JP2005232059A - Preventing/treating agent for hypoadiponectinemia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a preventing/treating agent for diseases caused by a low adiponectin state by a new mechanism of action. <P>SOLUTION: This preventing/treating agent for diseases caused by the low adiponectin state is provided by containing a compound having an anti-oxidation activity as an active ingredient. Since the preventing/treating agent elevates the production of the adiponectin, it is useful for the prevention and treatment of the diseases caused by the low adiponectin state such as hypoadiponectinemia, glucose tolerance disorder, diabetes mellitus, type 2 diabetes mellitus, insulin resistance syndrome, diabetic complications, hyperglycemia, arteriosclerosis, atherosclerosis, cardiovascular diseases, cerebrovascular accidents, vascular stenosis, peripheral vascular diseases, aneurysm, hyperlipidemia, hypercholesterolemia, obesity, or the like. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a preventive / therapeutic agent for diseases caused by a low adiponectin state, for example, hypoadiponectinemia, diabetes, arteriosclerosis and the like, which contains an antioxidative compound as an active ingredient.

Adiponectin is one of adipose tissue-derived endocrine factors that have been cloned in recent years (see Non-Patent Document 1), and it has been revealed that it functions as an important regulator of carbohydrate metabolism, lipid metabolism, and the like.
For example, adiponectin has been shown to increase sugar transport via IRS-1 signaling in muscles and the like, and further to increase fatty acid oxidation and clearance via a fatty acid transport protein to improve insulin resistance ( Non-patent document 2).
In addition, adiponectin suppresses the expression of adhesion molecules that increase in a TNF-α-dependent manner in vascular endothelial cells, inhibits adhesion between vascular endothelial cells and monocytes, reduces macrophage phagocytic ability and TNF-α production, It has been shown to have an anti-arteriosclerotic action, such as having an action of suppressing the growth of vascular smooth muscle cells by various growth factors (see Non-Patent Documents 3 to 6).

It has been shown that the low adiponectin state in the blood and the like caused by various causes is deeply related to the onset and progress of metabolic abnormalities such as abnormal carbohydrate metabolism and abnormal lipid metabolism, and diseases resulting therefrom.
For example, in patients with primary hypoadiponectinemia in humans due to genetic mutations, blood adiponectin levels are 25-30% of normal people, and most of these patients have arteries such as diabetes, myocardial infarction, angina He is a patient with sclerosis (see Non-Patent Document 7).
In adiponectin knockout mice, fatty acid transport protein-1 (FAT-1) in muscle is decreased, and systemic fatty acid clearance is decreased (see Non-Patent Document 2). Furthermore, adiponectin knockout mice exhibit strong insulin resistance and overt diabetes compared to wild-type mice in a short-term breeding with a high-fat and high-sucrose diet. In addition, adiponectin knockout mice have increased intimal smooth muscle proliferation after femoral artery abrasion, that is, intimal thickening, as compared to wild-type mice.
The blood concentration of adiponectin decreases in obese subjects, and conversely increases due to weight loss. In particular, the blood concentration decreases with the accumulation of visceral fat (see Non-Patent Document 8).
In arteriosclerotic disease and coronary artery disease, blood adiponectin is decreased independently of the degree of obesity (see Non-Patent Document 3).
The blood adiponectin concentration is reduced in proportion to the severity of diabetes in humans (see Non-Patent Document 9).
In monkeys, hypoadiponectinemia occurs before the onset of hyperinsulinemia and diabetes, which are indicators of insulin resistance, in the onset of obesity type 2 diabetes, which develops due to overeating and lack of exercise (see Non-Patent Document 10). ).
In humans and monkeys, blood adiponectin concentration strongly correlates with systemic insulin sensitivity calculated by the insulin clamp test (see Non-Patent Document 11).

A part has been clarified so far regarding the cause and mechanism of the decrease in the adiponectin concentration in the blood, the decrease in the expression level of adiponectin.
For example, one of the causes is the aforementioned gene mutation.
In addition, the obesity state induces a decrease in blood adiponectin concentration. This is because TNF-α whose expression is increased in hypertrophic adipocytes works in an autocrine / paracrine manner to suppress the expression of adiponectin gene. It is considered one of the beginnings (Non-patent Document 12).
However, there are still many unclear points regarding the cause and mechanism of the decrease in the expression level of adiponectin, and it is expected to clarify the mechanism.

As described above, since the low adiponectin state is deeply related to the onset and development of metabolic disorders such as abnormal carbohydrate metabolism and abnormal lipid metabolism and diseases resulting therefrom, adiponectin expression is enhanced in the blood and the like. Drugs that increase the concentration are important.
Examples of such drugs include PPAR (peroxisome proliferator-activated receptor) gamma agonists (see Non-Patent Document 12), but confirmation of safety such as side effects is necessary.
Attempts to introduce adiponectin recombinant proteins and adiponectin genes into living organisms using adenovirus vectors have also been conducted in animal experiments, but for practical use in terms of protein stability and safety, etc. There are many issues.
From the above, it is expected that the cause and mechanism of the decrease in the expression level of adiponectin will be clarified, and a drug that can safely improve the low adiponectin state by a new mechanism is expected.

On the other hand, regarding the prevention of type 2 diabetes, it is known that antioxidants are effective through the protective action of pancreatic β cells (see Non-Patent Documents 13 and 14). However, the involvement of an antioxidant in the expression of adiponectin is not disclosed.
Maeda et al., “Biochemical and Biophysical Research Communications” (USA), 1996, Vol. 221, p. 286-289 Maeda et al., “Nature Medicine” (UK), 2002, Vol. 8, p. 731-737 Ouchi et al., “Circulation”, (USA), 1999, Vol. 100, p. 2473-2476 Ouchi et al., “Circulation” (USA), 2000, 102, p. 1296-1301 Ouchi et al., “Circulation”, (USA), 2001, vol. 103, p. 1057-1063 Arita et al., “Circulation”, (USA), 2002, vol. 105, p. 2893-2898 Kondo et al., “Diabetes”, (USA), 2002, 51, p. 2325-2328 Arita et al., “Biochemical and Biophysical Research Communications” (USA), 1999, Vol. 257, p. 79-83 Hotta et al., “Arteriosclerosis, Thrombosis and Vascular Biology” (USA), 2000, Vol. 20, p. 1595-1599 Hotta et al., “Diabetes”, (USA), 2001, 50, p. 1126-1133 Weyer et al., “The Journal of Clinical Endocrinology and Metabolism, (USA), 2001, Vol. 86, p. 1930-1935. Maeda et al., “Diabetes”, (USA), 2001, 50, p. 2094-2099 Gorogawa et al., “Diabetes Research and Clinical Practice” (Netherlands), 2002, 51, p. 1-10 Kimoto et al., “Biochemical and Biophysical Research Communications” (USA), 2003, Vol. 303, p. 112-119

  The purpose of the present invention is to clarify the cause and mechanism of the decrease in the expression level of adiponectin, to provide a drug that can safely improve the low adiponectin state of mammals by a new mechanism, and to prevent diseases caused by the low adiponectin state It is to provide preventive and therapeutic agents.

  As a result of intensive studies to solve the above problems, the present inventors have clarified that oxidative stress such as reactive oxygen species is one of the causes of adiponectin production decrease, and a compound having an antioxidant action is adiponectin. As a result, the inventors have found that the production is increased and that it is highly effective in the prevention and treatment of diseases caused by a low adiponectin state, and the present invention has been completed.

That is, the present invention relates to the following (1) to (13).
(1) A prophylactic / therapeutic agent for a disease caused by a low adiponectin state comprising a compound having an antioxidant action as an active ingredient.
(2) The compound having an antioxidant action is represented by the general formula (I)

[Wherein R ¹ is a nitro group, a cyano group or a trihalo (lower) alkyl group, and R ² , R ³ and R ⁴ are the same or different and each represents a lower alkyl group] or a dihydropyridine compound or pharmaceutical The prophylactic / therapeutic agent according to the above (1), which is an acceptable salt thereof.
(3) The above compound (I) wherein the compound of the general formula (I) is isopropyl ester of 6-cyano-5-methoxycarbonyl-2-methyl-4- (3-nitrophenyl) -1,4-dihydropyridine-3-carboxylic acid ( 2) The preventive / therapeutic agent described.
(4) The preventive / therapeutic agent according to (1) above, wherein the compound having an antioxidant action is probucol and a derivative thereof.
(5) The prophylactic / therapeutic agent according to (1) above, wherein the compound having an antioxidant action is N-acetylcysteine and derivatives thereof.
(6) The prophylactic / therapeutic agent according to (1) above, wherein the compound having an antioxidant action is a NADPH oxidase inhibitor compound.
(7) The prophylactic / therapeutic agent according to (6) above, wherein the NADPH oxidase inhibitor compound is apocynin.
(8) The diseases caused by the low adiponectin state are hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications, hyperglycemia, arteriosclerosis, atherosclerosis, heart Any one of (1) to (7), wherein the disease is selected from the group consisting of vascular disease, cerebrovascular disorder, vascular stenosis, peripheral vascular disease, aneurysm, hyperlipidemia, hypercholesterolemia and obesity The preventive / therapeutic agent described.
(9) The preventive / therapeutic agent according to (8), wherein the disease caused by the low adiponectin state is hypoadiponectinemia.
(10) The prophylactic / therapeutic agent according to (8), wherein the disease caused by the low adiponectin state is diabetes.
(11) The prophylactic / therapeutic agent according to (8), wherein the disease caused by the low adiponectin state is arteriosclerosis.
(12) The preventive / therapeutic agent according to (8), wherein the disease caused by the low adiponectin state is hyperlipidemia.
(13) The preventive / therapeutic agent according to (8), wherein the disease caused by the low adiponectin state is obesity.

