JP2017200890A - Therapeutic agent used for diabetes or diabetic complication including protein having biological activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new therapeutic agent for use in diabetes or diabetic complications.SOLUTION: According to the present invention, there is provided a therapeutic agent used for diabetes or diabetic complications, the agent comprising Protein S or a protein having a 96% or more of homology with Protein S and biological activity equivalent thereto. Here, the diabetic complications include diabetic nephropathy, renal fibrosis, stroke, myocardial infarction, heart failure, peripheral vascular disorder and renal disorder, etc. The dosage form may be preferably selected from the group consisting of subcutaneous, intramuscular, intravenous, intrabronchial, intraperitoneal, intrahepatic, intrauterine, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.SELECTED DRAWING: Figure 16

Description

  The present invention contains a protein having biological activity such as protein S (in the present specification, sometimes referred to as “Protein S” or “PS”. HPS means human PS). The present invention relates to a therapeutic agent for diabetes or a therapeutic agent used for diabetic complications.

The world's diabetic population continues to explode, rising to 285 million in 2010 and projected to exceed 400 million by 2030. Mortality in diabetics is twice as high as in non-diabetics, and life expectancy in middle-aged diabetics is reported to be 5-10 years shorter. The increase in diabetes mortality is mainly due to the development of cardiovascular complications (stroke, myocardial infarction, heart failure, peripheral vascular disorders), renal disorders, and malignant diseases. An increasing number of patients are forced to introduce dialysis due to diabetic nephropathy, but only symptomatic therapeutic agents are used for nephropathy, and no fundamental therapeutic agents have been established.
Type 1 diabetes is caused by selective destruction of pancreatic beta cells due to genetic, environmental, and autoimmune factors. Type 2 diabetes develops as a result of relative insulin deficiency due to insulin resistance and insulin secretion failure, but it has been suggested that the amount of β cells decreases with time, leading to insulin secretion failure. β-cell apoptosis is thought to be a common mechanism in both type 1 and type 2 diabetes. However, a useful treatment for stopping apoptosis of β cells has not yet been established.

Special table 2011-514881 gazette

Takagi T, Taguchi O, Aoki S, Toda M, Yamaguchi A, Fujimoto H, Boveda-Ruiz D, Gil-Bernabe P, Ramirez AY, Naito M, Yano Y, D'Alessandro-Gabazza CN, Fujiwara A, Takei Y, Morser J, Gabazza EC: Direct effects of protein S in ameliorating acute lung injury. J Thromb Haemost 2009; 7: 2053-2063 Chelakkot-Govindalayathil AL, Mifuji-Moroka R, D'Alessandro-Gabazza CN, Toda M, Matsuda Y, Gil-Bernabe P, Roeen Z, Yasuma T, Yano Y, Gabazza EC, Iwasa M, Takei Y: Protein S exacerbates alcoholic hepatitis by stimulating liver natural killer T cells.J Thromb Haemost 2015; 13: 142-154 Gil-Bernabe P, D'Alessandro-Gabazza CN, Toda M, Boveda Ruiz D, Miyake Y, Suzuki T, Onishi Y, Morser J, Gabazza EC, Takei Y, Yano Y: Exogenous activated protein C inhibits the progression of diabetic nephropathy J Thromb Haemost 2012; 10: 337-346

In the present specification, the present inventors indicate that (1) diabetic patients have decreased blood PS and show an inverse correlation with the HbA1c value, and (2) hPS (human protein S) in an STZ-induced diabetic mouse model. Inhibition of pancreatic β-cell apoptosis by exogenous hPS administration and exogenous hPS administration, and (3) improvement of insulin resistance in type 2 diabetes mouse model has been shown to improve the pathology of diabetes.
It was shown that the development of diabetic glomerulosclerosis was suppressed by overexpression of hPS and exogenous hPS administration. From these results, protein S has an anti-apoptotic effect on β-cells and an insulin resistance-improving effect, and is also a breakthrough diabetes effective for diabetic nephropathy for which no effective treatment has yet been established. It can be applied as a therapeutic agent.
The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel therapeutic drug for diabetes and / or a therapeutic drug for combined use such as diabetic nephropathy.

