WO2009139459A1 - Method for prevention and treatment of metabolic syndrome through the inhibition of psgl-1 - Google Patents

Method for prevention and treatment of metabolic syndrome through the inhibition of psgl-1 Download PDF

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WO2009139459A1
WO2009139459A1 PCT/JP2009/059045 JP2009059045W WO2009139459A1 WO 2009139459 A1 WO2009139459 A1 WO 2009139459A1 JP 2009059045 W JP2009059045 W JP 2009059045W WO 2009139459 A1 WO2009139459 A1 WO 2009139459A1
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psgl
metabolic
expression
cells
gene
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Japanese (ja)
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賢一 四方
槇野 博史
千景 佐藤
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国立大学法人 岡山大学
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/41Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/41641,3-Diazoles
    • A61K31/41781,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52Purines, e.g. adenine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/66Phosphorus compounds
    • A61K31/675Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/04Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13Cyclosporins

Abstract

Disclosed is a prophylactic and/or therapeutic agent which can exhibit an effect by itself on the prevention and/or treatment of a basic condition of a lifestyle-related disease such as metabolic syndrome and type-2 diabetes.  Also disclosed is a method for the screening of the prophylactic and/or therapeutic agent. Specifically disclosed is a prophylactic and/or therapeutic agent for metabolic diseases, which comprises an inhibitor of PSGL-1 as an active ingredient.

Description

PSGL-1 prevention and therapy of metabolic syndrome by inhibition

The present invention is the prevention and / or therapeutic agents, and to a screening method for metabolic disorders. More particularly, the present invention contains an inhibitor against PSGL-1, metabolic syndrome, type 2 diabetes, impaired glucose tolerance, prevention of metabolic disorders such as obesity or dyslipidemia and / or therapeutic agent, and the prevention and / or methods of screening for therapeutic agents for metabolic disease index functional inhibition of PSGL-1.

In developed countries, lifestyle-related diseases has increased, especially sufferers of metabolic syndrome in recent years significantly, has become a serious problem in health care. In patients with metabolic syndrome, visceral fat obesity, hyperglycemia, hypertension, symptoms such as hyperlipidemia has intussusception, by stacking of these symptoms, arteriosclerotic diseases such as myocardial infarction and cerebral infarction which is likely to develop. Diagnostic criteria for metabolic syndrome has not been globally unified, for example, in Japan, the visceral fat obesity as essential item, metabolic if further applicable hypertension, two or more items of hyperglycemia and serum lipid abnormality It is diagnosed with the syndrome.

In recent years the prevalence of the metabolic syndrome, because there is largely due to satiation and lack of exercise, diet and exercise therapy have been emphasized so far as the treatment policy. However, these therapies are often force a fundamental improvement of the living habits of the patient's own, it is often therefore realize it is difficult. On the other hand, the major pathology of metabolic syndrome hypertension, drug therapy for each of hyperglycemia and serum lipid abnormality has been attempted, for example, high triglycerides / low HDL- cholesterolemia The fibrate or a statin; sugar metabolic disorders the sulfonylurea agents, rapid-acting insulin secretagogue, alpha-glucosidase inhibitor or insulin resistance-improving agent; for hypertension such as angiotensin converting enzyme inhibitors or adrenergic α receptor antagonists have been utilized. However, these medications, there is a problem such that it medical costs are expensive Ya not always reliable therapeutic effect. As the drug therapy for visceral fat obesity, only indirect prevention or treatment by central appetite inhibitors have been made.

In the development of lifestyle-related diseases, including metabolic syndrome and type 2 diabetes mellitus, the importance of insulin resistance has been widely recognized. The insulin resistance, skeletal muscle cells, liver cells, such as in adipocytes, by insulin sensitivity is reduced, although insulin secretion is maintained, a state in which its action is decreased. Insulin resistance hyperinsulinemia, then glucose intolerance, even believed to cause a high blood pressure and abnormal lipid metabolism (Non-Patent Document 1). Pathogenic mechanisms of insulin resistance are not being fully clear, accumulation and an increase in visceral fat caused by conventional over-nutrition and lack of exercise have been emphasized. Furthermore, in recent years, between the obesity and insulin resistance has been revealed that inflammation is interposed, is accumulation of macrophages surrounding bloated, necrosis and its coronary adipocyte, is widespread recognition that has led to inflammation and insulin resistance.

PSGL-1 is leukocytes and vascular endothelial cell membrane protein cloned as a ligand for certain P- selectin molecule to the cell surface involved in the adhesion of the (Patent Document 1, Non-Patent Document 2). PSGL-1 is a disulfide-linked homodimeric sialomucin of 220 kDa, most blood leukocytes (e.g., neutrophils, monocytes, macrophages, subsets of B cells, and all T cells) is to express PSGL-1 It has been known. Are involved a wide variety of cell adhesion molecules in very a key process migration and infiltration of leukocytes into damaged tissue in inflammatory, phenomenon called rolling observed in the early stages of inflammation, selectins and their through interaction with specific carbohydrate ligands (e.g. P- interaction selectin and PSGL-1) it was found to be possible to take place (non-Patent documents 3 and 4). However, in the development of lifestyle-related diseases, including metabolic syndrome and type 2 diabetes, the PSGL-1 plays an important role has not been known heretofore.

Kohyo 08-502886 JP

Reaven GM. , Diabetes; 37 (12): 1595-1607 (1988) Sako et al., Cell; 75 (6): 1179-1186 (1993) Tedder TF et al., FASEB J. ; 9 (10): 866-73 ( 1995) Yang J et al., Thromb Haemost. ; 81 (1): 1-7 (1999)

As can be seen from the state of the art described above, there is a need for easy novel therapeutic and prophylactic methods for the effectiveness and the patient against lifestyle-related diseases such as metabolic syndrome and type 2 diabetes. In particular, insulin resistance is the basis of their lifestyle diseases, inflammation of fat cells, and against obesity, expectations and importance to itself resulting in effective drug be said quite large. The present invention aims to provide a alone may lead to effect prophylaxis and / or therapeutic agent and the method for screening against prophylaxis and / or treatment of the underlying condition in lifestyle-related diseases such as metabolic syndrome and type 2 diabetes mellitus to.