  Since the preventive / therapeutic agent of the present invention increases adiponectin production, diseases caused by a low adiponectin state such as hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications, high Useful as a prophylactic / therapeutic agent for glycemia, arteriosclerosis, atherosclerosis, cardiovascular disease, cerebrovascular disorder, vascular stenosis, peripheral vascular disease, aneurysm, hyperlipidemia, hypercholesterolemia or obesity It is.

  In the present invention, the compound having an antioxidative action prevents oxidation by a compound that is oxidizable under physiological conditions (for example, reactive oxygen species), prevents production of reactive oxygen species, or depends on an oxidized compound. The compound which suppresses the influence (oxidative stress etc.) on a biological body is shown. Examples of those embodiments include the embodiments described below.

An embodiment of a compound that prevents oxidation by a compound that is oxidizable under physiological conditions is an anti-oxidant in the present invention when the compound captures and decomposes a compound that is oxidizing in vitro, for example, an endogenous oxygen group. The compound has an oxidizing action. In this case, compounds with antioxidant effects of reactive oxygen species, for example, singlet oxygen ⁽¹ O _2), lipid peroxide (LOOH, LOO ^·), halogenated oxygen ^- capturing the like (ClO etc.), decomposition or Prevents oxidative damage of lipids, proteins, nucleic acids, etc. by binding to reactive metals. The compounds having an antioxidant action in the present invention classified into this embodiment include the compounds described in the following classifications (1) to (15), but are not limited thereto. Various publications and documents cited in the following classifications (1) to (15) are incorporated herein by reference.

(1) Dihydropyridine compound As a suitable thing among dihydropyridine compounds, the dihydropyridine compound (I) of general formula (I) shown by said (2) is mentioned, for example. The dihydropyridine compound (I) of the general formula (I) is already known, and is described in, for example, British Patent No. 2036722 and Japanese Patent Laid-Open No. 4-128228, which discloses an active oxygen production inhibitory action. However, there is no disclosure regarding prevention / treatment of diseases caused by a low adiponectin state. With respect to the dihydropyridine compound (I), the definitions of R ¹ , R ² , R ³ and R ⁴ and preferred examples thereof will be described in detail as follows.

Preferable examples of the “trihalo (lower) alkyl group” represented by R ¹ include lower groups described later in R ² , R ³ and R ^{4 that} are trisubstituted with halogen (eg, chlorine, bromine, fluorine, iodine). An alkyl group is exemplified, and a preferred example thereof is a trifluoroalkyl group, and a most preferred example is a trifluoromethyl group.
Preferable examples of the “lower alkyl group” represented by R ² , R ³ and R ⁴ include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, pentyl, isopentyl, neopentyl, 1- or 2 - methylbutyl, include alkyl groups having 1 to 6 carbon atoms, hexyl, and the like, and preferred examples thereof are C ₁ -C ₄ alkyl group, the most preferred example of R ² is an isopropyl group, R ³ and A most preferred example of each R ⁴ is a methyl group.

  Suitable salts of the above-mentioned dihydropyridine compounds are conventional non-toxic and pharmaceutically acceptable salts such as alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, and inorganic bases such as ammonium salts. And organic amine salts such as triethylamine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, N, N′-dibenzylethyleneamine, and hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. Inorganic acid salts and organic carboxylic acid salts such as formic acid, acetic acid, trifluoroacetic acid, maleic acid and tartaric acid, and sulfonic acid addition salts such as methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid, and arginine and aspartic acid Basic or acidic amino acids such as glutamic acid and Salts or acid addition salts with bases Tsu and the like.

  Among the above-mentioned dihydropyridine compounds (I), particularly preferred is isopropyl ester of 6-cyano-5-methoxycarbonyl-2-methyl-4- (3-nitrophenyl) -1,4-dihydropyridine-3-carboxylic acid. And the compound represented by the generic name “nilvadipine”.

(2) Probucol and its derivatives The official chemical name of probucol is 4,4-[(1-methylethylidene) bis (thio)] bis [2,6-bis (1,1-dimethylethylethyl) phenol]. U.S. Pat. No. 3,862,332. US Pat. No. 5,262,439 also discloses soluble analogs of probucol in which one or both hydroxyl groups of probucol are substituted with ester groups that confer water solubility to the compound, such as mono- or di-succinic acid esters of probucol, Glutaric acid ester, adipic acid ester, subesic acid ester, sebacic acid ester, azelaic acid ester, maleic acid ester and the like are disclosed. Also disclosed are probucol derivatives that are mono- or di-esters, wherein the ester comprises an alkyl or alkenyl group having a functional group selected from the group consisting of carboxylic acid groups. U.S. Pat. No. 5,155,250 discloses 2,6-dialkyl-4-silylphenol. US Pat. No. 5,608,095 discloses alkylated-4-silylphenols.

(3) Dithiocarbamates Dithiocarbamates and related compounds are widely described in the patent and academic fields, for example, “Dithiocarbamates and related compounds”, Thorn et al., Elsevier, New York, 1962, etc.
Dithiocarbamates are members of the general class of compounds known as thiol antioxidants and are compounds having the structure of A-SC (S) -B, also referred to as carbodithiol, carbodithiolate. A and B may be any group that does not adversely affect the effectiveness or toxicity of the compound.
In an alternative embodiment, one or both of the sulfur atoms in the dithiocarbamate may be replaced with a selenium atom.

In one embodiment, A is hydrogen or a pharmaceutically acceptable cation including but not limited to: sodium, potassium, calcium, magnesium, aluminum, zinc, copper, cobalt, nickel, or Metal cations such as cadmium; salt-forming organic acids typified by carboxylic acids such as acetic acid, oxalic acid, tartaric acid, benzoic acid; or a nitrogen heterocycle or a part of the formula NR ⁵ R ⁶ R ⁷ R ⁸ , but R ⁵ , R ⁶ , R ⁷ and R ⁸ are independently hydrogen, C _1-6 linear, branched or (in the case of C _4-6 ) cyclic alkyl, hydroxy (C _1-6 ) alkyl (where 1 or The above hydroxyl groups are located on any carbon atom), or aryl, cations formed from ammonia or other nitrogen bases, and the like.

In another embodiment, A is a physiologically cleavable leaving group that can be cleaved in vivo from the molecule to which it is attached, such as acyl, alkyl, phosphate, sulfate or sulfonate. is there.
In one embodiment, B is for example alkyl, alkenyl, alkynyl, alkaryl, aralkyl, haloalkyl, haloalkenyl, haloalkynyl, aryl, hydrogen, C _1-6 alkoxy-C _1-10 alkyl, C _1-6 alkylthio —C _1-10 alkyl, NR ⁹ R ¹⁰ , — (CHOH) _n CH ₂ OH, where n is 0, 1, ₂ , 3, 4, 5 or 6, alkylacetyl, alkylpropionyl and alkylbutyryl -(CH ₂ ) _n CO ₂ R ¹⁴ , or hydroxy (C _1-6 ) alkyl- (wherein one or more hydroxyl groups are located on any carbon atom), and the like. R ¹⁴ is hydrogen or lower alkyl.

In another embodiment, B is NR ⁹ R ¹⁰ , where R ⁹ and R ¹⁰ are independently alkyl; — (CHOH) _n (CH ₂ ) _n OH; — (CH ₂ ) _n CO ₂ R ¹⁴ , — (CH ₂ ) _n CO ₂ R ¹⁵ ; hydroxy (C _1-6 ) alkyl-; alkenyl (eg, vinyl, allyl and CH ₃ CH═CH—CH ₂ —CH ₂ etc.); alkyl (CO ₂ H ), Alkenyl (CO ₂ H), alkynyl (CO ₂ H), or aryl, where the aryl group is substituted with, for example, a NO ₂ , CH ₃ , t-butyl, CO ₂ H, halo or p-OH group. R ⁹ and R ¹⁰ may be combined to form a bridge such as — (CH ₂ ) _m —, where m is 3, 4, 5, 6, 7, 8, 9 Or 10 and R ¹⁵ is acetyl, propionyl and butyryl. Alkyl, aryl, alkaryl or aralkyl, including

In yet another embodiment, B is a heterocyclic or alkyl heterocyclic group. The heterocycle may be optionally partly or wholly hydrogenated, and examples thereof include phenazine, phenothiazine, pyridine and dihydropyridine.
In yet another embodiment, B is a residue of a pharmaceutically active compound or drug. As used herein, the term drug refers to a substance used internally or externally as a medicament for the prevention or treatment of a disease or disorder. At this time, the -C (S) SA group can be directly bonded to the drug or can be bonded to the drug through an appropriate linking moiety.