The therapeutic agent used for diabetes and / or diabetic complications according to the invention for achieving the above object is protein S (Protein S), protein having a homology of 96% or more with protein S, or equivalent biological activity. It contains the protein which has.
In addition, a therapeutic drug for diabetic nephropathy according to another invention is characterized by containing protein S (Protein S) or a protein having 96% or more homology with protein S and having an equivalent biological activity. . The homology between two amino acid sequences can be calculated by NCBI <http://blast.ncbi.nlm.nih.gov/Blast.cgi>, for example. In general, it is known that if the homology is high, it has a similar biological activity, and if the homology is low, it has another biological activity. In the present invention, a protein having 96% or more homology with protein S or a protein having substantially the same biological activity is included.
The therapeutic agent is administered from the group consisting of subcutaneous, intramuscular, intravenous, intrabronchial, intraperitoneal, intrahepatic, intrauterine, intravaginal, rectal, buccal, sublingual, intranasal and transdermal. It is preferable that at least one selected.
The disease to which the therapeutic agent of the present invention can be administered is diabetes and / or diabetic complications. Examples of diabetic complications include diabetic nephropathy, renal fibrosis, pulmonary fibrosis, stroke, myocardial infarction, heart failure, peripheral vascular disorder and renal disorder.
Currently, various therapeutic agents are being administered for type 1 diabetes and type 2 diabetes, but all remain targeted treatments. In diabetic patients, as the diabetes progresses, apoptosis of β cells secreting insulin occurs, and the number of β cells gradually decreases. Currently, there is no drug that can prevent apoptosis of β-cells, so that the progression of diabetes and its complications cannot be suppressed. In the present invention, it was proved that protein S suppresses apoptosis of β cells by in vitro and in vivo experiments. Moreover, in this invention, it was shown that protein S is effective for diabetic nephropathy. Therefore, the use of protein S instead of or in addition to conventional drugs can prevent the progression of diabetes and / or diabetic nephropathy or diabetic complications.

Examples of drugs that can be used in combination include at least one drug selected from the group consisting of sulfonylurea, glinide, biguanide, thiazolidinedione, glycosidase, DPP-4, incretin, and sodium / glucose transporter 2. Specifically, (a) sulfonylurea (promoting insulin secretion) includes glimepiride, glipizide and glyburide; (b) glinide (promoting insulin secretion) includes nateglinide and repaglinide; (c) biguanide (liver sugar) (Formation reduction) as metformin, (d) thiazolidinedione (glitazone) (insulin resistance reduction) as pioglitazone and rosiglitazone, (e) glucosidase (sugar absorption inhibition) as acarbose and miglitol ( f) DPP-4 (dipeptidyl peptidase-4) inhibitors (increasing insulin secretion by sugar) are alogliptin, linagliptin, sitagliptin, saxagliptin, and (g) incretin preparation (increasing insulin secretion by sugar) is exenatide. , Liraglutide is (h) sodium-glucose transporter As 2 (promoting glucose excretion in the urinary tract), canagliflozin and dapagliflozin can be used as concomitant drugs, respectively.
The dose of the therapeutic agent for diabetes or the therapeutic agent for diabetic nephropathy of the present invention is not particularly limited, but is 0.1 mg / kg · day to 10 mg / kg · day (preferably 0.5 mg / kg · day to 5 mg / day) per person. kg / day).

  According to the present invention, a novel therapeutic drug for diabetes and / or a therapeutic drug for diabetic nephropathy can be provided.

