、これらに限定されない。 The cachexia treatment or prevention agent of the present invention may be a pharmaceutical composition containing any pharmaceutically acceptable additive. Examples of additives include carriers, excipients, stabilizers, suspending agents, emulsifiers, thickeners, dispersants, absorption enhancers, binders, lubricants, disintegrants, wetting agents, buffering agents, Examples include, but are not limited to, flavoring agents, preservatives, and coloring agents.
　本発明の悪液質の治療又は予防剤の経口投与のための剤形としては、例えば、錠剤、丸剤、カプセル剤、顆粒剤、シロップ剤、乳剤又は懸濁剤が挙げられ、これらは、公知の方法によって製造することができる。これらの剤形は、製剤分野において通常用いられる担体又は賦形剤等の添加剤を含有することができる。錠剤用の担体及び賦形剤としては、例えば、ラクトース、マルトース、サッカロース、澱粉又はステアリン酸マグネシウムが挙げられる。 Examples of the dosage form for oral administration of the cachexia treatment or prevention agent of the present invention include tablets, pills, capsules, granules, syrups, emulsions or suspensions, It can be produced by a known method. These dosage forms can contain additives such as carriers or excipients usually used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, maltose, saccharose, starch, and magnesium stearate.
　上記の悪液質の治療又は予防剤の非経口投与のための剤形としては、例えば、注射剤、坐薬、経鼻吸収剤、経肺吸収剤、経皮吸収剤又は局所徐放剤が挙げられ、これらは、公知の方法によって製造することができる。溶液製剤は、例えば、インターフェロン－β等のインターフェロンを、注射剤に用いられる無菌の水溶液に溶解又は抽出液に懸濁及び乳化して、リポソームに包埋させた状態で調製することができる。固体製剤は、例えば、インターフェロン－β等のインターフェロンに、マンニトール、トレハロース、ソルビトール、ラクトース又はグルコース等を賦形剤として加え、凍結乾燥物として調製することができる。さらにこれを粉体化して用いることもできる。また、これら粉体をポリ乳酸やグリコール酸等と混合し固体化して用いることもできる。ゲル剤は、例えば、インターフェロン－β等のインターフェロンを、グリセリン、ＰＥＧ、メチルセルロース、カルボキシメチルセルロース、ヒアルロン酸若しくはコンドロイチン硫酸等の増粘剤又は多糖に溶解して調製することができる。 Examples of the dosage form for parenteral administration of the cachexia treatment or prevention agent include injections, suppositories, nasal absorption agents, pulmonary absorption agents, transdermal absorption agents, and topical sustained release agents. These can be produced by known methods. The solution preparation can be prepared, for example, by interferon such as interferon-β dissolved in a sterile aqueous solution used for injection or suspended and emulsified in an extract and embedded in liposomes. A solid preparation can be prepared, for example, as a lyophilized product by adding mannitol, trehalose, sorbitol, lactose, glucose or the like as an excipient to an interferon such as interferon-β. Further, it can be used in the form of powder. Further, these powders can be mixed with polylactic acid, glycolic acid or the like and solidified for use. The gel can be prepared by, for example, dissolving interferon such as interferon-β in a thickener or polysaccharide such as glycerin, PEG, methylcellulose, carboxymethylcellulose, hyaluronic acid or chondroitin sulfate.
　いずれの剤形の製剤も、安定化剤として、ヒト血清アルブミン、ヒト免疫グロブリン、α２マクログロブリン又はアミノ酸等を含んでもよく、また、分散剤又は吸収促進剤として、インターフェロン活性を損なわない範囲でアルコール、糖アルコール、イオン性界面活性剤又は非イオン性界面活性剤等を含んでもよい。また、微量金属又は有機酸塩も必要に応じて含有してもよい。 The preparation of any dosage form may contain human serum albumin, human immunoglobulin, α2 macroglobulin, amino acid or the like as a stabilizer, and alcohol as a dispersant or absorption enhancer as long as it does not impair interferon activity. , Sugar alcohol, ionic surfactant or nonionic surfactant may be included. Moreover, you may contain trace metal or organic acid salt as needed.
　本発明の悪液質の治療又は予防剤は、有効成分であるインターフェロン－βの１０，０００Ｕ～１，８００万Ｕ／回の用量で、１日に１回又は２回の投与間隔で連日投与することができる。本発明の悪液質の治療又は予防剤はまた、２日以上の期間を隔てて１回の投与間隔で投与してもよく、さらに、２日～１ヵ月に１回の投与間隔で間歇投与してもよい。また、化学的に修飾したインターフェロン（例えば、インターフェロン－βとポリアルキレングリコールとの共有結合体）を有効成分とする本発明の悪液質の治療又は予防剤は、１週間に１回又は２回～１ヵ月に１回の投与間隔で間歇投与することが好ましく、１週間に１回又は２回の投与間隔で間歇投与することが特に好ましい。 The cachexia treatment or prevention agent of the present invention is administered at daily doses of once or twice a day at a dose of 10,000 U to 18 million U / dose of the active ingredient interferon-β. can do. The cachexia treatment or prevention agent of the present invention may also be administered at a single administration interval with a period of 2 days or more, and further, intermittent administration at an administration interval of once every 2 days to 1 month. May be. The cachexia treatment or prevention agent of the present invention comprising a chemically modified interferon (for example, a covalent conjugate of interferon-β and polyalkylene glycol) as an active ingredient is once or twice a week. It is preferable to administer intermittently at an administration interval of once a month, particularly preferably intermittently at an interval of once or twice a week.