In another embodiment, the dithiocarbamate is an amino acid derivative having an AO ₂ C—R ¹¹ —NR ¹² —C (S) SA structure, wherein R ¹¹ is a divalent B moiety that is a link moiety, or An internal residue of a natural amino acid (for example, CH ₃ CH for alanine, CH _{2 for} glycine, CH (CH ₂ ) ₄ NH _{2 for} lysine, etc.), and R ¹² is hydrogen or lower alkyl. .
B may also be a polymer having one or more dithiocarbamate groups attached directly or through a suitable linking moiety. The dithiocarbamate is preferably released from the polymer over a suitable period of time to provide a therapeutic effect under in vivo conditions. In a preferred embodiment, the polymer itself is also biodegradable in vivo and degrades within a period of 1 hour to several weeks depending on the application.
Many degradable polymers are known. For example, polymers and copolymers of amino acids such as lysine, arginine; poly (α-hydroxy acids), eg polylactic esters, including polylactic acid, polyglycolic acid, poly (lactide-co-glycolide), polyanhydrides, albumin or collagen And polysaccharides containing sugar units such as lactose, and peptides such as polycaprolactone, proteins, nucleoproteins, lipoproteins, glycoproteins, synthetic and natural polypeptides, and polyamino acids. The polymer may be a random or block copolymer.

B also groups to enhance the water solubility of the dithiocarbamate, for example - a lower alkyl -O-R ^13, provided that R ¹³ is -PO ₂ (OH) ^- a M ⁺ or PO ₃ (M ⁺⁾ _2, M ⁺ is It is a pharmaceutically acceptable _{cation; -C (O) (CH 2} ) 2 CO 2 - M + or -SO ₃ ^- M ^+; - lower alkylcarbonyl - lower alkyl; - carboxy lower alkyl; - lower alkylamino - N, N-disubstituted amino lower alkyl, wherein the substituents are each independently lower alkyl; pyridyl-lower alkyl; imidazolyl-lower alkyl-; imidazolyl-Y-lower alkyl, where Y is thio or amino Morpholinyl-lower alkyl; pyrrolidinyl-lower alkyl; thiazolinyl-lower alkyl; piperidinyl-lower alkyl; morpholinyl N-pyryl; piperazinyl-lower alkyl; N-substituted piperazinyl-lower alkyl, where the substituent is lower alkyl; triazolyl-lower alkyl; tetrazolyl-lower alkyl; tetrazolylamino-lower alkyl; or thiazolyl -Lower alkyl may be sufficient.
An alternative embodiment may be a dimer such as BC (S) S-SC (S) -B.

In the description in the classification of the (3), the term alkyl, unless otherwise specified, saturated linear C ₁ -C _10, branched or cyclic (C ₅ or more) a hydrocarbon. The alkyl group may be optionally substituted on any carbon with one or more moieties selected from the group consisting of hydroxyl, amino, and mono- or disubstituted amino, wherein the substituent is independently alkyl Aryl, alkaryl, or aralkyl; aryl, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, as known to those skilled in the art For example, as described in Greene et al., "Protecting groups in organic synthesis" John Wiley and Sons, 2nd edition, 1991, etc., it may or may not be protected.

In the description in the classification (3), the term alkenyl indicates a C ₂ -C ₁₀ linear or branched hydrocarbon having at least one double bond unless otherwise specified. The term alkynyl, unless otherwise specified, refers to a C ₂ -C ₁₀ straight or branched hydrocarbon having at least one triple bond. The term alkaryl refers to an aryl group having at least one alkyl substituent. The term aralkyl refers to an alkyl group having at least one aryl substituent. The term halo (alkyl, alkenyl, or alkynyl) denotes an alkyl, alkenyl, alkynyl group in which at least one of the hydrogens in the group has been replaced with a halogen atom.

In the description in the category (3), the term aryl indicates phenyl, biphenyl, or naphthyl, preferably phenyl, unless otherwise specified. The aryl group may be optionally substituted with one or more substituents selected from the group consisting of: alkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, Cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, CO ₂ H or a pharmaceutically acceptable salt thereof, CO ₂ (alkyl, aryl, alkaryl or aralkyl), and glucamine. These may or may not be protected by a protecting group as described above.

In the description in the classification (3), the term alkoxy indicates a moiety of the —O-alkyl structure unless otherwise specified. The term acyl refers to a group having the formula -C (O) R, where R is an alkyl, aryl, alkaryl, or aralkyl group. The term “heterocyclic” refers to an aromatic moiety comprising at least one sulfur, oxygen or nitrogen in the aromatic ring. The heterocyclic group may be optionally substituted in the same manner as aryl. The heterocyclic group may be partially or fully hydrogenated if desired. The term hydroxyalkyl represents any C ₁ -C ₆ alkyl group in which at least one is substituted with a hydroxy group of hydrogen bonded to the carbon atom.

In the description in the classification (3), a pharmaceutically acceptable cation is an organic or inorganic moiety that bears a positive charge and can be administered together with a drug, for example, as a counter cation in a salt. The term physiologically cleavable leaving group refers to a moiety that can be cleaved in vivo from an attached molecule, such as organic or inorganic anions, pharmaceutically acceptable cations, acyl (acetyl, propionyl and Including butyryl, including but not limited to (alkyl) C (O)), alkyl, phosphate, sulfate, sulfonate, and the like.
In the description in the classification (3), the “link moiety” means, for example, alkyl, alkenyl, alkynyl, aryl, polyalkyleneoxy (for example, — [(CH ₂ ) _n O—] _n —), —C _{1- A} divalent group that connects two chemical residues, such as ₆ alkoxy-C _1-10 alkyl-, -C _1-6 alkylthio-C _1-10 alkyl-, or-(CHOH) _n CH ₂ OH, etc. is there.

  Suitable dithiocarbamates include, for example, N-methyl-N-ethyldithiocarbamate, hexamethylenedithiocarbamate, sodium di (β-hydroxyethyl) dithiocarbamate, N-methylsodium-N-cyclobutylmethyldithiocarbamate, N-allyl -N-cyclopropylmethyl-dithiocarbamate sodium, cyclohexylamyl dithiocarbamate, dibenzyl-dithiocarbamate, sodium dimethylenedithiocarbamate, various pentamethylenedithiocarbamate salts, sodium pyrrolidine-N-carbodithioate, sodium piperidine-N-carbothioate , Morpholine-sodium N-carbothioate, α-furfuryl dithiocarbamate, imidazoline dithiocarbamate and the like.

(4) Radicut (Edaravone, MCI-186) and derivatives thereof The official name of Radicut is 3-methyl-1-phenyl-pyrazolin-5-one, which is described in European Patent Application No. 0208874. Radicut is a radical scavenger that has been developed as an agent for improving cerebral circulation / brain metabolism disorders. Examples of the derivatives include a group of compounds disclosed in the European patent application.

(5) N-acetylcysteine and derivatives thereof N-acetylcysteine is an N-acetylated derivative of cysteine. Cysteine is an amino acid with one chiral carbon atom and exists as an L-enantiomer, D-enantiomer, or a racemic mixture of L- and D-enantiomers. Similarly, N-acetylcysteine also has L-enantiomers, D-enantiomers, racemic mixtures of L- and D-enantiomers, and compositions in which one of the enantiomers is enantiomerically enriched. Any of these forms of N-acetylcysteine is included in the compounds having an antioxidant action of the present invention. Here, the composition in which one of the enantiomers is enantiomerically enriched means a composition containing at least 95% by weight, preferably at least 97% by weight, of one enantiomer of the compound. One embodiment is exemplified by one isomer of thioester or thioether of N-acetylcysteine or a salt thereof, preferably L-enantiomer.
N-acetylcysteine and its derivatives are described in, for example, WO95 / 26719, and all of N-acetylcysteine and its derivatives described in this patent publication are included in the compound having an antioxidant activity of the present invention. It is.

(6) Enzymes having antioxidant activity Examples of this class of compounds include catalase, superoxide dismutase (SOD), selenium glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and the like.

(7) Thiols Examples of this class of compounds include dithiothreitol, 2-mercaptomethanol, N-2-mercaptopropionylglycine, and ovothiol.

(8) Iron ion chelators Examples of this class of compounds include desferrioxamine (also referred to as deferoxamine or desferrol).

(9) Pyrazolopyrimidine compounds Pyrazolopyrimidine compounds interfere with xanthine oxidase. Examples of this class of compounds include allopurinol and oxypurinol.

(10) Aminosteroids Examples of this class of compounds include 21-aminosteroids referred to as lazaroids (U74006F).