The present inventors have, serious social problem to be going on diabetes, has been pouring earlier than the force in the prevention and treatment of lifestyle-related diseases such as hyperlipidemia and hypertension. Among them, rather than each pathology integrated with the lifestyle-related diseases is to prevent or treat separately, felt acutely the necessity of agents capable of preventing and / or treating the underlying pathology of lifestyle-related diseases in a single treatment , I went to focus the research for that. Consequently, by inhibiting the PSGL-1 is a cell adhesion-related molecules, they found that it is possible to prevent and / or treat the underlying pathology of lifestyle-related diseases, and have completed the present invention.

That is, the present invention is as follows:
[1] contains an inhibitor as an active ingredient for the PSGL-1, the prevention and / or therapeutic agent for metabolic diseases.
[2] metabolic diseases, metabolic syndrome, type 2 diabetes, impaired glucose tolerance, obesity or dyslipidemia, the [1] agent according.
[3] inhibitor against PSGL-1 is specific neutralizing antibodies against PSGL-1, or specifically inhibitable antisense nucleic acid or a small molecular weight interfering RNA expression PSGL-1, or these mammalian cells an expression vector capable of expressing a dosage of the above-mentioned [1] described in.
[4], comprising the following steps screening method for a substance capable of preventing or treating metabolic disorders:
(I) the test substance is assayed either inhibit the function of PSGL-1, and (II) PSGL-1 of the test substance that inhibits the function be selected as a substance capable of preventing or treating a metabolic disorder.
[5] metabolic diseases, metabolic syndrome, type 2 diabetes, impaired glucose tolerance, it is obesity or dyslipidemia, the method of the above-mentioned [4], wherein.

Agents of the present invention suppresses infiltration of macrophages into the visceral fat, may inhibit the development of inflammation and insulin resistance in adipose tissue I following. Thus, by using the agent of the present invention, without performing a diet or exercise therapy, metabolic syndrome, type 2 diabetes, impaired glucose tolerance, prevention of metabolic disorders such as obesity or dyslipidemia and / or treatment effect which can be obtained. By prevention and / or treatment of these metabolic diseases, it is possible to make prevention and / or suppression of progression of arteriosclerotic diseases caused by metabolic diseases according. The present invention also provides in combination methods of screening a substance capable of preventing or treating these metabolic diseases.

8-week-old db / db mice and BL6 mice weighing a diagram showing a measurement result of visceral fat weight and various metabolic data (n = 10, mean ± standard error, ★: P <0.0001, ★★ : P <0.005). (A) body weight (g), (B) is epididymal fat weight (g), (C) serum LDL (mg / dl), (D) from time to time blood glucose (mg / dl), (E) at any time IRI (ng / ml), shows measurement results of (F) is HbA1c (%). Is a view showing measurement results of quantitative real-time RT-PCR of various gene expression in 8-week-old db / db mice and BL6 mice visceral fat (n = 10, mean ± standard error, ★: P <0.005, ★: P <0.05). (A) is F4 / 80, (B) is MCP-1, (C) is PSGL-1, (D) is P- selectin, (E) is E- selectin, (F) the measurement results of ICAM-1 the shows. And the vertical axis represents the index value. 19 Shuyowaidaka fat diet mice and low-fat diet mice weighing a diagram showing a measurement result of visceral fat weight and various metabolic data (open circles: Low-fat diet mice (LF; n = 9), black triangles : high-fat diet mice (HF; n = 11), mean ± standard error, ★: P <0.0005, ★★ : P <0.001). (A) body weight (abscissa: Age-number (w), vertical axis: Weight (g)), (B) epididymal fat weight (g), (C) serum LDL (mg / dl), ( D) fasting glucose (mg / dl), (E) fasting IRI (ng / ml), it shows measurement results of (F) is HbA1c (%). 19 Shuyowaidaka is a diagram showing the results of the insulin tolerance test for fat diet mice and low-fat diet mice (ITT) and intraperitoneal glucose tolerance test (IPGTT) (open circles: Low-fat diet mice (LF; n = 9), black triangle: high fat diet mice (HF; n = 11), mean ± standard error, ★: P <0.005, ★★ : P <0.05). (A) is IPGTT blood glucose (mg / dl), shows (B) IPGTT plasma insulin value (ng / ml) is, (C) ratio on insulin preload blood glucose level of the ITT blood glucose level (%). In (A) ~ (C), showing the horizontal axis after the start load of minutes. In addition, it means that the "before" is a pre-load. 19 Shuyowaidaka is a graph showing measurement results of quantitative real-time RT-PCR of various gene expression in visceral adipose tissue fat diet mice (low fat diet mice (LF): n = 9, the high-fat diet mice ( HF): n = 10, mean ± standard error, ★: P <0.001). (A) is CD68, (B) is MCP-1, (C) PSGL-1, the (D) is P- selectin, (E) is E- selectin, (F) shows the measurement results of ICAM-1 ing. And the vertical axis represents the index value. Body weight in 17-week-old BL6 mice and PSGL-1 knockout mice after a high fat diet, is a diagram showing a measurement result of visceral fat weight and various metabolic data (high fat diet BL6 mice (WT-HF): n = 7, the high-fat diet PSGL-1 knockout mice (KO-HF): n = 8, mean ± standard error, ★: P <0.005, ★★ : P <0.05)). (A) body weight (g), (B) is epididymal fat weight / body weight ratio (%), (C) total cholesterol (mg / dl), (D) is LDL (mg / dl), (E) triglyceride (mg / dl) is, (F) serum FFA (μEq / l), (G) fasting glucose (mg / dl), (H) fasting IRI (ng / ml), (I ) is HbA1c (%), show the measurement results of (J) serum leptin (ng / ml is), (K) serum adiponectin (μg / ml). Is a diagram showing the results of the insulin tolerance test for BL6 mice and PSGL-1 knockout mice after a high fat diet (ITT) and intraperitoneal glucose tolerance test (IPGTT) (open circles: High fat diet BL6 mice (WT-HF ; n = 9), black triangle: high fat diet PSGL-1 knockout mice (KO-HF; n = 8 ), mean ± standard error, ★: P <0.05). (A) is IPGTT blood glucose (mg / dl), shows (B) IPGTT plasma insulin value (ng / ml) is, (C) ratio on insulin preload blood glucose level of the ITT blood glucose level (%). In (A) ~ (C), showing the horizontal axis after the start load of minutes. In addition, it means that the "before" is a pre-load. Visceral adipose tissue in BL6 mice and PSGL-1 knockout mice after a high-fat diet stained image, and shows the area of ​​the visceral fat cells. (A) a high-fat diet BL6 mice; visceral fat (WT-HF n = 1818 cells) PAS staining image (× 200), (B) a high-fat diet PSGL-1 knockout mice (KO-HF; n = 1834 cells) of visceral fat PAS stained image (× 200), (C) is visceral adipocytes area of ​​a high-fat diet BL6 mice (WT-HF) and PSGL-1 knockout mice (KO-HF) ( [mu] m 2; mean ± standard error, ★: shows P <0.0001). Is a view showing measurement results of quantitative real-time RT-PCR of various gene expression in visceral fat BL6 mice and PSGL-1 knockout mice after a high fat diet (high fat diet BL6 mice (WT-HF): n = 7, the high-fat diet PSGL-1 knockout mice (KO-HF): n = 7, mean ± standard error, ★: P <0.01, ★★ : P <0.05)). (A) is F4 / 80, and (B) shows a MCP-1, (C) is NOS 2 (iNOS), (D) the measurement result of leptin. And the vertical axis represents the index value. It is a diagram illustrating a BL6 mice (WT-HF) and PSGL-1 knockout mice (KO-HF) Liver weight and HE stained image of the liver triglyceride content as well as the liver in the post-high fat diet. (A) of the left graph of both mouse liver weight (unit: g) of shows the right graph liver triglyceride content of both mouse (unit: mg / g) shows. (B) shows the HE staining image (× 200) of each liver tissue for both mice.