　本発明の悪液質の治療又は予防剤は、単独で投与してもよいし、疾患の治療若しくは予防、又は症状の減少若しくは抑制に対して用いられる一種類若しくはそれ以上の他の任意の薬剤と組み合わせて投与してもよい。組み合わせる薬剤は、低分子化合物であってもよく、また、高分子のタンパク質、ポリペプチド、抗体又はワクチン等であってもよい。この場合、本発明の悪液質の治療又は予防剤は、組み合わせる薬剤と同時又は時間差をおいて投与することができる。なお、組み合わせて投与する場合、それぞれの薬剤を併用投与してもよいし、合剤として投与することもできる。組み合わせる薬剤の投与量は、それぞれ臨床上用いられる用量を基準として適宜選択することができる。合剤とする場合の本発明の悪液質の治療又は予防剤と組み合わせる薬剤との配合比は、投与対象、投与対象の年齢、体重、症状、投与時間、剤形、投与方法又は薬剤の組み合わせ等により、適宜選択することができる。 The cachexia treatment or prevention agent of the present invention may be administered alone, or one or more other arbitrary agents used for treatment or prevention of diseases, or reduction or suppression of symptoms. May be administered in combination. The drug to be combined may be a low molecular compound, or may be a high molecular protein, polypeptide, antibody, vaccine or the like. In this case, the cachexia treatment or prevention agent of the present invention can be administered simultaneously or with a time difference from the combined agents. In addition, when administering in combination, each chemical | medical agent may be administered together and can also be administered as a mixture. The dose of the drug to be combined can be appropriately selected based on the clinically used dose. The combination ratio of the cachexia treatment or prevention agent of the present invention when combined with the drug combined with the drug is the subject of administration, age of administration subject, body weight, symptom, administration time, dosage form, administration method, or combination of drugs. It can be selected as appropriate.
　本発明の悪液質の治療又は予防剤は、化学療法剤、免疫療法剤又は利尿剤等の薬剤と組み合わせて用いることもできる。 The agent for treating or preventing cachexia of the present invention can also be used in combination with drugs such as chemotherapeutic agents, immunotherapeutic agents or diuretics.
　化学療法剤としては、シクロホスファミド、イホスファミド、メルファラン、ブスルファン、ニムスチン、ラニムスチン若しくはテモゾロマイド等のアルキル化剤、メトトレキサート、フルオロウラシル、テガフール、カルモフール、ドキシフルリジン、カペシタビン、シタラビン、アンシタビン、エノシタビン、シタラビンオクホスファート、ゲムシタビン、メルカプトプリン若しくはフルダラビン等の核酸代謝拮抗薬、ドキソルビシン、ダウノルビシン、ピラルビシン、エピルビシン、イダルビシン、ミトキサントロン、マイトマイシンＣ、ブレオマイシン若しくはペプロマイシン等の抗腫瘍性抗生物質、ビンクリスチン、ビンブラスチン、ビンデシン、ビノレルビン、パクリタキセル若しくはドセタキセル等の微小管阻害剤、シスプラチン、カルボプラチン若しくはネダプラチン等の白金製剤、イリノテカン、ノギテカン若しくはエトポシド等のトポイソメラーゼ阻害薬、又は、トラスツズマブ、リツキシマブ若しくはイマニチブ等の分子標的治療薬等が挙げられる。 Examples of chemotherapeutic agents include cyclophosphamide, ifosfamide, melphalan, busulfan, nimustine, ranimustine, temozolomide and other alkylating agents, methotrexate, fluorouracil, tegafur, carmofur, doxyfluridine, capecitabine, cytarabine, ancitabine, phositabine, cytarabine, cytarabine Nucleic acid metabolism antagonists such as phart, gemcitabine, mercaptopurine or fludarabine, doxorubicin, daunorubicin, pirarubicin, epirubicin, idarubicin, mitoxantrone, mitomycin C, bleomycin or peplomycin, antitumor antibiotics, vincristine, vinblastine, vindesine, vinorelbine , Microtubule inhibitors such as paclitaxel or docetaxel, sysp Chin, platinum drugs such as carboplatin or nedaplatin, irinotecan, topoisomerase inhibitors such as Nogitekan or etoposide, or trastuzumab, targeted therapy, etc. of rituximab or imatinib, and the like.
　免疫療法剤としては、ムラミルジペプチド誘導体、レンチナン、シゾフィラン、ウベニメクス、ピシバニール、クレスチン、インターロイキン、顆粒球コロニー刺激因子又はエリスロポエチン等が挙げられる。 Examples of the immunotherapeutic agent include muramyl dipeptide derivatives, lentinan, schizophyllan, ubenimex, picibanil, krestin, interleukin, granulocyte colony stimulating factor or erythropoietin.