(11) Vitamins having antioxidant activity As this class of compounds, for example, vitamin C group (eg ascorbic acid etc.), vitamin E group (eg tocopherol, tocotrienol etc.), alpha riboic acid, or prodrugs thereof, or Analogs and the like.

(12) Flavonoids such as polyphenols Examples of this class of compounds include catechin, anthocyanin, rutin, chlorogenic acid, quercetin, tannin, and isoflavone.

(13) Carotenoids Examples of this class of compounds include β-carotene, canthaxanthin, astaxanthin, cryptoxanthin, lutein and the like.

(14) Other compounds having physiological antioxidant action Examples of this class of compounds include glutathione, cysteine, bilirubin, uric acid, quinone, ubiquinone, metal-binding protein, and the like.

(15) Antioxidant compounds used as additives, etc. This class of compounds includes, for example, BHA (dibutylhydroxytoluene), BHT (dibutylhydroxyanisole), Torolox (trade name), disodium EDTA, erythorbine Acid, isopropyl citrate, sulfur dioxide, propolis extract, rosemary extract, mannitol, dimethylthiourea (DMTU), butyl-α-phenylnitrone (BPN), hypoxanthine, xanthine, buthionine sulfoximine, maleic acid Examples include diethyl.

Embodiments of the compound that prevents the production of reactive oxygen species include a compound that interferes with cell function related to the production of reactive oxygen species, a compound that interferes with an enzyme related to the production of reactive oxygen species, and the like. For example, this embodiment includes compounds that suppress the activation of cells that produce reactive oxygen species (such as leukocytes) (for example, steroidal anti-inflammatory compounds, non-steroidal anti-inflammatory compounds, etc.). Further, as one of the active oxygen production systems, compounds that suppress the generation of active oxygen due to NADPH oxidase activation by angiotensin II stimulation (for example, ACE inhibitors, angiotensin II receptor blockers, NADPH oxidase inhibitors, etc.) include.
The compounds having antioxidant activity in the present invention classified into this embodiment include the compounds described in the following classifications (16) to (20), but are not limited thereto.

(16) Steroidal anti-inflammatory compounds This class of compounds includes, for example, hydrocortisone, hydroxyl triameinolone, α-methyl dexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, dezonide, dezoxymethazone, dezoxycorticosterone Acetoate, dexamethasone, dichlorizone, diflorazone diacetate, diflucortron valerate, fluadrenolone, fluchlorolone acetonide, fluorocortisone, flumethasone pivalate, norocinolone acetonide, fluoxynonide, flucortin butyl ester, Fluocortron, fluprednidene (fluprednidene) acetate, flulandrenolone, halcinonide, hydrocortisone acetate, hydrocor Zombylate, methylprednisolone, trianthinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluoroson diacetate, fluradrenolone acetonide, medrizone, ancinafel, ancinafide, betamaison, chloroprednisone, chlorprednisone acetate, crocorterone, Creacinolone, dichlorizone, diflupredionate, fluchloronide, flunizolide, fluorometallone, fluperolone, fluprecurnisone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, parameterzone, prednisolone, pradozone, beclomethasone dipropionate , Trianthinolone, cortico Steroid, and the like.

(17) Non-steroidal anti-inflammatory compounds Examples of this class of compounds include piroxicam, isoxicam, tenoxicam, sudoxicam, CP-14,304, aspirin, disarside, benolylate, trilysate, saphapurine, sorprine, diflunisal, fendsal, diclofenac, Fenclofenac, indomethacin, sulindac tolmethine, isoxepac, florenac, thiopinac, zidometacin, acemetacin, fenthiacac, zomepilac, clidanac, oxepinac, felbinac, mefenamic, meclofenamic, flufenamic, niflumic, torfenamic acid, profenamic acid, profenamic acid, profenamic acid , Beoxaprofen, flurbiprofen, ketoprofen, fenoprofen, Fenbufen, indoprofen, pyrprofen, carprofen, oxaprozin, pranoprofen, myloprofen, thixaprofen, suprofen, aluminoprofen, thiaprofen, phenylbutazone, oxyphenbutazone, feprazone, azapropazone, trimethasone, etc. It is done.

(18) ACE Inhibiting Compounds Examples of this class of compounds include enalapril maleate, setapril, captopril, imidapril hydrochloride, cilazapril, delapril hydrochloride, lisinopril, quinapril hydrochloride, alacepril, trandolapril, perindopril erbumine, benazepril hydrochloride, delapril hydrochloride Etc.

(19) Angiotensin II receptor blockers Examples of this class of compounds include losartan, candesartan cilexetil, valsartan, telmisartan, eprosartan, zolasartan, and the like.

(20) NADPH oxidase-inhibiting compounds Examples of this class of compounds include chemical formula (II)

A compound represented by the formula (apocynin) or chemical formula (III)

(Diphenyleneiodinium (DPI)) and the like. Preferably, the NADPH oxidase inhibitor compound is apocynin.

  Moreover, as an embodiment of a compound that suppresses the influence (oxidative stress etc.) on the living body by an oxidized compound, a compound that interferes with an enzyme (for example, JNK, p38MAP kinase, NFκB, PI3K, etc.) activated by oxidative stress, etc. Is included. The compounds having antioxidant activity in the present invention classified in this embodiment include the compounds described in the following classifications (21) to (24), but are not limited thereto.

(21) JNK-inhibiting compounds Examples of this class of compounds include anthrapyrazolone compounds such as SP600125.

(22) p38MAP kinase inhibitory compounds Examples of this class of compounds include SB203580, SB202190, SB239063, SB220025, and SC68376.

(23) NFκB inhibitory compounds Examples of this class of compounds include NFκB decoys and SP100030.

(24) PI3K inhibitor compound Examples of this class include wortmannin, LY294002, and quercetin.

  Further, in addition to the above-mentioned compounds that have been reported to have an antioxidant action, compounds that are newly found to have an antioxidant action in the future, whether known or new, can be used in the preventive / therapeutic agents of the present invention. It is a compound which has this.

  As the compound having an antioxidative action that can be used in the preventive / therapeutic agent of the present invention, any of the above-mentioned compounds can be used, but preferably a dihydropyridine compound, probucol and a derivative thereof, N-acetylcysteine And derivatives thereof or NADPH oxidase-inhibiting compounds.

  The compound having an antioxidative action contained in the preventive / therapeutic agent for diseases caused by the low adiponectin state in the present invention may be one type or two or more types. Here, when using the compound which has 2 or more types of antioxidant action, you may administer each with respect to an administration subject simultaneously, and may administer at a time difference. At this time, two or more kinds of compounds having an antioxidant action may be prepared as separate preparations or a single preparation.

Further, the preventive / therapeutic agent for diseases caused by the low adiponectin state of the present invention is an insulin sensitivity enhancer, a sugar absorption inhibitor, a biguanide drug, an insulin secretagogue, insulin or an insulin analog, a glucagon receptor antagonist, insulin Receptor kinase inhibitor, tripeptidyl peptidase II inhibitor, dipeptidyl peptidase IV inhibitor, protein tyrosine phosphatase-1B inhibitor, glycogen phosphorylase inhibitor, glucose-6-phosphatase inhibitor, fructose-bisphosphatase inhibitor, pyruvate Dehydrogenase inhibitor, hepatic gluconeogenesis inhibitor, D-kaileusitol, glycogen synthase kinase-3 inhibitor, glucagon-like peptide-1, glucagon-like peptide-1 analog, glucagon-like Peptide-1 agonist, amylin, amylin analog, amylin agonist, aldose reductase inhibitor, glycation end product inhibitor, protein kinase C inhibitor, γ-aminobutyric acid receptor antagonist, sodium channel antagonist, N-acetylated − α-linked-acid-dipeptidase inhibitor, insulin-like growth factor-I, platelet-derived growth factor, platelet-derived growth factor analog, epidermal growth factor, nerve growth factor, carnitine derivative, uridine, 5-hydroxy-1-methyl hydantoin, EGB-761, Bimokuromoru, sulodexide, Y-128, a hydroxymethyl glutaryl coenzyme A reductase inhibitors, fibrate compounds, beta ₃ - adrenergic receptor agonists, acyl-coenzyme A: cholesterol acyltransferase Element inhibitor, thyroid hormone receptor agonist, cholesterol absorption inhibitor, lipase inhibitor, microsomal triglyceride transfer protein inhibitor, lipooxygenase inhibitor, carnitine palmitoyltransferase inhibitor, squalene synthase inhibitor, low-density lipoprotein receptor potentiator Drugs, nicotinic acid derivatives, bile acid adsorbents, sodium-conjugated bile acid transporter inhibitors, cholesterol ester transfer protein inhibitors, appetite suppressants, angiotensin converting enzyme inhibitors, neutral endopeptidase inhibitors, angiotensin II receptor antagonists , endothelin converting enzyme inhibitors, endothelin receptor antagonists, diuretics, calcium antagonists, vasodilator antihypertensives, sympatholytic agents, central antihypertensives, alpha ₂ - adrenergic receptor agonists Antiplatelet agents, uric acid production inhibitor, a uricosuric agent and one member selected from the group consisting of urinary alkalization drug or two or more agents and can also be used in combination. At this time, the prophylactic / therapeutic agent for diseases caused by the low adiponectin state of the present invention and the aforementioned agent may be administered simultaneously to the administration subject, or may be administered with a time difference. Moreover, the preventive / therapeutic agent for a disease caused by the low adiponectin state of the present invention and the above-mentioned agent may be separate preparations or a single preparation.