The present invention contains an inhibitor against PSGL-1 as an active ingredient, a preventive and / or therapeutic agent for metabolic diseases.

The inhibition target of the agent of the present invention "PSGL-1" is a known adhesion molecules with O- glycosylation extracellular sialomucin. The PSGL-1, the human orthologs PSGL-1, or a variety of other mammals disclosed in Patent Document 1, for example, a mouse PSGL-1 (Yang J. others, Blood, May 15; 87 ( 10): 4176-86 (1996)), or the like can be given allelic form of their natural. The amino acid sequence of PSGL-1 and cDNA (mRNA) sequences are also known. The amino acid sequence and cDNA sequence of human PSGL-1 and mouse PSGL-1, exemplified in SEQ ID NO: 1 to 4 (SEQ ID NO 1: Human PSGL-1 mRNA sequence (NCBI Accession No. NM_003006); SEQ ID NO: 2: SEQ ID NO: amino acid sequence of the protein encoded by the mRNA sequence of 1 (NCBI accession number NP_002997); SEQ ID NO 3: mouse PSGL-1 mRNA sequence (NCBI Accession No. NM_009151); SEQ ID NO: 4: SEQ ID NO: 3 of the mRNA the amino acid sequence of the protein encoded by the sequence (NCBI accession number NP_033177)).

The active ingredient in the agent of the present invention, the "substance inhibiting PSGL-1" is a substance that inhibits the activity of PSGL-1 protein (hereinafter, also referred to as PSGL-1 activity inhibitor) to the expression of PSGL-1 inhibiting substance (hereinafter, also referred to as PSGL-1 expression inhibitor) and are included. Incidentally, the term "inhibit", as used herein, inhibition target (e.g., activity or expression) means that to fully or partially suppressed or reduced.

The PSGL-1 activity refers to the binding activity to selectins. The selectin, P- selectin, can be mentioned E- and L- selectin, PSGL-1 has highest affinity for P- selectin. The PSGL-1 activity inhibitors that can be used in the agent of the present invention, for example, specific neutralizing antibodies (anti-PSGL-1 polyclonal antibody, such as anti-PSGL-1 monoclonal antibody), and selectins (such as P- selectin) substance that inhibits the binding of (e.g., fucoidan, sulfatides, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, glycosaminoglycans, such as keratan sulfate), and low molecular substances that bind to the active center can be cited, preferably specific neutralizing antibodies, anti-PSGL-1 monoclonal antibody is more preferable. Further, specific neutralizing antibodies against selectin (anti-P- selectin polyclonal antibody, anti-P- selectin monoclonal antibodies, etc.), can be used as a PSGL-1 Inhibitor well as soluble selectins. PSGL-1 activity inhibitor can using the screening method described later, select, identify or confirm.

The PSGL-1 expression inhibitor may be used in the present invention, for example, specifically inhibitable antisense nucleic acid or a small molecular weight interfering RNA expression PSGL-1 (small interfering RNA; siRNA), PSGL-1 mRNA specifically cleavable ribozyme, these and the like expression vector capable of expressing in mammalian cells in. As examples of PSGL-1 expression inhibitor, infliximab (Remicade) anti TNFα antibody preparation, such as, a soluble TNFα receptor preparation, such as etanercept (Enbrel), methotrexate (Riumatorekkusu), tacrolimus; FK506 (Prograf), cyclosporine; CyA (Neoral), cyclophosphamide (Endoxan), azathioprine (Imuran), mizoribine (bredinin), and the like immunosuppressive drugs, such as MMF (Cellcept). PSGL-1 expression inhibitor may be by using the screening method described later, select, identify or confirm.

Examples of the antisense nucleic acids, and specifically hybridize with transcripts of PSGL-1 (mRNA or initial transcription product) refers to a nucleic acid capable of inhibiting the PSGL-1 translation. The nucleic acid, even DNA, even RNA, or may be a DNA / RNA chimera.
The length of the antisense nucleic acid is not particularly limited as long as it can PSGL-1 transcript specifically hybridizing, etc. in view of easiness and antigenic synthetic, usually 10 bases or more as the lower limit, preferably the include more than 15 bases, usually 100 bases or less, preferably not more than 30 bases or less, more preferably include the following 24 bases. Thus, the antisense nucleic acid, for example 10 or more bases 100 bases or less, preferably 15 bases or more 30 bases or less, more preferably be used the following oligonucleotide 15 bases or more 24 bases.
Further, the target sequence of the antisense nucleic acid also is not particularly limited as long as the sequence in which the antisense nucleic acid is PSGL-1 translation is inhibited by hybridizing, be the entire sequence of the mRNA partial sequence (usually 10 bases or more, preferably 15 bases or more, and usually 100 bases or less, preferably 30 bases or less, more preferably may be a 24 bases or less), or may be an intron portion of the initial transcription product but, when using oligonucleotides as antisense nucleic acid, the target sequence is preferably located from 5 'end of the mRNA of PSGL-1 to C-terminus of the coding region.
Specific examples of antisense nucleic acids, for example, in the case of human PSGL-1, SEQ ID NO: 1 contains the nucleotide sequence from position 1-1369 of mRNA described, usually 10 bases or more, preferably 15 bases or more, and Usually 100 bases or less, preferably 30 bases or less, more preferably include oligonucleotides having a sequence complementary to the nucleotide sequence of the following length 24 bases, or for example, in the case of mouse PSGL-1, SEQ ID NO: 3 contained in the nucleotide sequence to position 1-1412 of the described mRNA, usually 10 bases or more, preferably 15 bases or more, and usually 100 bases or less, preferably 30 bases or less, more preferably 24 bases or fewer in length bases oligonucleotides can be mentioned having a sequence complementary to the sequence.
Synthesis of the antisense nucleic acid may be carried out in a manner known per se, for example, it may be synthesized using a commercially available automated DNA / RNA synthesizer oligonucleotides having the desired base sequence.