　利尿剤としては、サリチル酸ナトリウムテオブロミン若しくはサリチル酸カルシウムテオブロミン等のキサンチン誘導体製剤、エチアジド、シクロペンチアジド、トリクロルメチアジド、ヒドロクロロチアジド、ヒドロフルメチアジド、ベンチルヒドロクロロチアジド、ペンフルチジド、ポリチアジド若しくはメチクロチアジド等のチアジド系製剤、スピロノラクトン若しくはトリアムテレン等の抗アルドステロン製剤、アセタゾラミド等の炭酸脱水素酵素阻害剤、クロルタリドン、メフルシド若しくはインダパミド等のクロルベンゼンスルホンアミド系製剤、アゾセミド、イソソルビド、エタクリン酸、ピレタニド、ブメタニド又はフロセミド等が挙げられる。
　本発明の悪液質の治療又は予防剤はまた、インターフェロン－β等のインターフェロンに加えて、組み合わせて用いる他の上記薬剤を含有してもよい。
　本発明は、本発明の悪液質の治療又は予防剤を上記投与対象に投与することを含む、悪液質の治療又は予防方法も提供する。 Examples of diuretics include xanthine derivative preparations such as sodium salicylate theobromine or calcium salicylate theobromine, ethiazide, cyclopenthiazide, trichloromethiazide, hydrochlorothiazide, hydroflumethiazide, benchylhydrochlorothiazide, penflutide, polythiazide, or methycrothiazide. Antialdosterone preparations such as spironolactone or triamterene, carbonic acid dehydrogenase inhibitors such as acetazolamide, chlorbenzenesulfonamide preparations such as chlorthalidone, mefluside or indapamide, azosemide, isosorbide, ethacrynic acid, piretanide, bumetanide or furosemide.
In addition to interferon such as interferon-β, the cachexia treatment or prevention agent of the present invention may also contain other above-mentioned agents used in combination.
The present invention also provides a method for treating or preventing cachexia, comprising administering the agent for treating or preventing cachexia of the present invention to the administration subject.
　以下、実施例を示して本発明を具体的に詳述するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
（実施例１）　インターフェロン－βとＰＥＧとの共有結合体の調製
　組換え型ヒト・インターフェロンβ（配列番号１）は、Ｇｏｅｄｄｅｌ　ｅｔ．ａｌ．，　Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｒｅｓｅａｒｃｈ（１９８０年）第８巻、ｐ．４０５７－４０７４に記載の方法に従って作製し、組換え型マウス・インターフェロン－β（ＮＣＢＩ　Ｐｒｏｔｅｉｎデータベース　アクセッション番号１ＩＦＡ＿Ａ）は、Ｍａｔｓｕｄａ　ｅｔ．ａｌ．，　Ｊｏｕｒｎａｌ　ｏｆ　Ｉｎｔｅｒｆｅｒｏｎ　Ｒｅｓｅａｒｃｈ（１９８６年）第６巻、ｐ．５１９－５２６に記載の方法に従って作製した。 Example 1 Preparation of Covalent Conjugate of Interferon-β and PEG Recombinant human interferon β (SEQ ID NO: 1) was prepared according to Goeddel et. al. Nucleic Acid Research (1980), Vol. 8, p. Recombinant mouse interferon-β (NCBI Protein database accession number 1IFA_A) was prepared according to Matsuda et. al. , Journal of Interferon Research (1986), Vol. 6, p. It was prepared according to the method described in 519-526.
　さらに、国際公開第２００５／０１９２６０号の実施例６に記載の方法に従って、組換え型ヒト・インターフェロンβのアミノ酸配列（配列番号１）の１３４番目のリジンのアミノ基に分子量４２，０００のＰＥＧが１分子共有結合した「インターフェロン－βとＰＥＧとの共有結合体」、及びに組換え型マウス・インターフェロンβに分子量４２，０００のＰＥＧが１分子共有結合した「マウス・インターフェロン－βとＰＥＧとの共有結合体」を調製した。具体的には、まず、０．５ｍｏｌ／Ｌ塩化ナトリウムを含む１００ｍｍｏｌ／Ｌ酢酸緩衝液（ｐＨ５．０）に溶解した組換え型ヒト・インターフェロン－β（最終濃度２００μｇ／ｍＬ）に、エチレングリコールを（最終濃度２０％で）添加した後、１ｍｏｌ／Ｌリン酸水素二ナトリウム溶液を用いてｐＨを７．６に調整した。これに、ヒドロキシスクシンイミドエステル活性化ＰＥＧ（分子量４２，０００、日油株式会社；品番６１Ｇ９９１２２Ｂ０１）を加え混合し、４℃で一晩、結合反応させた。この結合反応溶液に５倍容の１０ｍｍｏｌ／Ｌ酢酸緩衝液（ｐＨ４．５）を添加し、１０ｍｍｏｌ／Ｌ酢酸緩衝液（ｐＨ４．５）で平衡化した陽イオン交換カラム（ＴＯＹＯＰＥＡＲＬ　ＣＭ　６５０（Ｓ）；東ソー株式会社）に供した。下記の展開溶媒で、タンパク質を溶出及び分画した。
＜展開溶媒＞
　溶媒Ａ：１０ｍｍｏｌ／Ｌ酢酸緩衝液（ｐＨ４．５）