It is a graph which shows that hPS-TG mouse is resistant to STZ-induced diabetes. A: graph showing the transition of blood glucose level after administration of STZ, B: graph showing the transition of blood glucose level after administration of glucose, C: graph showing the transition of blood glucose level after administration of insulin, D: blood after administration of glucose It is a graph which shows transition of insulin concentration. It is a graph which shows the result of having investigated the insulin sensitivity when administering insulin or physiological saline to a db / db mouse. A is a graph showing the transition of blood glucose level after insulin administration, and B is a graph showing the results of the HOMA-IR test. It is a graph which shows the result of having investigated the change of the insulin sensitivity when hPS was added to L6 rat muscle cell, HepG2 human hepatocyte, and 3T3 L1 mouse adipocyte. A: graph showing glucose uptake or release with or without insulin and / or hPS; B: graph showing glucose uptake or release with or without insulin, hPS and / or anti-hPS antibody. It is the photograph figure (A) and graph (B) which show that hPS-TG mouse | mouth suppresses the apoptosis of (beta) cell. It is a graph which shows that hPS-TG mouse | mouth suppresses apoptosis of (beta) cell. It is a graph which shows the change of A: insulin dyeing | staining area | region, B: glucagon dyeing | staining area | region, C: apoptotic area | region. FIG. 2 is a micrograph (A) and graphs (B, C) showing that an hPS-TG mouse suppresses an inflammatory reaction and apoptosis of β cells. It is the result of the in vivo test | indication which shows that apoptosis of an islet is suppressed when hPS is administered. A: Graph showing changes in blood glucose level after STZ administration, B: Annexin V (showing the degree of apoptosis) and Propidium Iodide (nucleic acid staining dye of dead cells) in STZ / SAL group and STZ / hPS group It is a graph which shows intensity | strength, C: It is a graph which shows the dead cell ratio in STZ / SAL group and STZ / hPS group. It is a result which shows that hPS suppresses apoptosis of β cells. The upper left is a graph showing the intensity of Annexin V (Annexin V: indicating the degree of apoptosis) and Propidium Iodide (the nucleic acid staining dye of dead cells), the upper right is a graph showing the percentage of apoptotic cells, the center is a micrograph, the lower is hPS It is a gel filtration data and a graph which show that the cutting | disconnection of caspase 3 is suppressed by FIG. It is a result which shows that hPS suppresses apoptosis of β cells. It is a result which shows that phosphorylation of Akt / PKB and IκB of MIN6 cells is increased. A: A diagram showing the phosphorylation of Akt / PKB and IκB by flow cytometry, a graph, a diagram showing the results of Western blotting, B: a graph showing the results of examining the expression of BIRC3 by Western blot, C : It is a graph which shows the result of having investigated the expression of BIRC3 by RT-PCR method. Data showing that phosphorylation of Akt / PKB and IκB in primary β cells is promoted by hPS. A: A graph showing the results of examining Akt / PKB by flow cytometry, and B: A graph showing the results of examining IκB by flow cytometry. It is a graph which shows the result of having investigated the relationship between three types of TAM PS receptors (Tyro3, Axl, Mer) and apoptosis by hPS by flow cytometry. It is a graph which shows the result of having investigated the relationship with three types of TAM PS receptors (Tyro3, Axl, Mer) and the apoptosis by hPS by the flow cytometry using the antibody. It is a graph which shows the result of having investigated the change of expression of all the genes (BIRC1a, BIRC1b, BIRC2, BIRC3, BIRC4, BIRC5, BIRC6 and BIRC7) which belong to a mouse BIRC (IAP) family. It is a graph which shows the result of having investigated the change of apoptosis when specifically suppressing a BIRC gene using siRNA. It is a graph which shows the result of having investigated the relationship between hPS and the expression level of Blc2, Bax, BclxL, and Apaf1. It is data which shows the result which confirmed that hPS showed the improvement effect of diabetic nephropathy. A: test protocol, B: graph showing changes in body weight and blood glucose level during the test, C: graph showing collagen, hydroxyproline and blood creatinine concentrations in tissue, D: graph showing urinary L-FABP, E : It is the graph which investigated the expression level of TGF (beta) 1 and podosin. It is the microscope picture figure and graph which investigated glomerular mesangial expansion (Mesangial expression), fibrotic area (Fibrotic area), and apoptosis area (Apoptotic area). It is a graph which shows the change of the amount of blood TAT (coagulation activation marker). It is data which shows another test result which confirmed that hPS showed the improvement effect of diabetic nephropathy. A: Test protocol, B: Graph showing changes in blood glucose level during the test, C: Graph showing hydroxyproline, blood creatinine concentration and urinary L-FABP, D: Glomerular mesangial expansion and fibrosis area were examined. It is a microscope picture figure and a graph. It is a graph which shows the change of coagulation factor (TF), fibrinolysis (PAI-1), and t-PA.

Next, embodiments of the present invention will be described with reference to the drawings. The technical scope of the present invention is not limited by these embodiments, and can be implemented in various forms without changing the gist of the invention.
PS is known as an anticoagulant and is also involved in inflammation and apoptosis. The effect of PS on diabetes has not been known so far. The inventor compared the progression of diabetes between wild-type mice and transgenic mice overexpressing hPS (hPS-TG). In addition, wild-type mice were used to examine the effect of the presence or absence of hPS administration on the progression of diabetic nephropathy, and whether the progression of diabetic nephropathy differs between wild-type mice and hPS-TG. hPS-TG showed significantly superior results in blood glucose concentration, glucose tolerance, insulin sensitivity and insulin secretion compared to wild type mice. PS is administered to db / db mice, mouse adipocytes, rat muscle cells, and human hepatocytes, which are non-insulin-dependent (type 2) diabetes model animals and homozygous individuals that develop symptoms similar to human obese diabetes Then, insulin sensitivity improved. When PS was given to pancreatic β cells, significant suppression of apoptosis was observed with increased expression levels of BIRC3 and BcL-2 and increased activity of Akt / PKB. Diabetic nephropathy progression was significantly suppressed in diabetic wild-type mice and hPS-TG diabetic mice administered with PS compared to the control group. Compared to healthy subjects, patients with type 2 diabetes had significantly lower blood free PS levels. According to this study, it was found that PS has the effect of suppressing β-cell apoptosis and inhibiting the progression of diabetic nephropathy.

  PS is a glycoprotein produced in the liver with a molecular weight of 69,000 in a vitamin K-dependent manner. It works as an anticoagulant by increasing the inhibitory activity of active protein C (APC) several times. APC directly inhibits the activity of the prothrombinase complex and tenase, whereas PS itself stimulates inhibition of the tissue factor pathway. The physiological importance of PS as an anticoagulant is explained by the fact that patients with congenital PS deficiency suffer from severe thrombotic complications. Apart from anticoagulant effects, PS regulates inflammatory responses and apoptotic pathways via Tyro3, Axl and Mer (TAM) tyrosine kinase receptors. In addition, PS suppresses the expression of inflammatory cytokines and exhibits the neuroprotective action of APC in various cells in vitro and a mouse model of LPS-induced acute lung injury (Non-patent Document 1). In plasma, PS exists as a free form and a complex form bound to C4b-binding protein (C4BP), which is an inhibitor of the classical complement activation pathway. The possibility that PS suppresses inflammation caused by activation of complement by the localization of C4BP in the cell membrane has been reported. In addition, PS promotes the clearance of apoptotic cells by macrophages and suppresses inflammation and immune responses. Furthermore, PS suppresses apoptosis directly through activation of the Akt transmission pathway. The present inventors focused on the anti-apoptotic effect of PS and proved the hypothesis that PS works to protect against diabetes by inhibiting the apoptosis of pancreatic β cells and further suppresses the development of diabetic nephropathy. .