In the present invention, the low adiponectin state is characterized by, for example, obesity or visceral fat accumulation due to overeating or lack of exercise, etc., obesity or visceral fat accumulation due to gene mutation, or adiponectin production decrease caused by gene mutation State.
This low adiponectin state includes, for example, a decrease in adiponectin gene expression in cells, tissues, or living bodies; a decrease in adiponectin protein production in cells, tissues, or living bodies; a decrease in adiponectin protein concentration in plasma, tissues, or living bodies; Examples thereof include a decrease in adiponectin activity in medium, tissue, or living body; an increase in clearance of adiponectin protein in a cell, tissue, living body, or the like.

  In the present invention, a disease caused by a low adiponectin state refers to a disease characterized in that a part or all of causes such as onset, progress or formation of the disease are in a low adiponectin state.

  Diseases caused by the low adiponectin state include, for example, hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome (insulin receptor abnormality, Rabson-Mendenhall syndrome, lebriconism, Koverling-Dunnigan syndrome, Sepip Syndrome, Lawrence syndrome, Cushing syndrome, acromegaly, etc.), glomerular disease (eg, diabetic glomerulosclerosis), diabetic complications (eg, diabetic gangrene, diabetic arthropathy, diabetic glomerulosclerosis, diabetes) Skin disorders, diabetic neuropathy, diabetic cataract, diabetic nephropathy, diabetic retinopathy, diabetic osteopenia), hyperglycemia (eg characterized by abnormal glucose metabolism such as eating disorders) , Osteoporosis (especially diabetic or pre-sugar Due to disease symptoms), glaucoma, polycystic ovary syndrome, arteriosclerosis, atherosclerosis, cardiovascular disease (angina, heart failure, myocardial infarction, etc.), cerebrovascular disorder (eg cerebral infarction, stroke, etc.) , Stenosis (eg after percutaneous angioplasty), peripheral vascular disease, aneurysm, xanthoma, restenosis after PCTA, hypertension, pulmonary hypertension, renal failure, nephritis, cachexia (eg cancer / Tuberculosis, endocrine diseases and chronic diseases such as AIDS, progressive weight loss due to lipolysis, muscle degeneration, anemia, edema, anorexia, etc.), hyperlipidemia, hypercholesterolemia, obesity, etc. Preferably hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications, hyperglycemia, arteriosclerosis, atherosclerosis, cardiovascular disease, cerebrovascular disorder, vascular narrowing , Peripheral vascular disease, aneurysm, hyperlipidemia, include hypercholesterolemia or obesity.

  In the present invention, hypoadiponectinemia refers to a disease characterized by a decrease in adiponectin protein concentration and / or adiponectin activity in plasma, particularly in a low adiponectin state.

The prophylactic / therapeutic agent for a disease caused by a low adiponectin state of the present invention can be used before or after the onset of the disease.
Among them, the present invention is particularly excellent when the living body (for example, mammals such as humans and mice) is in a low adiponectin state but has not yet developed a disease caused by the low adiponectin state. By administering a prophylactic / therapeutic agent, the low adiponectin state can be improved and the onset of the disease can be prevented.

  The preventive / therapeutic agent for diseases caused by a low adiponectin state according to the present invention is applied to mammals including humans, capsules, microcapsules, tablets, granules, powders, troches, pills, ointments, suppositories. In addition, it can be administered orally or parenterally in the form of pharmaceutical preparations known per se, such as injections and syrups.

The agent for preventing or treating a disease caused by a low adiponectin state according to the present invention is an excipient such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, such as cellulose, methylcellulose. , Hydroxymethylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants such as stearin Lubricants such as magnesium acid, aerosil, talc, sodium lauryl sulfate, etc., and taste-masking agents such as citric acid, menthol, glycine, such as sodium benzoate Preservatives such as um, sodium bisulfite, methyl paraben, propyl paraben, for example, stabilizers such as citric acid, sodium citrate, acetic acid, suspending agents such as methyl cellulose, polyvinyl pyrrolidone, aluminum stearate, such as hydroxypropyl Manufactured by a method known per se using various organic or inorganic carriers commonly used for formulation such as a dispersant such as methyl cellulose, a diluent such as water, and a base wax such as cacao butter, white petrolatum, polyethylene glycol and the like. be able to.
The dosage of various agents containing the compound having an antioxidative action of the present invention as an active ingredient depends on the kind of the compound used as the active ingredient and / or the weight and / or age of the patient and / or the degree of the disease and further the administration. Usually, 0.5 to 1000 mg, preferably 1 to 500 mg is administered per day by oral administration, although it varies depending on various factors such as the route. An effective single dose is selected within the range of 0.01-20 mg / kg of patient body weight, preferably 0.05-2 mg.

  In addition, by applying the present invention and examining the antioxidant action of candidate compounds, it is possible to screen for compounds that are effective in the prevention and treatment of diseases caused by the low adiponectin state. Here, as a method for “inspecting the antioxidant activity” of a candidate compound, for example, the step of measuring the activity of a candidate compound that prevents oxidation by a compound that is oxidative under physiological conditions, and “preventing production of reactive oxygen species” Examples of the method include one or two or more steps such as “the step of measuring the activity” and “the step of measuring the activity of preventing the influence of the oxidized compound on the living body”.

An embodiment of the step of measuring the activity of preventing the candidate compound from being oxidized by a compound that is oxidizing under physiological conditions includes, for example, a compound in which the candidate compound is oxidizing in vitro, such as singlet oxygen ( ¹ O ₂ ), lipid peroxides (LOOH, LOO ^·), halogenated oxygen (ClO ^-, etc.), oxygen, superoxide anion, hydrogen peroxide, superoxide radical, Ripookishido group, capturing the active oxygen species such as hydroxyl groups, decomposition The process of measuring the activity to be equal.
As an embodiment of the step of measuring the activity that hinders the production of reactive oxygen species, the step of measuring the activity that hinders the cell function related to the production of reactive oxygen species, the activity that hinders the enzyme etc. related to the production of reactive oxygen species are measured Process etc. are included. As one of the active oxygen production systems, generation of active oxygen by NADPH oxidase activation by angiotensin II stimulation is exemplified, and therefore, a method for measuring an activity that prevents the binding of angiotensin to an angiotensin II receptor, an activity that prevents NADPH oxidase activity The method of measuring can be used.
Examples of the step of measuring the activity that prevents the influence of the oxidized compound on the living body include the step of measuring the activity that blocks the enzyme activated by oxidative stress (for example, JNK, p38MAP kinase, NFκB, PI3K, etc.). I can do it.

By combining the method for investigating the antioxidant activity of the above-mentioned candidate compounds and the method for examining the effects of candidate compounds on adiponectin promoter activity, it is possible to make more effective compounds effective in the prevention and treatment of diseases caused by low adiponectin status Screening.
The compound isolated by this screening becomes a candidate for a compound that promotes the expression of an adiponectin gene, and a candidate for a compound that is effective for prevention / treatment of a disease caused by a low adiponectin state.
EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

(Experimental example 1)
Enhancement of oxidative stress and suppression of antioxidant enzyme expression in adipose tissue of KKAy mice (test method)
Plasma, liver and visceral fat (periorbital fat, mesenteric fat) were removed from 7-week-old and 13-week-old female C57BL / 6 mice (normal mice) and female KKAy mice (obese / diabetic mice), respectively, and weighed. And then frozen with liquid nitrogen.
The glucose concentration in plasma was measured using L type Wako Glu2 (manufactured by Wako Pure Chemical Industries, Ltd.). Plasma insulin concentration was measured using Shionoria insulin RIA kit (manufactured by Shionogi & Co.). The plasma concentration of triacylglycerol was measured using L-type Wako TG • H (manufactured by Wako Pure Chemical Industries, Ltd.). The amount of lipid peroxide in plasma was measured using LPO Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). For the amount of lipid peroxide in tissue, first, frozen tissue pieces were ultrasonically disrupted in 50 mM Tris (hydroxymethyl) aminomethane-1.15% potassium chloride buffer (pH 7.4) and centrifuged at 3000 rpm for 15 minutes. Then, the amount of lipid peroxide in the supernatant was measured using the kit described above. The measured value was corrected by the protein concentration in the supernatant. The protein concentration was measured using a BCA protein assay kit (Pierce).
For the measurement of mRNA expression level in tissue, first, frozen tissue pieces were homogenized in 1 mL of RNA-STAT60 solution (manufactured by TEL-TEST “B”) using a Polytron homogenizer, and RNA was extracted according to the protocol. CDNA was synthesized using ThermoScript RT-PCR System (manufactured by Invitrogen) using 400 ng of extracted RNA as a template. The expression level of various genes in cDNA was measured using a light cycler (manufactured by Roche Diagnostics) by real-time PCR, and calculated as a relative value corrected with the value of cyclophilin mRNA expression level as a control. did. The PCR conditions in the measurement are 95 ° C., 10 minutes → (95 ° C., 15 seconds → 65 ° C., 5 seconds → 72 ° C., 15 seconds) × 40 cycles, and the sequence of the PCR primer of each gene used for the measurement Is as follows.