　溶媒Ｂ：１ｍｏｌ／Ｌ塩化ナトリウムを含む１０ｍｍｏｌ／Ｌ酢酸緩衝液（ｐＨ４．５） Furthermore, according to the method described in Example 6 of International Publication No. 2005/019260, PEG having a molecular weight of 42,000 is added to the amino group of the 134th lysine of the amino acid sequence of recombinant human interferon β (SEQ ID NO: 1). “Covalent conjugate of interferon-β and PEG” in which one molecule is covalently bonded, and “Molecular interferon-β and PEG in which one molecule of PEG having a molecular weight of 42,000 is covalently bonded to recombinant mouse interferon β. A “covalent conjugate” was prepared. Specifically, first, ethylene glycol was added to recombinant human interferon-β (final concentration 200 μg / mL) dissolved in 100 mmol / L acetate buffer (pH 5.0) containing 0.5 mol / L sodium chloride. After addition (at a final concentration of 20%), the pH was adjusted to 7.6 using a 1 mol / L disodium hydrogen phosphate solution. To this, hydroxysuccinimide ester activated PEG (molecular weight 42,000, NOF Corporation; product number 61G99122B01) was added and mixed, and a binding reaction was carried out at 4 ° C. overnight. A cation exchange column (TOYOPEARL CM 650 (S)) was added to this binding reaction solution by adding 5 volumes of 10 mmol / L acetate buffer (pH 4.5) and equilibrated with 10 mmol / L acetate buffer (pH 4.5). ; Tosoh Corporation). The protein was eluted and fractionated with the following developing solvent.
<Developing solvent>
Solvent A: 10 mmol / L acetate buffer (pH 4.5)
Solvent B: 10 mmol / L acetate buffer (pH 4.5) containing 1 mol / L sodium chloride
　上記溶媒Ａ、Ｂの混合液を陽イオン交換カラムの樹脂量に対して４０倍量通液させ、その通液の間に溶媒Ｂの送液比率を０％から６５％に連続的に上昇させることにより溶出を行った。溶出された分画は、さらに、陽イオン交換カラムＳＰ－５ＰＷ（東ソ株式会社）に供し、組換え型ヒト・インターフェロン－βのアミノ酸配列（配列番号１）の１３４番目のリジンのアミノ基に分子量４２，０００のＰＥＧが１分子共有結合した「ヒト・インターフェロン－βとＰＥＧとの共有結合体」（以下、「ＰＥＧ－ＩＦＮ－β」）を単離した。また、ＰＥＧ－ＩＦＮ－βの調製方法と同じ方法で、組み換え型マウス・インターフェロンβに分子量４２，０００のＰＥＧが１分子共有結合した「マウス・インターフェロン－βとＰＥＧとの共有結合体」（以下、「ＰＥＧ－ｍＩＦＮ－β」）を調製した。 The mixed solution of the solvents A and B is passed through 40 times the resin amount of the cation exchange column, and the solvent B feed rate is continuously increased from 0% to 65% during the passage. Elution. The eluted fraction was further subjected to a cation exchange column SP-5PW (Tosoh Corporation), and the amino group of the 134th lysine of the amino acid sequence of recombinant human interferon-β (SEQ ID NO: 1) was used. A “covalent conjugate of human interferon-β and PEG” (hereinafter, “PEG-IFN-β”) in which one molecule of PEG having a molecular weight of 42,000 was covalently bonded was isolated. In addition, a “covalent conjugate of mouse interferon-β and PEG” in which one molecule of PEG having a molecular weight of 42,000 is covalently bonded to recombinant mouse interferon β in the same manner as the preparation method of PEG-IFN-β , “PEG-mIFN-β”).
（実施例２）悪液質モデルにおけるインターフェロン－βの低アルブミン血症改善作用及び高ＩＬ－６血症改善作用
　Ｈｕｙｎｈらの方法（Ｍｏｌｅｃｕｌａｒ　ｃａｎｃｅｒ　ｔｈｅｒａｐｅｕｔｉｃｓ、２００７年、第６巻、Ｐ．２９５９－２９６６）に従って、卵巣癌細胞の腹膜播種による悪液質モデルマウスを作製し、これを用いて、悪液質における低アルブミン血症及び高ＩＬ－６血症に対する、インターフェロン－βの作用を検討した。インターフェロン－βとして、実施例１で調製したＰＥＧ－ｍＩＦＮ－βを用いた。 (Example 2) Hypoalbuminemia-improving effect and hyper-IL-6-emia-improving effect of interferon-β in a cachexia model The method of Huynh et al. (Molecular cancer therapeutics, 2007, Vol. 6, P. 2959-2966) ), A cachexia model mouse was prepared by peritoneal seeding of ovarian cancer cells, and the effect of interferon-β on hypoalbuminemia and hyper-IL-6 in the cachexia was examined. As interferon-β, PEG-mIFN-β prepared in Example 1 was used.