1. Test method <Subject>
Blood samples were collected from 6 type 1 diabetics, 26 type 2 diabetics, and 37 healthy individuals undergoing glycemic treatment. PS and C4BP concentrations in each blood sample were measured (Table 1). None of the diabetic patients developed diabetic or other neurological disorders. All patients and healthy subjects received informed consent, and the study protocol was approved by the Mie University Ethics Committee (No. 2194).

<Test animal>
The present inventor produced homozygous human PS transgenic mice (hPS-TG) from C57BL / 6 strain mice (Non-patent Document 2). Briefly, it is as follows. Full-length hPS cDNA was integrated into the pCAG plasmid (cytomegalovirus enhancer + chicken + β-actin promoter) having a CAG promoter and a rabbit β-globin polyadenylation site. This plasmid was cleaved at a predetermined position, purified and microinjected into fertilized eggs obtained from C57BL / 6J mice, and transformed mice were detected by Southern blotting. Many organs of hPS-TG expressed hPS, and the plasma hPS concentration was 85 ± 3 μg / ml (Non-patent Document 2). Wild type (WT) littermates were used as controls. There was no difference in islet mass between WT and hPS-TG. 19-22 g male wild type mice (8-10 weeks old) and 31-33 g db / db male mice (6 weeks old) were purchased from Japan SLC (Hamamatsu City). All animals were maintained in an environment free of specific pathogens and kept in the Mie University Animal Facility with a 12-hour light-dark cycle. The studies herein were performed according to ARRIVE guidelines for animal studies. Each test protocol was approved by the Mie University Animal Research Committee (Approval No: 24-50) and the animal treatment procedures were performed according to institutional guidelines. The mice were randomly grouped and the measurer was not informed of the details of each group.

<Induction of diabetes>
In order to evaluate the sensitivity to induction of diabetes, 200 μL (40 mg / kg body weight) of streptozotocin (STZ: Sigma) was used for hPS-TG (hPS-TG / STZ) and wild-type littermates (WT / STZ). ) Was administered intraperitoneally for 5 consecutive days. Control mice were intraperitoneally administered 200 μL saline for 5 consecutive days (hPS / SAL, WT / SAL).
<Evaluation of diabetes>
After one week of STZ administration (hPS TG / STZ, WT / STZ) or saline administration (hPS / SAL, WT / SAL) for 4 weeks, fasting blood glucose concentration was measured . At 3 weeks after administration of STZ or saline, an intraperitoneal glucose tolerance test (IPGT) was performed 16 hours after administration of intraperitoneal glucose (1 g / kg body weight). Blood samples were collected from the tail vein on days 0, 7, 14, 21 and 28 for blood glucose measurement. Insulin sensitivity test is performed by administering intraperitoneal insulin (1U / kg body weight) to non-fasted mice, and insulin secretion test is performed by administering intraperitoneal glucose (3g / kg body weight) to mice after 16 hours fasting It was. Blood samples were taken from the tail vein to measure glucose or insulin concentrations. All groups of animals were sacrificed 4 weeks after STZ or saline administration and the pancreatic tissue was excised for use in histochemical staining.

<Induction of diabetic neuropathy>
After unilateral nephrectomy for hPS-TG mice and WT littermates, after 4 weeks of recovery, WT (WT / STZ) and hPS-TG (hPS-TG / STZ) continued for 5 days Then, STZ was intraperitoneally administered. Further, for hPS-TG, STZ was intraperitoneally administered for 5 days in addition to induce diabetes similar to that of WT. For the control groups (hPS TG / SAL, WT / SAL), the same amount of physiological saline was administered intraperitoneally. Mice were sacrificed 8 weeks after administration of STZ or saline. In another study, WT mice that underwent unilateral nephrectomy were administered intraperitoneally with STZ or physiological saline for 5 consecutive days after a 4-week recovery period, and osmotic pressure over 4 weeks. HPS or physiological saline was transdermally administered from a minipump.
<Tissue processing and staining>
Mice were euthanized by pentobarbital overdose, then the pancreas and kidney were removed, dehydrated and paraffin-embedded, cut into 3 μm sections, and the pancreas was hematoxylin / eosin (H & E) stained and immunostained For the kidney, periodate Schiff (PAS) and Masson trichrome staining were performed, respectively. Using an Olympus BX50 microscope, Olympus DP70 digital camera and WinROOF image processing software using an Olympus BX50 microscope to examine the area of islets stained with H & E and glomeruli stained with PAS or trichrome (> 30 / mouse). Calculated in combination.