The siRNA is a partial sequence of the coding region of the transcript of PSGL-1 (mRNA or initial transcription product) (usually 18 bases or more, preferably 21 bases or more, and usually 30 bases or less, preferably 27 bases, and more preferably 23 bases or less; in the case of the initial transcription product is a double stranded oligo RNA having a sequence complementary to including intron), the expression of PSGL-1 by cutting specifically recognizes the transcript as long as it can inhibit not particularly limited. SiRNA length is usually 21 to 23 bases. Those skilled in the art, SEQ ID NO: human PSGL-1 mRNA sequence or SEQ ID NO: described in 1: Based on the mRNA sequence of mouse PSGL-1 set forth in 3, to inhibit the expression of PSGL-1 in human or mouse it can be determined sense strand and the sequence of the antisense strand of siRNA may be used for.
Synthesis of siRNA may be carried out in a manner known per se, for example, the sense and antisense strands were synthesized respectively an automated DNA / RNA synthesizer, may be synthesized by them anneal.

As the above expression vector, it is possible to use any of those used in the art, e.g., plasmids from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (e.g. , pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), bacteriophages such as λ phage, retroviruses, vaccinia virus, other animal viruses such as baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, and the like pcDNAI / Neo.

As used herein, the term "metabolic disease", various nutrients (e.g., proteins, lipids, carbohydrates, inorganic salts, vitamins, etc.) in the metabolic pathways, various works have congenital or acquired causes occurs failure stage, as a result, it means a disease or condition caused by metabolites blood or specific organs or insufficient or may become excessive. Examples of metabolic disorders, metabolic syndrome, diabetes mellitus (type 1 diabetes, type 2 diabetes, gestational diabetes etc.), impaired glucose tolerance, obesity, diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, dyslipidemia diseases (hypertriglyceridemia, hypercholesterolemia, low HDL cholesterolemia, etc. postprandial hyperlipemia), and the like hypertension.

For diagnostic criteria for the metabolic syndrome, in Japan, established by the metabolic syndrome diagnostic criteria Review Committee, which is composed of members of the 8 Society, such as the Japanese Society of Internal Medicine in 2005, it was reported.
According to this report, the metabolic syndrome, visceral fat accumulation (waist circumference is male 85cm or more, women 90cm or more) as an essential item, it in addition to lipid metabolism (hypertriglyceridemia (150 mg / dl or higher ) and / or low HDL cholesterolemia (less than 40 mg / dl)), high blood pressure (systolic blood pressure above 130 mmHg, and / or diastolic blood pressure more than 85 mmHg) and hyperglycemia (fasting blood glucose 110 mg / dl or higher) 3 more than two items of the items to be refers to the if applicable.

Diagnostic criteria for metabolic syndrome, in Western US hyperlipidemic guidelines: National Cholesterol Education Program of Adult Treatment Panel III (NCEP-ATP III) and has been proposed from the WHO (JAMA 2001; 285: 2486-97 and, .. see 539-553): 15; Diabet Med 1998. In NCEP-ATP III obesity (waist circumference measurement), high triglycerides, is defined as where applicable three or more low HDL-cholesterol, the following conditions of hypertension and hyperglycemia.
1) West (abdominal circumference) is 102cm or more in men, 88cm or more in women;
2) fasting triglyceride is 150mg / dl or more;
3) less than 40mg / dl HDL cholesterol in men and less than women in the 50mg / dl;
4) blood pressure 85mmHg or more 130mmHg or more, or diastolic blood pressure at maximum pressure;
5) fasting blood glucose level is 110mg / dl or more.
On the other hand, in the diagnostic criteria of WHO, hyperinsulinemia (top 25% of non-diabetic patients) or fasting blood glucose 110 mg / dl or more addition, visceral obesity, dyslipidemia, hypertension, the following conditions of the micro-albuminuria is defined as where applicable two or more.
1) visceral obesity: waist / hip ratio> 0.9 (male),> 0.85 (woman) or BMI is greater than 30;
2) abnormal lipid metabolism: plasma triglycerides 150mg / dl or more;
3) Hypertension: systolic blood pressure 140 / diastolic blood pressure 90mmHg higher or antihypertensive agents in oral;
4) Micro albuminuria: urinary albumin excretion rate 20 [mu] g / min or more or urinary albumin / creatinine ratio 30 mg / g or more.
Agents of the present invention may be used as a prophylactic and / or therapeutic agent for metabolic syndrome, which is determined by the diagnostic criteria described above.

For the criterion of diabetes, new criteria from the Japan Diabetes Society in 1999 have been reported.
According to this report, diabetes is a condition showing a fasting blood glucose level (glucose concentration in venous plasma) is 126 mg / dl or more, 75 g oral glucose tolerance test (75 g OGTT) 2 h level (glucose concentration in venous plasma) is 200 mg / dl or higher is a state in which from time to time blood glucose level (glucose concentration of intravenous plasma) of not less than 200 mg / dl. A condition not falling under the above-mentioned diabetes and different from "a fasting blood glucose level (glucose concentration in venous plasma) is 110 mg / dl or less than 75g oral glucose tolerance test (75 g OGTT) 2 h level (glucose concentration in venous plasma) is 140 mg / the state is not a state "(normal type) showing less than dl, it is called a" borderline type ".