　ヒト卵巣癌細胞株ＯＶ－９０（ＣＲＬ－１１７３２；Ａｍｅｒｉｃａｎ　ｔｙｐｅ　ｃｕｌｔｕｒｅ　ｃｏｌｌｅｃｔｉｏｎ）を、１５％ウシ胎仔血清を含むＭＥＭ培地を用いて、３７℃、５％ＣＯ２インキュベーターで培養し維持した。ＯＶ－９０細胞をトリプシン／ＥＤＴＡにて回収後、リン酸緩衝液（ＰＢＳ）で洗浄し、ＯＶ－９０細胞懸濁液を調製した。雌性ＳＣＩＤマウス（Ｃ．Ｂ．－１７／ｌｃｒ－ｓｃｉｄ／ｓｃｉｄ－Ｊｃｌ；日本クレア）の腹腔内に、ＯＶ－９０細胞懸濁液（５×１０６細胞／匹）を移植した。なお、ヒト卵巣癌細胞を移植しなかった同一週齢の雌性ＳＣＩＤマウスを、対照群とした（ｎ＝５）。 Human ovarian cancer cell line OV-90 (CRL-11732; American type culture collection) was cultured and maintained in a MEM medium containing 15% fetal calf serum at 37 ° C. in a 5% CO 2 incubator. OV-90 cells were collected with trypsin / EDTA and then washed with phosphate buffer (PBS) to prepare an OV-90 cell suspension. An OV-90 cell suspension (5 × 10 6 cells / mouse) was transplanted into the abdominal cavity of female SCID mice (CB-17 / lcr-scid / scid-Jcl; Japan Claire). In addition, the female SCID mouse of the same age which did not transplant a human ovarian cancer cell was made into the control group (n = 5).
　細胞懸濁液移植の４４日後から被験物質の投与を開始した（この日を投与開始日とする）。実施例１で作製したＰＥＧ－ｍＩＦＮ－βの溶液（溶媒：１５０ｍｍｏｌ／Ｌ塩化ナトリウムを含む２０ｍｍｏｌ／Ｌ酢酸緩衝液（ｐＨ４．５））を、１０万単位（Ｕ）／匹／回の用量となるように、皮下投与した。投与間隔は、ヒトにおける週１回の投与に相当する隔日投与とした（ｎ＝１１；ＰＥＧ－ｍＩＦＮ－β投与群）。溶媒投与群には、ＰＥＧ－ｍＩＦＮ－βの代わりに、同容量の、１５０ｍｍｏｌ／Ｌ塩化ナトリウムを含む２０ｍｍｏｌ／Ｌ酢酸緩衝液（ｐＨ４．５）を、同様に投与した（ｎ＝１１）。 Administration of the test substance was started 44 days after cell suspension transplantation (this day is taken as the administration start date). A solution of PEG-mIFN-β prepared in Example 1 (solvent: 20 mmol / L acetate buffer (pH 4.5) containing 150 mmol / L sodium chloride) with a dose of 100,000 units (U) / animal / dose As such, it was administered subcutaneously. The dosing interval was every other day equivalent to once a week in humans (n = 11; PEG-mIFN-β administration group). In the solvent administration group, instead of PEG-mIFN-β, the same volume of 20 mmol / L acetate buffer (pH 4.5) containing 150 mmol / L sodium chloride was similarly administered (n = 11).
　３回目の投与が終了した翌日（投与開始日から５日後）に、対照群、溶媒投与群及びＰＥＧ－ｍＩＦＮ－β投与群の各個体の腋下静脈からイソフルラン吸入麻酔下にて採血した。採取した血液に抗凝固剤としてＥＤＴＡを添加した後、血漿を得た。 On the next day after completion of the third administration (5 days after the administration start date), blood was collected under the anesthesia by inhalation of isoflurane from the arm of the individual of the control group, the solvent administration group, and the PEG-mIFN-β administration group. Plasma was obtained after adding EDTA as an anticoagulant to the collected blood.
　採血した個体は、麻酔下にて安楽死させ、開腹した後、腹腔内の腫瘍塊をピンセットとハサミを用いて摘出し、その重量を測定した。 The collected blood sample was euthanized under anesthesia, and after laparotomy, the tumor mass in the abdominal cavity was removed using tweezers and scissors, and its weight was measured.
　血漿中のアルブミン濃度測定は、ｍｏｕｓｅ　Ａｌｂｕｍｉｎ　ＥＬＩＳＡ　Ｑｕａｎｔｉｔａｔｉｏｎ　Ｓｅｔ（ＢＥＴＨＹＬ　Ｌａｂ）を用い、キットに添付の説明書に従って行った。なお、血漿は希釈液（Ｓａｍｐｌｅ　Ｄｉｌｕｅｎｔ：５０ｍｍｏｌ／Ｌ　Ｔｒｉｓ、０．１４％　ＮａＣｌ、１％ＢＳＡ、０．５％Ｔｗｅｅｎ２０）を用いて、１００万倍に希釈して測定に使用した。 The albumin concentration in plasma was measured using a mouse Albumin ELISA Quantitation Set (BETHYL Lab) according to the instructions attached to the kit. The plasma was diluted 1 million times with a diluent (Sample Diluent: 50 mmol / L Tris, 0.14% NaCl, 1% BSA, 0.5% Tween 20) and used for measurement.