<Immunohistochemistry>
Insulin, glucagon and F4 / 80 immunostaining were performed using biotinology studies according to standard methods using specific antibodies obtained from DAKO Corporation (California, USA) and Novus Biologicals (Colorado, USA). It was measured at the place. As described above, the immune response area of insulin or glucagon in all visible islets was measured from 4 pancreatic sections (40-50 fields / mouse) by a researcher who was informed of the experimental group.
<Biochemical analysis>
Blood glucose was measured by the glucose oxidase method, and insulin was measured using a kit for ALPCO diagnosis (New Hampshire, USA). Thrombi-antithrombin complex (TAT: Sedralene Institute (Ontario, Canada)) and total plasminogen activator inhibitor 1 (PAI-1: E1 / PAI-1, R & D, Minnesota, USA) Measured using kit, total collagen and hydroxyproline. Complement C4BP was measured using an EIA kit from Assay Pro (Missouri, USA). Total hPS was measured according to the previous report (Non-patent Document 2). To measure free hPS, coat a 96-well microplate with C4BP (ATGen: Sampyeong-dong, Korea), and after washing and blocking, add biotin-labeled polyclonal rabbit anti-hPS antibody (DakoCytomation: Danish Glostrap) did. The urinary liver fatty acid binding protein (L-FABP), a marker for assessing the severity of diabetic nephropathy, uses the EIA kit (R & D), and the tissue factor is EIA (Santa Cruz Biotechnology) using a primary biotinylated antibody. : California, USA) and tissue plasminogen activator activator (tPA: Nottingham, UK) was measured using a chromogenic substrate (S-2288). The homeostasis model assessment (HOMA-IR) for evaluating insulin resistance was determined according to the previous report as HOMA-IR = [fasting insulin (μU / mL) × fasting glucose (mmol / L)] / 22.5.

<Cell culture>
Mouse pancreatic β cell line MIN6 was obtained from Osaka University, L6 rat skeletal muscle cells were obtained from Kobe University, HepG2 cells were obtained from Riken Cell Bank, and rodent 3T3 L1 and rat RAW264 cells were obtained from ATCC. The cells were cultured in DMEM (Sigma Aldrich) containing non-activated FCS. Adipocyte differentiation was induced by treatment with 0.5 mmol / l 3-isobutyl-1-methylxanthine, 4 mg / ml dexamethasone, 1 mg / ml insulin and 10% FCS.
<Glucose uptake / release test>
Cells were incubated in DMEM (low concentration glucose) + 1% BSA for 6 hours, transferred to DMEM containing high concentration glucose (1 g / L), and then plasma-derived hPS (20 μg / ml: Enzyme Research Laboratories, USA) Indiana: purity of 95% or higher) was added, and after 30 minutes, insulin (200 nM) was added and cultured for 4 hours. The amount of glucose in the culture supernatant was measured using a glucose colorimetric assay kit (Biovision).
<Isolation of primary mouse islet cells>
Pancreatic tissue was excised from mice (WT and hPS-TG) euthanized 4 weeks after intraperitoneal administration of STZ or physiological saline, cut into 1-2 mm, and then in 1 mg / ml collagenase Incubated at 37 ° C for 30 minutes. Prior to use in the assay, after centrifugation and resuspension, the cells are placed on a discontinuous Percoll gradient, centrifuged, and then the islet cell layer is recovered and washed and dispersed with a trypsin / EDTA solution. It was.

<Evaluation of apoptosis>
Apoptosis in islet histological samples was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) using a commercially available kit (Chemicon International, California, USA). As described above, the number of TUNEL positive cells in the islets was counted in 6 sections (40-50 fields) per mouse by a researcher who did not know the contents of the sample. Primary pancreatic β cells and MIN6 β cell lines were stained with FITC annexin V (BD Pharmingen) and propidium iodide, and then analyzed by flow cytometry (BD Biosciences: Oxford, England). Furthermore, apoptosis of the MIN6 β cell line was evaluated by immunofluorescence microscopy after staining as described above. Measures mRNA expression of inhibitors of apoptotic repeat-containing baculovirus inhibitor (BIRC) 3 / apoptotic inhibitor (IAP), B cell lymphoma 2 (Bcl-2) regulatory protein family and apoptotic protease activator 1 (APAF1) did.
<In vivo evaluation of insulin sensitivity and beta cell apoptosis>
The effect of externally administered hPS on insulin sensitivity was evaluated in db / db mice. Insulin sensitivity test and HOMA-IR measurement were performed after 0, 1, 2, 4 and 6 hours on fasted db / db mice administered subcutaneously with hPS (2 mg / kg; n = 5) or physiological saline (n = 5). Carried out. To evaluate the effect of hPS on β-cell apoptosis induced by STZ in vivo, exogenous hPS (2 mg / kg: n = 6) or saline (n = 6) was applied to STZ in wild-type mice. Transdermal administration was performed for 5 days 1 hour before administration, and euthanized 9 days after the last STZ administration. Islets were harvested, incubated in trypsin-containing EDTA solution at 37 ° C, and after gentle dispersion, apoptosis was assessed by flow cytometry.