Adiponectin Fw primer: 5'-GATGGCAGAGATGGCACTCC-3 '[SEQ ID NO: 1]
Rv primer: 5'-CTTGCCAGTGCTGCCGTCAT-3 '[SEQ ID NO: 2]
TNF alpha
Fw primer: 5'-GCCACCACGCTCTTCTG-3 '[SEQ ID NO: 3]
Rv primer: 5'-GGTGTGGGTGAGGAGCA-3 '[SEQ ID NO: 4]
PAI-1
Fw primer: 5'-TCAGCCCTTGCTTGCCTCAT-3 '[SEQ ID NO: 5]
Rv primer: 5'-GCATAGCCAGCACCGAGGA-3 '[SEQ ID NO: 6]
PPAR gamma
Fw primer: 5′-CCAGAGTCTGCTGATCTGCG-3 ′ [SEQ ID NO: 7]
Rv primer: 5'-GCCACCTCTTTGCTCTGCTC-3 '[SEQ ID NO: 8]
gp91phox
Fw primer: 5'-TTGGGTCAGCACTGGCTCTG-3 '[SEQ ID NO: 9]
Rv primer: 5′-TGGCGGTGTGCAGTGCTATC-3 ′ [SEQ ID NO: 10]
p22phox
Fw primer: 5'-GTCCACCATGGAGCGATGTG-3 '[SEQ ID NO: 11]
Rv primer: 5′-CAATGGCCAAGCAGACGGTC-3 ′ [SEQ ID NO: 12]
p67phox
Fw primer: 5'-CTGGCTGAGGCCATCAGACT-3 '[SEQ ID NO: 13]
Rv primer: 5'-AGGCCACTGCAGAGTGCTTG-3 '[SEQ ID NO: 14]
p47phox
Fw primer: 5'-GATGTTCCCCATTGAGGCCG-3 '[SEQ ID NO: 15]
Rv primer: 5'-GTTTCAGGTCATCAGGCCGC-3 '[SEQ ID NO: 16]
p40phox
Fw primer: 5′-GCCGCTATCGCCAGTTCTAC-3 ′ [SEQ ID NO: 17]
Rv primer: 5′-GCAGGCTCAGGAGGTTCTTC-3 ′ [SEQ ID NO: 18]
Superoxide dismutase Fw primer: 5'-CAGCATGGGTTCCACGTCCA-3 '[SEQ ID NO: 19]
Rv primer: 5′-CACATTGGCCACACCGTCCT-3 ′ [SEQ ID NO: 20]
Catalase Fw primer: 5′-CCAGCGACCAGATGAAGCAG-3 ′ [SEQ ID NO: 21]
Rv primer: 5'-CCACTCTCTCAGGAATCCGC-3 '[SEQ ID NO: 22]
Cyclophilin Fw primer: 5'-CAGACGCCACTGTCGCTTT-3 '[SEQ ID NO: 23]
Rv primer: 5'-TGTCTTTGGAACTTTGTCTGCAA-3 '[SEQ ID NO: 24]
The results are shown in [FIG. 1] to [FIG. 18].

(result)
As is apparent from [FIG. 1] and [FIG. 2], the body weight and visceral fat weight of KKAy mice were already significantly increased at 7 weeks of age as compared with C57BL / 6 mice. In addition, as is clear from [FIG. 3] and [FIG. 4], KKAy mice have already shown significantly higher plasma insulin and plasma triacylglycerol concentrations from 7 weeks of age than C57BL / 6 mice. Insulin resistance and hyperlipidemia were confirmed. Furthermore, it was revealed that lipid peroxide in plasma was elevated and oxidative stress was already increased at 7 weeks of age (see [FIG. 5]). Furthermore, when lipid peroxides in adipose tissue were measured, it was found that oxidative stress was also increased in adipose tissue (see [FIG. 6]). However, since hyperglycemia has not yet occurred at the age of 7 weeks (see [FIG. 7]), it was confirmed that oxidative stress is enhanced by obesity and hyperlipidemia.
When the expression levels of various genes in adipose tissue were measured, the expression of adiponectin was reduced in KKAy mice, as is clear from (see [FIG. 8]). At this time, the expression of PPAR gamma, which is one of the regulators of adiponectin transcription, also decreased from the 7th week of age (see [FIG. 9]). On the contrary, the expression of TNF alpha and PAI-1 which are inflammatory cytokines was increased (see [FIG. 10] and [FIG. 11]). NADPH oxidase is composed of five subunits, gp91phox, p22phox, p67phox, p47phox, and p40phox, and the expression of all these subunits was increased in the adipose tissue of KKAy mice ([FIG. 12] to FIG. 12). [See FIG. 16].
In addition, the expression of superoxide dismutase and catalase, which are enzymes that remove reactive oxygen species, was decreased in the adipose tissue of KKAy mice (see FIG. 17 and FIG. 18). From the above results, it was suggested that oxidative stress is increased in obese adipose tissue due to increased expression of enzymes that produce reactive oxygen species and decreased expression of enzymes that remove them.
In addition, the same study was conducted using db / db mice, which are obese / diabetic mice of different strains, and the same tendency as KKAy mice, that is, increased lipid peroxide in adipose tissue, increased protein peroxidation. Oxidative stress such as: adiponectin gene expression decreased, reactive oxygen species producing enzymes (NADPH oxidase subunits gp91, p67) gene expression increased, reactive oxygen species removing enzymes (SOD, catalase) genes A decrease in expression was confirmed, and reactive oxygen species increased in adipose tissue, suggesting the possibility that the expression of adiponectin was suppressed by an increase in oxidative stress.

(Example 1)
Effect of reactive oxygen species on 3T3-L1 cells (test method)
A 6-well plate was seeded with 3 × 10 ⁴ cells / well of 3T3-L1 cells, and cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum. The medium was changed every other day, and 8 days later, 5 mg / mL insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine were added to the medium to induce differentiation. Two days after the induction of differentiation, the medium was returned to Dulbecco's modified Eagle medium containing 10% fetal bovine serum, and the culture was further continued while changing the medium every other day. Eight days after induction of differentiation, reactive oxygen species were added to 3T3-L1 cells differentiated into adipocytes, and 24 hours later, mRNA expression levels of various genes in the cells were measured. Reactive oxygen species were generated by adding 600 μM hypoxanthine and 0-20 mU / mL xanthine oxidase to the medium. RNA extraction was performed using RNA-STAT60 (manufactured by TEL-TEST “B”), and the mRNA expression levels of various genes in the cells were measured in the same manner as the measurement of mRNA expression levels in tissues. In addition, the mRNA expression level of various genes was calculated as a relative value corrected with the value of the cyclophilin mRNA expression level as a control.
The action of antioxidants on 3T3-L1 cells is to differentiate 3T3-L1 cells under the above-mentioned conditions, and after 8 days of differentiation induction, 0.2 mM hydrogen peroxide and 10 mM antioxidant N-acetylcysteine are added for 24 hours. Later, mRNA expression levels of various genes in the cells were measured. It was calculated as a relative value corrected with the value of the cyclophilin mRNA expression level.
The results are shown in [FIG. 19] to [FIG. 23].

(result)
When the action of reactive oxygen species on 3T3-L1 adipocytes was examined, it was found that the expression of adiponectin decreased depending on the dose of reactive oxygen species (see [FIG. 19]). At this time, the expression of PPAR gamma also decreased (see [FIG. 20]). On the other hand, PAI-1 expression was increased by the action of reactive oxygen species (see [FIG. 21]). From the above results, it became clear that the production of adiponectin is suppressed by oxidative stress such as reactive oxygen species.
Here, when N-acetylcysteine, which is an antioxidant, is allowed to act, the expression levels of adiponectin and PPARγ in 3T3-L1 cells are increased by N-acetylcysteine ([FIG. 22], [FIG. 23]).
From the results of Examples 1 and 2 above, oxidative stress in adipose tissue is increased in obesity and the like, and as a result, the expression of adiponectin is suppressed, and a compound having an antioxidant action is associated with obesity and the like. It was shown that the reduction of adiponectin production can be suppressed.