　血漿中のマウスＩＬ－６の濃度測定は、Ｑｕａｎｔｉｋｉｎｅ　ｍｏｕｓｅ　ＩＬ－６　ｋｉｔ　（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ）を用い、また、血漿中のヒトＩＬ－６の濃度測定は、Ｑｕａｎｔｉｋｉｎｅ　ｈｕｍａｎ　ＩＬ－６　ｋｉｔ（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ）を用い、キットに添付の説明書に従って行った。なお、血漿は生理食塩水（大塚製薬工場）を用いて、５倍希釈して測定に使用した。 The concentration of mouse IL-6 in plasma was measured using Quantikine mouse IL-6 kit (R & D Systems), and the concentration of human IL-6 in plasma was measured using Quantikine human IL-6 kit (R & D Systems). Used according to the instructions attached to the kit. The plasma was diluted 5-fold with physiological saline (Otsuka Pharmaceutical Factory) and used for measurement.
　統計解析は、溶媒投与群とＰＥＧ－ｍＩＦＮ－β投与群との間の２群間で行い、等分散性の検定（Ｆ検定）によりＳｔｕｄｅｎｔのｔ検定又はＷｅｌｃｈ検定を選択して行った。統計解析の結果、Ｐ値が０．０５未満の場合に統計学的に有意であると判断した。 Statistical analysis was performed between two groups, the solvent administration group and the PEG-mIFN-β administration group, and the Student t-test or Welch test was selected by an equidispersity test (F test). As a result of statistical analysis, when the P value was less than 0.05, it was judged to be statistically significant.
　その結果を図１～４に示す。図１の縦軸は、血漿中アルブミン濃度（平均値±標準誤差；ｎ＝５～１１）、図２の縦軸は、血漿中マウスＩＬ－６濃度（平均値±標準誤差；ｎ＝５～１１）、図３の縦軸は、血漿中ヒトＩＬ－６濃度（平均値±標準誤差；ｎ＝５～１１）、図４の縦軸は、腹腔内の腫瘍重量（平均値±標準誤差；ｎ＝５～１１）をそれぞれ示し、横軸の「対照」は対照群、「溶媒」は溶媒投与群、「ＰＥＧ－ｍＩＦＮ－β」はＰＥＧ－ｍＩＦＮ－β投与群を示す。また、これらの図中の記号＊（アスタリスク）は、溶媒群と比較して統計学的に有意（Ｐ＜０．０５）であることを示す。 The results are shown in Figs. The vertical axis of FIG. 1 is the plasma albumin concentration (mean ± standard error; n = 5 to 11), and the vertical axis of FIG. 2 is the plasma mouse IL-6 concentration (mean ± standard error; n = 5 to 11). 11), the vertical axis in FIG. 3 is the plasma human IL-6 concentration (mean value ± standard error; n = 5 to 11), and the vertical axis in FIG. 4 is the tumor weight in the abdominal cavity (mean value ± standard error; n = 5 to 11), “control” on the horizontal axis indicates the control group, “solvent” indicates the solvent administration group, and “PEG-mIFN-β” indicates the PEG-mIFN-β administration group. In addition, the symbol * (asterisk) in these figures indicates that it is statistically significant (P <0.05) compared to the solvent group.
　溶媒投与群は対照群と比較して、血漿中アルブミン濃度の低下（図１）、及び、血漿中マウスＩＬ－６濃度の増加（図２）が認められ、ヒト卵巣癌細胞を移植したマウス（溶媒投与群）は悪液質を発症していることが示された。ＰＥＧ－ｍＩＦＮ－β投与により、この血漿中アルブミン濃度の低下は改善し（溶媒投与群と比較して統計学的に有意（Ｐ＜０．０５））（図１）、また、血漿中マウスＩＬ－６濃度の増加は抑制された（図２）。したがって、ＰＥＧ－ｍＩＦＮ－βは、悪液質における低アルブミン血症及び高ＩＬ－６血症を改善することが示された。 In the solvent administration group, a decrease in plasma albumin concentration (FIG. 1) and an increase in plasma mouse IL-6 concentration (FIG. 2) were observed compared to the control group, and mice transplanted with human ovarian cancer cells (FIG. 2). It was shown that the solvent administration group) developed cachexia. PEG-mIFN-β administration improved this decrease in plasma albumin concentration (statistically significant (P <0.05) compared to the vehicle-administered group) (FIG. 1), and plasma mouse IL The increase in -6 concentration was suppressed (Figure 2). Thus, PEG-mIFN-β has been shown to improve hypoalbuminemia and hyper IL-6 in cachexia.
　一方、移植したヒト卵巣癌細胞由来である血漿中ヒトＩＬ－６濃度及び腹腔内の腫瘍重量は、溶媒投与群とＰＥＧ－ｍＩＦＮ－β投与群との間で有意な変化は認められなかった（図３及び図４）。 On the other hand, plasma human IL-6 concentration and intraperitoneal tumor weight derived from transplanted human ovarian cancer cells were not significantly changed between the solvent administration group and the PEG-mIFN-β administration group ( 3 and 4).