As for the criteria of diabetes, from the ADA (American Diabetes Association) in 1997, from the WHO in 1998, and new diagnostic criteria were reported.
According to these reports, diabetes is a condition showing a fasting blood glucose level (glucose concentration in venous plasma) is not less 126 mg / dl or more, 75 g oral glucose tolerance test 2 h level (glucose concentration in venous plasma) is 200 mg / dl or higher which is a state that shows.
Further, according to the above reports, impaired glucose tolerance, fasting blood glucose level (glucose concentration in venous plasma) is 110 mg / dl or more and less than 126 mg / dl, and, 75 g oral glucose tolerance test 2 h level (veins glucose concentration) in the plasma of not less than 140 mg / dl or higher 200 mg / dl. Furthermore, according to the report of ADA, a condition referred to as a fasting blood glucose level (glucose concentration in venous plasma) is less than 110 mg / dl or higher 126 mg / dl state IFG (Impaired Fasting Glucose). On the other hand, according to the report of WHO, 75 g oral glucose tolerance test 2 h level (glucose concentration in venous plasma) is 140 mg / dl or more, the state is less than 200 mg / dl is called a IGT (Impaired glucose tolerance).
Agents of the present invention, diabetes is determined by the new criteria mentioned above, it may also be used as a prophylactic and / or therapeutic agent for impaired glucose tolerance (IFG, IGT).

Agents of the present invention, by blending as it is or a pharmacologically acceptable carrier, can be administered orally or parenterally. The carrier pharmacologically acceptable, various organic or inorganic carrier substances in common use as pharmaceutical materials, an excipient, lubricant, binder, disintegrant for solid preparations; and solvent, solubilizing agents, suspending agents, isotonizing agents, buffers and soothing agents for liquid preparations. If necessary, preservatives, antioxidants, coloring agents, can also be used pharmaceutical additives such as sweeteners.

The medicament of the present invention, the dosage form for oral administration (including soft capsule, a microcapsule), for example, tablets (including dragees and film-coated tablets), pills, granules, powders, capsules, syrups, emulsions, include such suspensions, also, as the dosage form for parenteral administration include injections, infusions, drip infusions, and the like suppositories. Further, suitable base (e.g., polymer of butyric acid, polymer of glycolic acid, butyric acid - copolymers of glycolic acid, a mixture of the polymers of glycolic acid butyric, polyglycerol fatty acid esters, etc.) it is also effective in combination to sustained release formulations.

The content of the inhibitor against PSGL-1 in the medicament of the present invention varies depending on the form of pharmaceutical preparations, 2 to 85 wt% based on the total usual formulation, preferably 5 to 70 wt%.

Inhibitors against PSGL-1 as a way of Seisuru the above dosage forms can be applied to known production methods generally used in the art. Further, when Seisuru the above dosage forms will optionally contain excipients conventionally used in pharmaceutical field in Seisuru in the dosage form, binders, disintegrating agents, carriers such as lubricants, sweetening agents, surfactants, suspending agents, various pharmaceutical additives such as emulsifiers and the like as appropriate, can be prepared by incorporating an appropriate amount.

For example, in the case where Seisuru inhibitors against PSGL-1 in tablet, excipient, binder, disintegrating agent, can be produced by containing lubricants and the like, if Seisuru pills and granules the, excipient, binder, can be produced by incorporating a disintegrating agent and the like. Further, if the Seisuru the powders and capsules such excipients, and the like sweetening agents when Seisuru Syrups, suspending agents when Seisuru emulsion or suspension, a surfactant , it can be prepared by incorporating the like emulsifier.

Examples of the excipient include lactose, sucrose, glucose, starch, sucrose, microcrystalline cellulose, powdered glycyrrhiza, mannitol, sodium bicarbonate, calcium phosphate, and calcium sulfate.

Examples of binders include 5 to 10 wt% starch glue liquid, 10 to 20% by weight gum arabic solution or gelatin solution, 1 to 5 wt% tragacanth solution, carboxymethylcellulose solution, sodium alginate solution, glycerin and the like.

Examples of the disintegrant include starch and calcium carbonate.

Examples of lubricants include magnesium stearate, stearic acid, calcium stearate, purified talc.

Examples of the sweetener include glucose, fructose, invert sugar, sorbitol, xylitol, glycerin, simple syrup and the like.

Examples of surfactants are sodium lauryl sulfate, polysorbate 80, sorbitan mono fatty acid esters, stearic acid polyoxyl 40.

Examples of suspending agents include gum arabic, sodium alginate, sodium carboxymethylcellulose, methylcellulose, bentonite and the like.

Examples of the emulsifier include gum arabic, tragacanth, gelatin, and the like polysorbate 80.

Furthermore, an appropriate amount in the production of inhibitors against PSGL-1 in the above-mentioned dosage form, optionally, coloring agents conventionally used in the field of pharmaceutical preparations, preservatives, fragrances, flavoring agents, stabilizers, and the like thickener it can be added.

The medicament of the present invention containing the inhibitor against PSGL-1 can be used safely in a stable and low toxicity. Dose the patient's condition and body weight of the day, type of inhibitor against PSGL-1, varies depending on the administration route, one skilled in the art can determine the appropriate amount in consideration of these factors. Moreover, administration can be administered once a day, or may be administered by dividing into 2 to 3 times.

When administering an inhibitor parenterally for PSGL-1 are generally administered in the form of a liquid (e.g., injection). The single dose varies depending on subject to be administered, target organ, symptom, route for administration, etc .; for example, in the form of an injection, to administer from about 0.1 mg ~ about 100mg per normal body weight 1kg intravenously it is convenient. The injections, in addition to intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, include such drip injections, As the depot preparation, and the like iontophoresis transdermal agent. Such injections are prepared by methods known per se, i.e., dissolution inhibitors against PSGL-1 in a sterile aqueous or oily solution is prepared by suspending or emulsifying. As the aqueous solution for injection, physiological saline and isotonic solutions containing glucose and other auxiliary agents (e.g., D- sorbitol, D- mannitol, sodium chloride, etc.) may be used in combination with an appropriate dissolution aid such , alcohols (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80, HCO-50) and the like can be used in combination. The oily liquid, sesame oil, include soybean oil, solubilizing agents (e.g., benzyl benzoate, benzyl alcohol etc.) may be used in combination with such. Further, a buffer (e.g., phosphate buffer, sodium acetate buffer), soothing agent (e.g., benzalkonium chloride, procaine hydrochloride), stabilizers (e.g., human serum albumin and polyethylene glycol), preservatives (eg, benzyl alcohol, phenol etc.) may be such a formulation. The prepared injection solution is usually filled in ampoules.