<Macrophage phenotype evaluation test>
RAW264.7 cells were treated for 30 minutes with or without hPS (20 μg / ml), and cultured for 24 hours with or without high-concentration glucose. The expression of NO synthase-induced M1 marker and M2 marker arginase 1 was analyzed by RT-PCR.
<Evaluation of TAM receptor mediation>
After treating MIN6 cells with anti-Tyro3, anti-Axl, anti-Mer or isotype IgG for 30 minutes, 20 μg / ml hPS and 2 mM STZ were added, cultured for 24 hours, and then examined for apoptosis. All antibodies were goat IgG and purchased from R & D (Minnesota, USA).
<Effect of RIRC3 knockdown>
33 nmol BIRC3 siRNA or scrambled siRNA was introduced into MIN6 cells, cultured for 30 minutes with or without hPS, and cultured for 24 hours with or without 3 mM STZ. Apoptotic cells were examined by flow cytometry.

<Akt / PKB and NFκB activity>
Activating Akt / PKB and NFκB activity in MIN6 cells with antibodies against p-Akt / PKB, Akt / PKB, p-IκB, cleaved caspase 3 and β-actin (cell signaling: Massachusetts, USA) and human / mouse cIAP After performing a general method using an antibody against -2 / HIAP-1 (R & D: Minnesota, USA), it was examined by Western blot. The activity of Akt / PKB and NFκB when hPS was added to primary pancreatic β cells was examined by flow cytometry with or without anti-hPS.
<Gene expression analysis>
Total RNA was extracted from pancreatic tissue with Trizol reagent (Invitrogen, California, USA), treated with reverse transcriptase using oligo dT primer, and Superscript Preamplification System Kit (Invitrogen) Amplified by the PCR used. Quantitative amplification reactions were performed using Applied Biosystems Step One Real-Time PCR System, Tackman Master Mix and SYBR Green. Table 2 shows the base sequences of the primers. Data were analyzed with Applied Biosystems 7500 software and gene expression was normalized at the GAPDH transcription level.

<Statistical analysis>
Data were expressed as mean ± standard error (SEM) unless otherwise specified. Statistical differences between groups were calculated by ANOVA using the Tukey method and post hoc analysis, and differences between the two groups were calculated by Mann-Whitney U test. StatView 4.1 package software (Abacus concept: California, USA) was used for statistical analysis. A risk rate of less than 5% (p <0.05) was considered statistically significant.

2. Test results <There is less free hPS in the blood of diabetic patients>
There was no significant difference in BMI and age between patients with type 2 diabetes and healthy subjects (Table 1). The blood free hPS concentration was significantly lower among healthy subjects in both type 1 and type 2 diabetics, and the C4BP level was significantly higher in type 2 diabetics. There were no significant differences in gender differences. In patients with type 2 diabetes, there was a significant negative correlation between free PS and hemoglobin A1c (HbA1C), and a significant positive correlation between C4BP and HbA1c (Table 3). . In patients with type 2 diabetes, age was significantly correlated with free PS and C4BP. In type 1 diabetic patients, there was no significant difference between parameters (Table 4).