　インターフェロン－βには癌細胞の増殖を抑制する作用（以下、抗腫瘍作用）を有することが知られている。しかし、ＰＥＧ－ｍＩＦＮ－βの投与により、血漿中ヒトＩＬ－６濃度及び腹腔内の腫瘍重量には変化がなかった。すなわち、ＰＥＧ－ｍＩＦＮ－βの投与で認められた悪液質改善作用は、ヒト卵巣癌細胞に対する抗腫瘍作用が十分に現れない場合において認められたことから、それがインターフェロン－βの抗腫瘍作用の結果として二次的に起こる作用ではなく、抗腫瘍作用とは独立した作用であることが示された。 Interferon-β is known to have an action of suppressing the growth of cancer cells (hereinafter referred to as an antitumor action). However, administration of PEG-mIFN-β did not change plasma human IL-6 concentration and intraperitoneal tumor weight. That is, the cachexia ameliorating effect observed with the administration of PEG-mIFN-β was observed when the antitumor effect on human ovarian cancer cells was not sufficiently exhibited. As a result of this, it was shown that the action was not a secondary action but an action independent of the antitumor action.
　したがって、インターフェロン－βは、抗腫瘍作用とは独立した、悪液質改善作用を有することが示された。 Therefore, it was shown that interferon-β has a cachexia improving action independent of the antitumor action.
（実施例３）悪液質モデルマウスにおけるインターフェロン－βの体重低下改善作用及び延命作用
　悪液質被験体における体重低下及び生存日数減少に対する、インターフェロン－βの作用を検討した。インターフェロン－βとして、ＰＥＧ－ＩＦＮ－β及びＰＥＧ－ｍＩＦＮ－βを用いた。 (Example 3) Weight loss improving effect and life prolonging effect of interferon-β in cachexia model mice The effect of interferon-β on weight loss and days of survival in cachexic subjects was examined. As interferon-β, PEG-IFN-β and PEG-mIFN-β were used.
　実施例２と同様の方法で、ヒト卵巣癌細胞株ＯＶ－９０の細胞懸濁液（５×１０６細胞／匹）を雌性ＳＣＩＤマウス（Ｃ．Ｂ．－１７／ｌｃｒ－ｓｃｉｄ／ｓｃｉｄ－ｊｃｌ；日本クレア）の腹腔内に移植した。なお、ヒト卵巣癌細胞を移植しなかった同一週齢の雌性ＳＣＩＤマウスを対照群とした（ｎ＝４）。 A cell suspension of human ovarian cancer cell line OV-90 (5 × 10 6 cells / mouse) was treated with female SCID mice (CB-17 / lcr-scid / scid-jcl) in the same manner as in Example 2. Transplanted intraperitoneally; In addition, the female SCID mouse | mouth of the same week age which did not transplant a human ovarian cancer cell was made into the control group (n = 4).
　細胞懸濁液移植の４６日後から被験物質の投与を開始した（この日を投与開始日とする）。被験物質として、実施例１で作製したＰＥＧ－ＩＦＮ－βとＰＥＧ－ｍＩＦＮ－βとを混合して調製した混合液（以下、ＰＥＧ－ＩＦＮ－βｓ）を、ＰＥＧ－ＩＦＮ－βとＰＥＧ－ｍＩＦＮ－βのそれぞれが５００００Ｕ／匹／回の用量となるように、皮下投与した。投与間隔は、ヒトにおける週１回の投与に相当する隔日投与とした（ｎ＝６；ＰＥＧ－ＩＦＮ－βｓ投与群）。溶媒投与群には、同容量の、１５０ｍｍｏｌ／Ｌ塩化ナトリウムを含む２０ｍｍｏｌ／Ｌ酢酸緩衝液（ｐＨ４．５）を同様に投与した（ｎ＝７）。被験物質及び溶媒の投与は、１２日間に６回行った。 Administration of the test substance was started 46 days after cell suspension transplantation (this day is taken as the administration start date). As a test substance, a mixed solution prepared by mixing PEG-IFN-β prepared in Example 1 and PEG-mIFN-β (hereinafter referred to as PEG-IFN-βs) was obtained by combining PEG-IFN-β and PEG-mIFN. Subcutaneous administration was performed so that each of -β was a dose of 50000 U / animal / dose. The dosing interval was every other day equivalent to once a week in humans (n = 6; PEG-IFN-βs administration group). The same volume of 20 mmol / L acetate buffer (pH 4.5) containing 150 mmol / L sodium chloride was similarly administered to the solvent administration group (n = 7). The test substance and the solvent were administered 6 times in 12 days.
　投与開始日から６日後に、対照群、溶媒投与群、ＰＥＧ－ＩＦＮ－βｓ投与群の各個体の体重を測定した。また、投与開始日から１２日後に対照群、溶媒投与群、ＰＥＧ－ＩＦＮ－βｓ投与群の生存率（投与開始日から１２日後の生存個体数／投与開始日の生存個体数）を評価した。 6 days after the start of administration, the body weight of each individual in the control group, the solvent administration group, and the PEG-IFN-βs administration group was measured. In addition, the survival rate of the control group, the solvent administration group, and the PEG-IFN-βs administration group (number of surviving individuals 12 days after the administration start date / number of surviving individuals on the administration start date) was evaluated 12 days after the administration start date.