Agents of the present invention, metabolic diseases such as those exemplified above (e.g., metabolic syndrome, type 2 diabetes, impaired glucose tolerance, obesity, dyslipidemia (hypertriglyceridemia, hypercholesterolemia, low HDL cholesterolemia for postprandial hyperlipemia, etc.), etc.), it can lead to the prevention and / or therapeutic effect alone. However, inhibitors against PSGL-1 in applying to each disease as described above, can be used together to such diseases as appropriate and normal drugs used or therapy. When used in combination inhibitors and other drugs on PSGL-1, dosage forms of the combination drug and inhibitor against PSGL-1 is not particularly limited, at the time of administration, have been combined and the combination drug and inhibitor against PSGL-1 it may be Re. Such dosage forms, for example, (1) administration of a single preparation obtained by simultaneously formulating the concomitant drug and an as inhibitors against PSGL-1, and the combination drug and inhibitor for (2) PSGL-1 separately simultaneous administration by the same administration route of two kinds of preparations obtained by formulating, (3) two identical route of administration of preparations obtained by separate formulation of the combination drug and inhibitor against PSGL-1 administration at an interval via the, (4) simultaneous administration of different administration routes of two preparations obtained by separately formulating the combination drug and inhibitor against PSGL-1, for (5) PSGL-1 administration (eg inhibitors and combination drug and the time staggered in two different administration routes preparations separately obtained by formulating the order of administration, or the reverse of the order of agent → combination drug of the present invention of administration) and in It is below. The dose of the concomitant drug can be appropriately selected on the clinically employed dose as a reference. The proportion of the combination drug and inhibitor for PSGL-1 on the administration subject, can be appropriately selected depending on such administration route, target disease, condition, combination. For example, when the administration subject is a human, with respect to inhibitor 1 part by weight with respect to PSGL-1, the combination drug may be used in an amount of 0.01 to 100 parts by weight.

The present invention also provides a method of screening for substance capable of preventing or treating a metabolic disorder. The screening method comprises the following steps:
(I) the test substance is the function of PSGL-1 (i.e., activity or expression) assaying or inhibiting, and (II) a test substance that inhibited the function of PSGL-1 may prevent or treat metabolic disorders It is selected as a substance.
In the screening method, the term "metabolic disease" is synonymous with that described in the description of the prophylactic and / or therapeutic agent.
Hereinafter, it details the screening method of the present invention.

Test substance subjected to the screening method may be any known compounds and novel compounds, for example, nucleic acids, carbohydrates, lipids, proteins, peptides, organic low molecular compounds, compounds made using combinatorial chemistry techniques libraries, solid phase synthesis or the phage display method random peptide library produced by, or the microorganism, animals and plants, natural ingredients such as derived from marine organisms and the like. The test substance may be an unlabeled be labeled, also mixtures comprising label and unlabelled member at a predetermined ratio can also be used as the test substance. The labeling substance, for example, FITC, a fluorescent substance FAM such as luminol, luciferin, luminescent substances such as lucigenin, 3 H, 14 C, 32 P, 35 S, 123 radioactive isotopes such I, biotin, streptavidin e.g., affinity substance and the like.

Screening methods of the PSGL-1 Inhibitor]
As a substance that inhibits the PSGL-1 activity (PSGL-1 activity inhibitor), PSGL-1 and selectins (e.g., P- selectin), and the substance that inhibits the binding between. Hereinafter, a substance that inhibits the binding of PSGL-1 and selectins (hereinafter, also referred to as PSGL-1 binding inhibitor) for the screening method is described for.
PSGL-1 screening method of binding inhibitor (hereinafter, also referred to as a screening method A) comprises the following steps (a), (b) and (c):
(A) the presence of a test substance, contacting the selectin PSGL-1;
(B) the ability to bind the selectins and PSGL-1 in the presence of a test substance is measured, comparing the binding ability with the ability to bind to selectins and PSGL-1 in the absence of the test substance process;
(C) the based on the comparison result of (b), the product of step of selecting a substance that the test substance may prevent or treat metabolic disorders results in a reduction of binding ability to selectins and PSGL-1.

In step screening method A (a), the test substance, either selectin is placed in contact under conditions with PSGL-1. Contact to PSGL-1 selectin in the presence of the test substance in the presence of a test substance, selectins or the expressing cells may be done by contacting the PSGL-1 or its expression cells. Further, a test substance with respect to PSGL-1, the order of contacting the selectin is not particularly limited, be previously brought into contact with the PSGL-1 either, may be contacted simultaneously.
Selectin and PSGL-1 can be prepared by a method known per se. They are, for example, expressing cells (respectively, e.g., vascular endothelial cells and white blood cells) of the selectin or PSGL-1 can be isolated and purified from. However, rapid, and easily prepared and large quantities these proteins, also, to prepare them for human protein is preferably prepared a recombinant protein by a genetic engineering technique. Recombinant protein, cell lines may be one prepared by any of the cell-free system.
Selectin or PSGL-1 expressing cells are not particularly limited as long as it expresses a selectin or PSGL-1, selectin or PSGL-1 expressing cells described above were transformed with selectin or PSGL-1 expression vector cells and the like. The cells, one skilled in the art can readily identify or prepared primary cells, the primary culture cell lines derived from cells, commercially available cell lines, and the like can be cell lines obtained from cell banks can be used.

In step screening method A (b), first, the presence of the test substance, the ability to bind to selectins and PSGL-1 is measured. As "binding ability" to be measured is not particularly limited as long as it can evaluate the binding between selectins and PSGL-1, the amount of binding, binding strength (affinity constant, association rate constant, the parameters such as the dissociation rate constant including), the binding mode (including the concentration dependent binding) can be mentioned. Measurement of the binding ability, for example, be carried out by a per se known method such as flow cytometry using a selectin labeled with a labeling substance described above. Furthermore, using surface plasmon resonance binding capacity assay (Biacore, etc.) is also suitably used.
Then, ability to bind selectins and PSGL-1 in the presence of a test substance is compared with the ability to bind to selectins and PSGL-1 in the absence of the test substance. Comparison of the binding capacity is preferably performed on the basis of the presence or absence of a significant difference. Ability to bind selectins and PSGL-1 in the absence of the test substance, compared measurements of binding capacity of the selectin and PSGL-1 in the presence of a test substance, even binding capacity measured in advance, at the same time it may be measured binding capacity, but the accuracy of the experiments, it is preferred that the binding capacity measured simultaneously from the viewpoint of reproducibility.

In step screening method A (c), a test substance which results in a reduction of binding ability to selectins and PSGL-1 is selected as a substance capable of preventing or treating a metabolic disorder.