(Example 2)
Effect of reactive oxygen species on human adiponectin promoter activity (test method)
HEK293 cells were seeded in a 96-well multiplate (manufactured by Nunk) and cultured overnight at 37 ° C. under 5% carbon dioxide. A plasmid for measuring human adiponectin promoter activity was 50 ng / well, a plasmid for expression of a nuclear receptor was each 15 ng / well, and introduced transiently into the cells using the calcium phosphate method. Eight hours after gene transfer, hypoxanthine 100 μM and 20 mU / mL xanthine oxidase were added to the culture solution. After 16 hours, the supernatant was removed, 50 μL of cell lysate * was added, and the luminescence activity was measured with an Lmax microplate luminometer (Molecular Device).
The composition of the cell lysate * is as follows.
30 mM Tricine (pH 7.8), 8 mM MgOAc, 0.2 mM EDTA, 1% Triton X-100, 0.5 mM Luciferin, 1.5 mM ATP, 0.5 mM CoA, 0.7% 2-Mercaptoethanol
The plasmid for measuring human adiponectin promoter activity is, for example, the plasmid pGL3 (Promega) by the method described in “Sambrook et al., Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, 1989”. The sequence of the region from the upstream -908 base to the downstream 14 base of the transcription start site of the human adiponectin locus is functionally recombined and prepared on the 5 'side of the luciferase gene (manufactured by Kogyo Co., Ltd.). .
Moreover, the plasmid for nuclear receptor expression is, for example, a plasmid described in “Sambrook et al., Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, 1989”. A human PPAR gamma gene and a human RXR alpha gene are functionally recombined and prepared downstream of the CMV promoter of pcDNA3.1 (manufactured by Invitrogen).
3T3-L1 cells were seeded in 6-well plates, and differentiation into adipocytes was induced by the method described above. Four days after differentiation, the plasmid for measuring human adiponectin promoter activity was transiently introduced into cells using 2 μg / well, Lipofetoamine 2000 (Invitrogen). Three hours and 30 minutes after the gene introduction, 0.2 mM hydrogen peroxide and 10 mM antioxidant N-acetylcysteine were added and further cultured. After 20 hours, the culture supernatant was removed, the cells were lysed with 400 μL of passive lysis buffer (Promega), centrifuged at 12,000 × g for 3 minutes, and then the supernatant of Luciferase Assay System (Promega) was added to 50 μL of the supernatant. Luminescence activity was measured by adding 250 μL of the solution.
The results are shown in [FIG. 25] and [FIG. 26].

(result)
It was confirmed that reactive oxygen species suppress human adiponectin promoter activity in HEK293 cells and 3T3-L1 cells differentiated into adipocytes (see FIG. 25 and FIG. 26). In addition, by causing N-acetylcysteine to act, the promoter activity suppressed by the reactive oxygen species could be restored (see [FIG. 26]). Using this system, it is considered possible to screen for antioxidants, signal transduction inhibitors involved in the reduction of adiponectin production, and transcriptional regulators.

(Example 3)
Action of NADPH oxidase inhibitor on KKAy mice (test method)
From the results of Example 1 and Example 2, the compound having an antioxidant action suppresses adiponectin production reduction associated with obesity and the like, and diseases caused by the low adiponectin state, such as hypoadiponectinemia, diabetes, insulin resistance The possibility of being useful as a prophylactic / therapeutic agent for hyperlipidemia was shown. In obese adipose tissue, it is considered that oxidative stress is enhanced by the increased expression of NADPH oxidase, and therefore the effect of NADPH oxidase inhibitor on KKAy mice was examined. Apocynin was used as a NADPH oxidase inhibitor.
Water or 5 mM apocynin aqueous solution was administered as drinking water for 6 weeks to 5-week-old female C57BL / 6 mice and female KKAy mice, respectively. After 6 weeks, plasma and periovar fat were removed from each group of mice and frozen in liquid nitrogen.
The glucose concentration in plasma was measured using Glucose Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Plasma insulin concentration was measured using a mouse insulin ELISA kit (manufactured by Shiba Goat). The triacylglycerol concentration in plasma was measured using TG Test Wako (manufactured by Wako Pure Chemical Industries). The amount of lipid peroxide in tissue and the amount of mRNA expression in tissue were measured using the same method as in Example 1.
The results are shown in [FIG. 27] to [FIG. 34].

(result)
In both C57BL / 6 mice and KKAy mice, there was no difference in body weight between the control group and the apocynin administration group (see [FIG. 27]). However, in KKAy mice, apocynin administration significantly reduced blood glucose levels, plasma insulin, and plasma triacylglycerol (see [FIG. 28], [FIG. 29], [FIG. 30]). In C57BL / 6 mice, these changes were not observed by apocynin administration.
In KKAy mice, apocynin administration significantly reduced the amount of lipid peroxide in adipose tissue (see [Fig. 31]), and NADPH oxidase was suppressed to reduce oxidative stress in adipose tissue. I was told.
Furthermore, in KKAy mice, adiponectin concentration in plasma was increased by administration of apocynin (see [FIG. 32]). At this time, the adiponectin mRNA expression level in the adipose tissue was increased, and the TNF-alpha mRNA expression level was decreased (see [FIG. 33] and [FIG. 34]).
From the above results, NADPH oxidase inhibitors can increase adiponectin production and reduce diabetes, insulin resistance, and hyperlipidemia by reducing oxidative stress in adipose tissue that is promoted in obesity. Indicated.

  Since the preventive / therapeutic agent of the present invention increases adiponectin production, diseases caused by a low adiponectin state such as hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications, high Useful as a prophylactic / therapeutic agent for glycemia, arteriosclerosis, atherosclerosis, cardiovascular disease, cerebrovascular disorder, vascular stenosis, peripheral vascular disease, aneurysm, hyperlipidemia, hypercholesterolemia or obesity It is.