　体重測定結果に関する統計解析は、溶媒投与群と、ＰＥＧ－ＩＦＮ－βｓ投与群との間の２群間で行い、等分散性の検定（Ｆ検定）によりＳｔｕｄｅｎｔのｔ検定又はＷｅｌｃｈ検定を選択して行った。統計解析の結果、Ｐ値が０．０５未満の場合に統計学的に有意であると判断した。 Statistical analysis of the body weight measurement results was performed between two groups, the solvent administration group and the PEG-IFN-βs administration group, and the Student's t test or Welch test was selected by the homodispersity test (F test). I went. As a result of statistical analysis, when the P value was less than 0.05, it was judged to be statistically significant.
　体重測定結果を図５に示す。図５の縦軸は、体重（平均値±標準誤差；ｎ＝４～７）を示し、横軸の「対照」は対照群、「溶媒」は溶媒投与群、「ＰＥＧ－ＩＦＮ－βｓ」はＰＥＧ－ＩＦＮ－βｓ投与群を示す。また、図中の記号＊（アスタリスク）は、溶媒群と比較して統計学的に有意（Ｐ＜０．０５）であることを示す。 The results of weight measurement are shown in FIG. The vertical axis in FIG. 5 represents the body weight (mean value ± standard error; n = 4 to 7), and the horizontal axis “control” is the control group, “solvent” is the solvent administration group, and “PEG-IFN-βs” is The PEG-IFN-βs administration group is shown. Moreover, the symbol * (asterisk) in the figure indicates that it is statistically significant (P <0.05) compared to the solvent group.
　溶媒投与群は、対照群と比較して体重の低下が認められ、ヒト卵巣癌細胞を移植したマウス（溶媒投与群）は悪液質を発症していることが示された。 In the solvent administration group, a decrease in body weight was observed compared to the control group, and it was shown that mice transplanted with human ovarian cancer cells (solvent administration group) developed cachexia.
　これに対して、ＰＥＧ－ＩＦＮ－βｓ投与群では、溶媒投与群と比較して体重が統計学的に有意に増加した（図５）。したがって、ＰＥＧ－ＩＦＮ－βｓは、悪液質における体重低下に対する改善作用を有することが示された。 In contrast, the body weight of the PEG-IFN-βs administration group increased statistically significantly compared to the solvent administration group (FIG. 5). Therefore, PEG-IFN-βs was shown to have an improving effect on weight loss in cachexia.
　投与開始日から１２日後の生存率の評価結果を、表１に示す。表１中の生存率は、「投与開始日から１２日後の生存個体数／投与開始日の生存個体数」として示されている。 Table 1 shows the evaluation results of the survival rate 12 days after the administration start date. The survival rate in Table 1 is shown as “the number of surviving individuals 12 days after the administration start date / the number of surviving individuals on the administration start date”.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１に示すように、溶媒投与群では、投与開始日から１２日後には７例中４例が死亡したが、ＰＥＧ－ＩＦＮ－βｓ投与群では、投与開始日から１２日後まで全６例が生存した。したがって、ＰＥＧ－ＩＦＮ－βｓは、悪液質における生存日数減少を改善し、延命作用を有することが示された。 As shown in Table 1, in the solvent administration group, 4 out of 7 cases died 12 days after the administration start date, whereas in the PEG-IFN-βs administration group, all 6 cases died from the administration start date to 12 days later. Survived. Thus, PEG-IFN-βs has been shown to improve survival and reduce life in cachexia.
　実施例２及び３の結果から、インターフェロン－βが、抗腫瘍作用とは独立して悪液質改善作用を有し、悪液質の治療及び予防に有効であることが示された。 From the results of Examples 2 and 3, it was shown that interferon-β has a cachexia improving action independent of the antitumor action and is effective in the treatment and prevention of cachexia.
　本発明の悪液質の治療又は予防剤は、慢性疾患の患者における悪液質の治療又は予防のために用いることができる。
　本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書にとり入れるものとする。 The cachexia treatment or prevention agent of the present invention can be used for the treatment or prevention of cachexia in patients with chronic diseases.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
　配列番号１：　ヒト・インターフェロン－βのアミノ酸配列 SEQ ID NO: 1: amino acid sequence of human interferon-β

Claims (4)

  1. 　インターフェロン－βを有効成分として含有する、悪液質の治療又は予防剤。 A cachexia treatment or prevention agent containing interferon-β as an active ingredient.
  2. 　前記インターフェロン－βは、ポリアルキレングリコールと共有結合している、請求項１記載の悪液質の治療又は予防剤。 The cachexia treatment or prevention agent according to claim 1, wherein the interferon-β is covalently bonded to polyalkylene glycol.
  3. 　前記ポリアルキレングリコールは、ポリエチレングリコールである、請求項２記載の悪液質の治療又は予防剤。 3. The cachexia treatment or prevention agent according to claim 2, wherein the polyalkylene glycol is polyethylene glycol.
  4. 　前記悪液質は、がん悪液質である、請求項１～３のいずれか一項記載の悪液質の治療又は予防剤。 The cachexia treatment or prevention agent according to any one of claims 1 to 3, wherein the cachexia is cancer cachexia.
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