Screening methods of the PSGL-1 expression inhibitor]
Next, the screening method of a substance (PSGL-1 expression inhibitor) that inhibits the expression of PSGL-1 (hereinafter, also referred to as a screening method B) will be described. Screening Method B, following steps (a), including (b) and (c):
(A) contacting a measurable cellular expression of a gene test substance and PSGL-1 protein or encoding the same;
(B) the step of measuring the expression level in the cells contacted with the test substance, compared to the expression level in control cells the expression level not contacted with the test substance;
(C) the based on the comparison result of (b), the test substance reduced the expression level may prevent or treat metabolic diseases step of selecting as the material.

In the screening method B of step (a), the test substance is brought into contact with measurable cellular expression of PSGL-1 proteins. Contact of the test substance the expression of PSGL-1 protein to measurable cell may be carried out in a culture medium.
"PSGL-1 protein or measurable cell expression of the gene encoding" and, PSGL-1 gene products, e.g., transcripts, translation product (i.e., protein) directly or indirectly the level of expression of It refers to the evaluable cells. Directly evaluable cells the expression level of PSGL-1 gene product, obtained there a PSGL-1 gene in an expressible cell in nature, whereas, indirectly evaluable expression levels of PSGL-1 gene product such cells can be cells that allow a reporter assay for PSGL-1 gene transcription regulatory region.
PSGL-1 gene cells capable of expressing the naturally is, PSGL-1 gene are not potentially particularly limited as long as it expresses, cells constitutively expressing the PSGL-1 gene, PSGL-1 gene the inducing conditions (e.g., treatment with drugs) and the like cells expressed. The cells, one skilled in the art can readily identify, primary cells, the primary culture cell lines derived from cells, commercially available cell lines, and the like can be cell lines obtained from cell banks can be used. The PSGL-1 gene Examples of cells capable naturally expressed leukocyte (e.g., neutrophil, monocyte, macrophage, a subset of B cells, and all T cells) can be mentioned.
Cells that allow a reporter assay for PSGL-1 gene transcription regulatory region is a cell that contains a operably linked reporter gene PSGL-1 gene transcription regulatory region, to the region. PSGL-1 gene transcription regulatory region, a reporter gene is inserted into an expression vector.
PSGL-1 gene transcription regulatory region is not particularly limited as long as it is a region capable of controlling the expression of PSGL-1 gene, for example, the region from the transcription initiation point to upstream about 2kbp or 1 or more in the nucleotide sequence of the region, nucleotides are deleted, substituted or added in the nucleotide sequence, and the like can be given areas with the ability to control the transcription of the PSGL-1 gene.
Reporter gene may be any gene encoding a detectable protein or enzyme, for example GFP (green fluorescent protein) gene, GUS (beta-glucuronidase) gene, LUS (luciferase) gene, CAT (chloramphenicol acetyl transferase ) gene and the like.
Cells PSGL-1 gene transcription regulatory region, a reporter gene that is operably linked to the region is introduced, as long as possible to evaluate the PSGL-1 gene transcription regulatory function, i.e., the expression level of the reporter gene is quantitatively It not particularly limited as long as it can be analyzed. However, expressing physiological transcriptional regulatory factor for PSGL-1 gene, it is considered to be appropriate by the evaluation of the regulation of the expression of PSGL-1 gene, the cells to be the introduction of natural and PSGL-1 gene in expressible cells are preferred.
Culture medium the expression of the test substance and PSGL-1 gene and cell measurable contacted is suitably selected depending on the type of cells used, for example, about 5 to 20% fetal calf serum minimal essential medium with (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI1640 medium, 199 medium and the like. Culture conditions are also be appropriately determined depending on the type of cells used, eg, pH of the medium is about 6 to about 8, the culture temperature is usually from about 30 to about 40 ° C., incubation time from about 12 to about 72 hours.

In step (b) of the screening method B, submitted firstly, the expression level of PSGL-1 gene in the cells contacted with the test substance is measured. Measurement of expression level, such as the taking into account the type of cells used, may be carried out by a method known per se.
For example, a measurable cellular expression of PSGL-1 gene, if the PSGL-1 gene using cells capable of expressing in nature, expression level, PSGL-1 gene product, e.g., a transcription or translation product It can be measured by a method known per se as a target. For example, the expression level of transcripts, total RNA was prepared from cells, RT-PCR, can be measured by Northern blotting. In addition, the expression level of the translation product, prepared extracts from cells can be measured by an immunological technique. The immunological technique, radioisotope immunoassay (RIA method), ELISA method (Methods in Enzymol 70:. 419-439 (1980)), such as a fluorescent antibody technique can be used.
On the other hand, as measurable cellular expression of PSGL-1 gene, the use of cells that allow a reporter assay for PSGL-1 gene transcription regulatory region, expression levels can be measured on the basis of the signal intensity of the reporter.
Then, the expression level of PSGL-1 gene in the cells contacted with the test substance is compared with the expression level of PSGL-1 gene in a control cell not contacted with the test substance. Comparison of expression levels is preferably performed on the basis of the presence or absence of a significant difference. Expression of PSGL-1 gene in a control cell not contacted with the test substance, compared measurement of the expression amount of PSGL-1 gene in the cells contacted with the test substance, be an expression amount measured beforehand, at the same time measurement may be an expression amount was, but the accuracy of the experiments, it is preferable that the expression amount measured simultaneously from the viewpoint of reproducibility.

In step (c) of the screening method B, submitted test substance that reduces the expression level of PSGL-1 gene is selected as a substance capable of preventing or treating a metabolic disorder.

Hereinafter, a more detailed explanation of the present invention by way of test examples, the present invention is not intended to be limited to these.

Test Example 1: Macrophages in metabolic data and visceral fat diabetic model mice, examination of the expression of adhesion molecules
[Experiment]
8-week-old db / db mice (c57BL / KsJ-db / db) and C57 / BL6 mice weighing was measured fat mass and various metabolic data. RNA was extracted from visceral adipose tissue, by real-time RT-PCR, F4 / 80, a marker of macrophages, MCP-1, a marker of inflammation, is the adhesion molecule P- selectin, E- selectin, such as ICAM-1 It was examined gene expression level. Moreover, using DNA microarrays to analyze gene expression profiles in visceral fat tissue.
[result]
Compared to the control group in the db / db mice, weight, observed visceral fat weight, LDL, a significant increase in time to time blood glucose and IRI, differences in HbA1c was observed (Figure 1). The DNA microarray, gene showed increased expression of more than two-fold in db / db mice compared to the control group is 1001, a result of further performing cluster analysis, 40 of cells including L- selectin and PSGL-1 It showed increased expression of adhesion molecules associated gene (Table 1). Also, by real time RT-PCR, in db / db mice showed a significant increased expression of F4 / 80, MCP-1, PSGL-1 and P- selectin (Fig. 2).