<HPS TG mice have strong resistance to STZ-induced diabetes>
Diabetes was induced with STZ in hPS-TG mice and wild-type mice, and the severity of diabetes was compared. Mice were injected intraperitoneally with STZ or saline for 5 consecutive days. A. Change in blood glucose level after injection of STZ or saline (WT / SAL n = 3, hPS / SAL n = 4, WT / STZ n = 7, hPS TG / STZ n = 6), B. STZ injection Intraperitoneal glucose tolerance test (WT / SAL n = 3, hPS / SAL n = 3, WT / STZ n = 7, hPS TG / STZ n = 7) under fasting for 16 hours after 3 weeks, C. insulin Sensitivity test (WT / SAL n = 3, hPS / SAL n = 3, WT / STZ n = 5, hPS TG / STZ n = 3), D. Serum insulin concentration after intraperitoneal administration of glucose under fasting for 16 hours ( WT / SAL n = 3, hPS / SAL n = 4, WT / STZ n = 7, hPS TG / STZ n = 5). All data were expressed as mean ± SEM, and more representative results were obtained from three independent experiments. In the graph, WT represents wild type; STZ represents streptozotocin; hPS represents protein S; SAL represents physiological saline. Statistical significance was shown as * p <0.005 vs hPS / STZ; ‡ p <0.01 vs WT / SAL.
As shown in FIG. 1A, the blood glucose concentration at the time of non-fasting 14, 21, and 28 days after the first STZ administration was significantly decreased in the hPS-TG mice compared to the wild-type mice. Seven days after STZ administration, the blood glucose concentration when STZ was administered to wild-type mice was significantly increased compared to control wild-type mice administered with physiological saline. On the other hand, 21 days after STZ administration, the group in which STZ was administered to hPS-TG mice showed a significant increase compared to the hPS-TG mice to which physiological saline was administered. As a result of the IPGT test, the blood glucose level of STZ-administered hPS-TG mice significantly decreased compared to STZ-administered wild-type mice at all points. In addition, the hPS-TG mice and wild type mice administered with STZ had significantly increased blood glucose levels compared to mice administered with physiological saline (FIG. 1B). In the insulin sensitivity test after glucose administration, wild-type mice administered with STZ have significantly higher blood glucose levels than hPS-TG mice administered with STZ (FIG. 1C), and blood 30 minutes after intraperitoneal glucose administration. The intermediate insulin concentration was significantly higher in hPS-TG mice than in STZ-administered wild-type mice (FIG. 1D).
<Insulin sensitivity improves when hPS is administered>
In db / db mice to which hPS was administered, insulin sensitivity was significantly improved 15 minutes after insulin administration, and the state persisted compared to control mice. The db / db mice administered with hPS showed an improvement trend in HOMA-IR, but no significant difference was observed (FIG. 2).
FIG. 3A shows L6 rat muscle cells, 3T3-L1 adipocytes HepG2 cells in low glucose medium and high glucose (1 g / L) medium with insulin alone (200 nM), hPS alone (20 μg / ml) or both. The results of measuring the glucose concentration in the medium after culturing for 4 hours were shown in B, and the results of adding anti-hPS antibody (20 μg / ml) before stimulation with insulin and hPS were shown in B. Representative data from two independent experiments are shown. In the figure, group 1 is n = 3, and * p <0.005 vs insulin (-), hPS (-); ** p <0.05 vs insulin (+), hPS (-); ‡ p <0.02 vs insulin ( -), hPS (-), anti-hPS (-); ¶p <0.01 vs insulin (+), hPS (-), anti-hPS (-); §p <0.001 vs insulin (+), hPS (+ ), anti-hPS (-).
When hPS was added to L6 rat muscle cells, HepG2 human hepatocytes and 3T3 L1 mouse adipocytes, insulin sensitivity was improved (FIG. 3A). Since the effect of hPS was inhibited by the anti-hPS antibody, it was confirmed to be specific (FIG. 3B).

<HPS-TG mice suppress inflammatory reaction and apoptosis of islet β cells>
In FIG. 5, the pancreas was removed 28 days after intraperitoneal injection of STZ or physiological saline, and double immunostaining of insulin (green) and glucagon (red) was performed (n = 3 for each group). Apoptotic cells were stained by the TUNEL method (WT / SAL n = 3; hPS / SAL n = 3; WT / STZ n = 6; hPS / STZ n = 6). The area of insulin, glucagon, and apoptosis-positive cells was quantified using software (WinROOF). For comparison, the average value of WT / SAL was set to 100%. All data are shown as mean ± SEM and representative data from 3 experiments. In the figure, WT, wild type; STZ, streptozotocin; hPS, protein S; SAL, physiological saline are shown. Statistical differences indicate * p <0.05 vs WT / SAL and hPS / SAL groups; ** p <0.05 vs WT / STZ groups.
STZ-administered wild-type mice that were sacrificed 28 days after STZ administration had significantly greater islet area and insulin-specific staining area compared to saline-administered wild-type mice, saline-administered hPS mice, and STZ-administered hPS mice. Decreased (FIG. 4) and the area of apoptotic cells increased significantly (FIG. 5). STZ-administered wild-type mice showed significantly decreased insulin-stained areas and increased glucagon-stained areas compared to saline-administered wild-type mice, but saline-treated hPS-TG mice and STZ-treated hPS- There was no significant difference between TG mice (FIG. 5).
Compared to STZ-administered wild-type mice and saline-administered wild-type mice, infiltration of macrophages into pancreatic islets was significantly reduced in STZ-administered hPS mice, but between saline-administered hPS mice and STZ-administered hPS mice. There was no significant difference (FIGS. 6A and B). In vitro, hPS promoted differentiation of M2-type macrophages (FIG. 6C).
<In vivo, administration of hPS suppresses islet apoptosis>
C57BL / 6 wild-type mice were examined for apoptosis of pancreatic cells after subcutaneous administration of hPS or physiological saline during and after STZ administration. In mice administered with hPS, apoptosis of β cells was significantly suppressed compared to controls administered with physiological saline (FIGS. 7A, B, and C).
<HPS suppresses apoptosis and activates Akt / PKB and NFκB of pancreatic β cells>
FIG. 8 and FIG. 9 show that apoptosis of mouse β cell line MIN6 is induced by STZ in the presence of hPS (20 μg / ml) or physiological saline, and stained with annexin-FITC and iodopropidium and flow cytometry. The result of evaluation was shown. The results of flow cytometry (upper left of FIG. 8) and fluorescence microscope (center of FIG. 8) are shown. After treatment with hPS (20 μg / ml) for 30 minutes and 60 minutes, phosphorylation of Akt / PKB and IκB was evaluated by flow cytometry (FIG. 9A) and Western blotting (FIG. 9B). In addition, expression of mBIRC3 was analyzed by Western blotting (FIG. 9B). Islet cells were isolated after induction of diabetes from wild-type and hPS TG mice, and expression of mBIRC was evaluated by quantitative RT-PCR (9C). All data are shown as mean ± SEM, and representative data were shown in 3 independent experiments (n = 3 for each group). In the figure, STZ, streptozotocin; hPS, protein S; SAL, physiological saline SAL / SAL, physiological saline only; pretreated with hPS / SAL, protein S and administered with physiological saline; SAL / STZ, physiological saline It shows that STZ was administered after pretreatment; STZ was administered after pretreatment with hPS / STZ, hPS. Statistical markers indicate * p <0.05 vs SAL / SAL and hPS / SAL groups; ** p <0.05 vs SAL / STZ group; ¶ p <0.05 vs hPS (−).
When MIN6 cells were pretreated with hPS, apoptosis was significantly suppressed when STZ was administered, and caspase 3 cleavage was significantly suppressed by hPS, compared to control (FIG. 8). Phosphorylation of Akt / PKB and IκB in MIN6 cells was significantly increased when treated with hPS compared to control (FIG. 9A). The phosphorylation of Akt / PKB and IκB in primary β cells was significantly promoted by hPS (FIGS. 10A and B). MIN6 cells expressed three types of TAM PS receptors (FIG. 11), and only Mer of these mediated the inhibitory effect of apoptosis by hPS (FIGS. 12A and B).