It is the figure which showed the average body weight at the time of 7 weeks old (7W) and 13 weeks old (13W) of the C57BL / 6 mouse (C57) and the KKAy mouse (KKAy) as an average value ± standard error (g). Mean value ± standard error of liver, periovarial fat, and mesenteric fat tissue weight at 7 weeks of age (7W) and 13 weeks of age (13W) of C57BL / 6 mice (C57) and KKAy mice (KKAy) ( It is the figure which showed g). It is the figure which showed the average value +/- standard error (microU / mL) of the plasma insulin density | concentration in the C57BL / 6 mouse (C57) and the KKAy mouse (KKAy) at the age of 7 weeks (7W) and 13 weeks of age (13W). . The figure which showed the average value +/- standard error (mg / dl) of the plasma triacylglycerol density | concentration at the time of 7 weeks age (7W) and 13 weeks age (13W) of C57BL / 6 mouse (C57) and KKAy mouse (KKAy) It is. Plasma lipid peroxide concentrations at 7 weeks of age (7W) and 13 weeks of age (13W) of C57BL / 6 mice (C57) and KKAy mice (KKAy) were expressed as mean values of thiobarbituric acid-reactive substrate concentrations ± standard error It is the figure shown as (nmol malondialdehyde / ml). The lipid peroxide concentration in adipose tissue at 7 weeks of age (7 W) and 13 weeks of age (13 W) of C57BL / 6 mice (C57) and KKAy mice (KKAy) is the mean value of thiobarbituric acid-reactive substrate concentrations ± standard It is the figure shown as an error (nmol malondialdehyde / mg protein). It is the figure which showed the average value +/- standard error (mg / dl) of the blood glucose level in the C57BL / 6 mouse (C57) and the KKAy mouse (KKAy) at the age of 7 weeks (7W) and 13 weeks of age (13W). The figure which showed the average value +/- standard error of the relative value of the adiponectin mRNA expression level in the structure | tissue at the time of 7 weeks age (7W) and 13 weeks age (13W) of C57BL / 6 mouse (C57) and KKAy mouse (KKAy) It is. Average values ± standard error of relative expression levels of PPAR gamma mRNA in tissues at 7 weeks of age (7W) and 13 weeks of age (13W) of C57BL / 6 mice (C57) and KKAy mice (KKAy) are shown. FIG. Shown are mean values ± standard error of relative values of TNF alpha mRNA expression levels in tissues at 7 weeks of age (7W) and 13 weeks of age (13W) of C57BL / 6 mice (C57) and KKAy mice (KKAy) FIG. Shows the mean ± standard error of the relative value of the expression level of PAI-1 mRNA in tissues at 7 weeks of age (7W) and 13 weeks of age (13W) of C57BL / 6 mice (C57) and KKAy mice (KKAy) It is a figure. The figure which showed the average value +/- standard error of the relative value of the expression level of gp91phox mRNA in a tissue at the time of 7 weeks old (7W) and 13 weeks old (13W) of C57BL / 6 mouse (C57) and KKAy mouse (KKAy) It is. The figure which showed the average value +/- standard error of the relative value of the expression level of p22phox mRNA in a tissue at the time of 7 weeks old (7W) and 13 weeks old (13W) of C57BL / 6 mouse (C57) and KKAy mouse (KKAy) It is. The figure which showed the average value +/- standard error of the relative value of the expression level of p67phox mRNA in a tissue at the time of 7 weeks old (7W) and 13 weeks old (13W) of C57BL / 6 mouse (C57) and KKAy mouse (KKAy) It is. The figure which showed the average value +/- standard error of the relative value of the expression level of p47phox mRNA in a tissue at the time of 7 weeks old (7W) and 13 weeks old (13W) of C57BL / 6 mouse (C57) and KKAy mouse (KKAy) It is. The figure which showed the average value +/- standard error of the relative value of the expression level of p40phox mRNA in a tissue at the time of 7 weeks old (7W) and 13 weeks old (13W) of C57BL / 6 mouse (C57) and KKAy mouse (KKAy) It is. Shows the mean value ± standard error of the relative value of the superoxide dismutase mRNA expression level in the tissues at 7 weeks of age (7W) and 13 weeks of age (13W) of C57BL / 6 mice (C57) and KKAy mice (KKAy) It is a figure. The figure which showed the average value +/- standard error of the relative value of the catalase mRNA expression level in the structure | tissue at the time of 7 weeks age (7W) and 13 weeks age (13W) of C57BL / 6 mouse (C57) and KKAy mouse (KKAy) It is. It is the figure which showed the average value +/- standard error of the relative value of the adiponectin mRNA expression level in 3T3-L1 cell. The horizontal axis shows the concentration (mU / ml) of the added xanthine oxidase. It is the figure which showed the average value +/- standard error of the relative value of the PPAR gamma mRNA expression level in 3T3-L1 cell. The horizontal axis shows the concentration (mU / ml) of the added xanthine oxidase. It is the figure which showed the average value +/- standard error of the relative value of the PAI-1 mRNA expression level in 3T3-L1 cell. The horizontal axis shows the concentration (mU / ml) of the added xanthine oxidase. It is the figure which showed the average value +/- standard error of the relative value of the adiponectin mRNA expression level in 3T3-L1 cell. It is the figure which showed the average value +/- standard error of the relative value of the PPAR gamma mRNA expression level in 3T3-L1 cell. It is the figure which showed typically the structure of the plasmid for a human adiponectin promoter activity measurement. LUC refers to the luciferase gene. It is the figure which showed the adiponectin promoter activity in HEK293 cell as an average value +/- standard error of the relative value of luciferase activity. The horizontal axis shows the addition (+) / non-addition (−) of the plasmid for nuclear receptor expression (PPARγ / RXRα / LRH-1) and the addition (+) / non-addition (−) of active oxygen species. . It is the figure which showed the adiponectin promoter activity in the 3T3-L1 cell differentiated to the fat cell as the average value +/- standard error of the relative value of the luciferase activity. The horizontal axis shows the addition (+) / non-addition (−) of N-acetylcysteine and the addition (+) / non-addition (−) of active oxygen species. It is the figure which showed the body weight in the control group (white column) and the apocynin administration group (black column) of C57BL / 6 mouse (C57BL / 6) and KKAy mouse (KKAy) by the average value +/- standard error (g). The figure which showed the blood glucose level in the control group (white column) and the apocynin administration group (black column) of a C57BL / 6 mouse (C57BL / 6) and a KKAy mouse (KKAy) by the average value +/- standard error (mg / dL). is there. The figure which showed the plasma insulin density | concentration in the control group (white column) and the apocynin administration group (black column) of C57BL / 6 mouse (C57BL / 6) and KKAy mouse (KKAy) by the average value +/- standard error (ng / mL). It is. The plasma triacylglycerol concentrations in the control group (white column) and the apocynin administration group (black column) of C57BL / 6 mice (C57BL / 6) and KKAy mice (KKAy) are shown as mean ± standard error (mg / dL). It is a figure. The lipid peroxide concentration in adipose tissue in the control group (white column) and the apocynin administration group (black column) of C57BL / 6 mice (C57BL / 6) and KKAy mice (KKAy) is the mean value of the thiobarbituric acid-reactive substrate concentration ± It is the figure shown as standard error (nmol malondialdehyde / mg protein). The figure which showed the plasma adiponectin density | concentration in the control group (white column) and the apocynin administration group (black column) of a C57BL / 6 mouse (C57BL / 6) and a KKAy mouse (KKAy) by the average value +/- standard error (microgram / mL). It is. It is the figure which showed the average value +/- standard error of the relative value of the adiponectin mRNA expression level in a fat tissue in the control group (left column) and apocynin administration group (right column) of a KKAy mouse | mouth (KKAy). It is the figure which showed the average value +/- standard error of the relative value of the expression level of TNFalpha mRNA in the fat tissue in the control group (left column) and the apocynin administration group (right column) of a KKAy mouse (KKAy).

SEQ ID NO: 1: Oligonucleotide designed to act as a PCR primer for detecting an adiponectin gene SEQ ID NO: 2: Oligonucleotide designed to act as a PCR primer for detecting an adiponectin gene SEQ ID NO: 3: TNF alpha Oligonucleotide designed to act as a PCR primer for detecting the gene SEQ ID NO: 4: Oligonucleotide designed to act as a PCR primer for detecting the TNF alpha gene SEQ ID NO: 5: detecting the PAI-1 gene Oligonucleotide designed to act as a PCR primer for sequencing SEQ ID NO: 6: Oligonucleotide designed to act as a PCR primer for detecting the PAI-1 gene SEQ ID NO: 7 Oligonucleotide designed to act as a PCR primer for detecting the R gamma gene SEQ ID NO: 8: Oligonucleotide designed to act as a PCR primer for detecting the PPAR gamma gene SEQ ID NO: 9: gp91phox gene detected Oligonucleotide designed to act as a PCR primer for sequencing SEQ ID NO: 10: oligonucleotide designed to act as a PCR primer for detecting gp91phox gene SEQ ID NO: 11 as a PCR primer for detecting p22phox gene Oligonucleotide designed to act SEQ ID NO: 12: oligonucleotide designed to act as a PCR primer for detecting the p22phox gene SEQ ID NO: 13: p Oligonucleotide designed to act as a PCR primer for detecting the 7phox gene SEQ ID NO: 14: Oligonucleotide designed to act as a PCR primer for detecting the p67phox gene SEQ ID NO: 15 to detect the p47phox gene Oligonucleotide designed to act as a PCR primer for SEQ ID NO: 16: Oligonucleotide designed to act as a PCR primer for detecting the p47phox gene SEQ ID NO: 17 acts as a PCR primer for detecting the p40phox gene Specifically designed oligonucleotide SEQ ID NO: 18: oligonucleotide designed to act as a PCR primer for detecting the p40phox gene SEQ ID NO: 19: super Oligonucleotide designed to act as a PCR primer for detecting the xidodismutase gene SEQ ID NO: 20: Oligonucleotide designed to act as a PCR primer for detecting the superoxide dismutase gene SEQ ID NO: 21: Catalase gene Oligonucleotide designed to act as a PCR primer for detection SEQ ID NO: 22: Oligonucleotide designed to act as a PCR primer for detecting a catalase gene SEQ ID NO: 23: PCR for detecting a cyclophilin gene Oligonucleotide designed to act as a primer SEQ ID NO: 24: oligonucleotide designed to act as a PCR primer for detecting the cyclophilin gene

Claims (13)

  A prophylactic / therapeutic agent for a disease caused by a low adiponectin state comprising a compound having an antioxidant action as an active ingredient. Compounds having antioxidant activity are represented by the general formula (I)

[Wherein R ¹ is a nitro group, a cyano group or a trihalo (lower) alkyl group, and R ² , R ³ and R ⁴ are the same or different and each represents a lower alkyl group] or a dihydropyridine compound or pharmaceutical The preventive / therapeutic agent according to claim 1, which is an acceptable salt thereof.   The compound of general formula (I) is isopropyl ester of 6-cyano-5-methoxycarbonyl-2-methyl-4- (3-nitrophenyl) -1,4-dihydropyridine-3-carboxylic acid Prophylactic / therapeutic agent.   The prophylactic / therapeutic agent according to claim 1, wherein the compound having an antioxidant action is probucol and a derivative thereof.   The prophylactic / therapeutic agent according to claim 1, wherein the compound having an antioxidant action is N-acetylcysteine and its derivatives.   The prophylactic / therapeutic agent according to claim 1, wherein the compound having an antioxidative action is a NADPH oxidase inhibitory compound.   The preventive / therapeutic agent according to claim 6, wherein the NADPH oxidase inhibitor compound is apocynin.   Diseases caused by low adiponectin status are hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications, hyperglycemia, arteriosclerosis, atherosclerosis, cardiovascular disease, The prophylactic / therapeutic agent according to any one of claims 1 to 7, which is a disease selected from the group consisting of cerebrovascular disorder, vascular stenosis, peripheral vascular disease, aneurysm, hyperlipidemia, hypercholesterolemia and obesity. .   The prophylactic / therapeutic agent according to claim 8, wherein the disease caused by the low adiponectin state is hypoadiponectinemia.   The prophylactic / therapeutic agent according to claim 8, wherein the disease caused by the low adiponectin state is diabetes.   The prophylactic / therapeutic agent according to claim 8, wherein the disease caused by the low adiponectin state is arteriosclerosis.   The prophylactic / therapeutic agent according to claim 8, wherein the disease caused by the low adiponectin state is hyperlipidemia.   The prophylactic / therapeutic agent according to claim 8, wherein the disease caused by the low adiponectin state is obesity.

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