Table 1 shows the results of DNA microarray using visceral fat db / db mice and BL6 mice. Compared to wild type (BL6 mouse), a total of 1001 genes showed increased expression of more than two-fold in db / db mice were grouped according to Gene Ontology category.

Figure JPOXMLDOC01-appb-T000001

Test Example 2: Study of high-fat diet mouse macrophage in metabolic data and visceral fat, the expression of adhesion molecules
[Experiment]
The low-fat diet and high-fat diet respectively 12 weeks C57 / BL6 mice 7 weeks old, underwent insulin tolerance test (ITT) and intraperitoneal glucose tolerance test (IPGTT). The In ITT insulin 0.7 units / kg was administered intraperitoneally, preload and 30 minutes after loading, blood glucose levels were measured 60 minutes and 120 minutes, were compared the rate of change of the load before the blood glucose level. Glucose 1.2 g / kg was administered intraperitoneally in IPGTT, preload and 30 minutes after loading, blood glucose was measured and IRI of 60 minutes and 120 minutes. In addition, gene expression in measurement and visceral adipose tissue similarly various metabolic data as in Test Example 1 was examined by real-time RT-PCR and DNA microarray.
[result]
In high-fat diet mice compared to low-fat diet group showed body weight, visceral fat weight, LDL, fasting glucose, a significant increase in fasting IRI and HbA1c (Figure 3). Also at ITT and IPGTT, showed impaired glucose tolerance and insulin resistance in the high-fat diet group (Fig. 4). The result of DNA microarray, observed genes that were found increased expression of more than double in the high-fat diet group than in the control group 566, in cluster analysis, increased expression of 37 of the cell adhesion molecule-related genes including PSGL-1 was observed (Table 2). Real-time RT-PCR, was a significant increased expression of CD68, MCP-1, PSGL-1 and P- selectin in db / db mice (FIG. 5).

Table 2 shows the results of a DNA microarray using visceral fat of the high fat diet mice and low-fat diet mice. The genes showed expression of more than double in high fat diet mice compared to low fat diet mice were classified according Gene Ontology category.

Figure JPOXMLDOC01-appb-T000002

Test Example 3: Analysis of PSGL-1 knockout mice
[Experiment]
The high fat diet was loaded 10 weeks 7 weeks old PSGL-1 knockout mice and C57 / BL6 mice, in the same manner as in Test Example 2 underwent ITT and IPGTT, was measured for various metabolic data. The RNA was extracted from visceral fat was examined gene expression levels of inflammation-related genes by real-time RT-PCR. Furthermore, performs PAS staining of visceral fat tissue, was measured the area of ​​visceral fat cells. Furthermore, was measured in liver weight and liver triglyceride content was also performed HE staining of liver tissue was studied changes in liver tissue.
[result]
In a high-fat diet PSGL-1 knockout mice and C57 / BL6 mice, weight, fat weight, but no difference was observed in the fasting blood glucose and HbA1c, T-Cho, LDL, TG, FFA, fasting IRI and serum leptin concentration was a significant reduction in the PSGL-1 knockout mice (Fig. 6). Was a significant improvement in glucose tolerance in the ITT in PSGL-1 knockout mice, but was not observed difference in blood glucose level even IPGTT, showed a significantly decrease in the IRI in PSGL-1 knockout mice (Fig. 7). In addition, we measured the area of ​​fat cells in PAS staining of visceral fat tissue showed a decrease of adipocyte area in PSGL-1 knockout mice compared to C57 / BL6 mice (FIG. 8). In real time RT-PCR, showed reduced expression of F4 / 80, MCP-1, NOS2 and significant mRNA of leptin in PSGL-1 knockout mice (Figure 9). The PSGL knockout mice observed a significant decrease small liver weight, tend to decrease also the liver triglyceride content was observed (FIG. 10 (A)). Further, in the HE stained image, hepatocellular damage (ballooning change) has been clearly suppressed in PSGL-1 knockout mice (FIG. 10 (B)).

[Conclusion]
PSGL-1 is expressed on leukocytes and vascular endothelial cells, P-, and E- and L- selectin function as a ligand is a molecule that induces leukocyte infiltration into inflammatory foci. Our results, by inhibiting PSGL-1, macrophage infiltration into visceral fat tissue of obese mice reduced, inflammation is suppressed insulin resistance in visceral fat was shown to improve. By inhibiting the PSGL-1, the prevention of metabolic syndrome, and it is possible to improve the onset of diabetes suppression of insulin resistance in obese individuals.

Agents of the present invention suppresses infiltration of macrophages into the visceral fat, may inhibit the development of inflammation and insulin resistance in adipose tissue I following. Thus, by using the agent of the present invention, without performing a diet or exercise therapy, metabolic syndrome, type 2 diabetes, impaired glucose tolerance, prevention of metabolic disorders such as obesity or dyslipidemia and / or treatment effect which can be obtained. By prevention and / or treatment of these metabolic diseases, it is possible to make prevention and / or suppression of progression of arteriosclerotic diseases caused by metabolic diseases according. The present invention also provides in combination methods of screening a substance capable of preventing or treating these metabolic diseases.

The present invention Japanese Patent Application No. filed in Japan 2008-128086: the (filing date May 15, 2008) has been the basis, the contents of which are incorporated in full herein by this reference.

Claims (5)

  1. Containing inhibitors against PSGL-1 as an active ingredient, the prevention and / or therapeutic agent for metabolic diseases.
  2. Metabolic diseases, metabolic syndrome, type 2 diabetes, impaired glucose tolerance, obesity or dyslipidemia, agent according to claim 1.
  3. PSGL-1 inhibitors for the specific neutralizing antibodies to PSGL-1, or specifically inhibitable antisense nucleic acid or a small molecular weight interfering RNA expression PSGL-1, or these expressible in mammalian cells a Do expression vector, agent according to claim 1.
  4. Comprising the following steps, a screening method for a substance capable of preventing or treating metabolic disorders:
    (I) the test substance is assayed either inhibit the function of PSGL-1, and (II) PSGL-1 of the test substance that inhibits the function be selected as a substance capable of preventing or treating a metabolic disorder.
  5. Metabolic diseases, metabolic syndrome, type 2 diabetes, impaired glucose tolerance, is obesity or dyslipidemia, The method of claim 4.
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