<HPS regulates IAP and Bcl2 proteins>
Changes in expression of all genes belonging to the mouse BIRC (IAP) family were examined in MIN6 cells treated with STZ alone, hPS alone and both STZ and hPS. BIRC1b and BIRC3 mRNA expression levels were significantly increased in cells treated with hPS (hPS / SAL) compared to cells treated with physiological saline (SAL / SAL). In cells treated with hPS (hPS / STZ), the expression level of BIRC3 increased significantly, and BIRC1b increased (p = 0.05) compared to cells treated with saline prior to STZ administration (SAL / STZ) (FIG. 13). According to Western blot, the mRNA expression level of BIRC3 was significantly increased in MIN6 cells (hPS / SAL, hPS / STZ) treated with hPS compared to the control (FIG. 9B). When BIRC3 was specifically blocked with siRNA, the effect of hPS on apoptosis of MIN6 cells was reduced (FIGS. 14A, B, C). Compared with cells isolated from STZ-treated wild-type mice (WT / STZ), BIRC3 mRNA expression levels were significantly higher in cells isolated from STZ-treated hPS-TG mice (hPS-TG / STZ). Increased (FIG. 9C). In an in vitro test using MIN6 cells, hPS increased the expression of BcL2, which suppresses apoptosis, but suppressed the expression of Bax, which is an intracellular protein that promotes apoptosis (FIG. 15).

<HPS improves diabetic glomerulosclerosis>
Next, in addition to the β-cell apoptosis-suppressing effect of hPS, it was investigated whether or not it showed an improving effect on diabetic nephropathy.
As shown in FIG. 16A, STZ (40 mg / kg) was intraperitoneally injected into a single nephrectomized mouse, and hPS was administered by subcutaneous pump implantation after 4 weeks. Changes in body weight and blood glucose level (Fig. 16B), results of colorimetric analysis of collagen and hydroxyproline in kidney tissue (Fig. 16C), enlargement of glomerular mesangial region, PAS staining (upper four in Fig. 17) ), Collagen deposition was evaluated by Masson-Trichrome staining (four middle plates in FIG. 17), and apoptosis was evaluated by the TUNEL method (four bottom plates in FIG. 17). Quantification was performed using software (WinROOF). For comparison, the SAL / SAL group was taken as 100%. In the kidney tissue, TGFβ1 and podocin mRNA expression was measured by RT-PCR (FIG. 16E), and plasma TAT was measured by ELISA (FIG. 18). All data are shown as mean ± SEM. The scale bar in the micrograph is 20 μm. Two experiments were performed to show representative data. N = 5 for each group. In the figure, WT, wild type; STZ, streptozotocin; hPS, protein S; SAL, and physiological saline are shown, respectively.

  Nephrectomy was performed on one side of wild type C57BL / 6 mice. After a complete recovery period after surgery, diabetes due to STZ was induced. As a control, physiological saline was administered. STPS-administered hPS group (STZ / p-hPS) and STZ-administered saline group (STZ / p-SAL) showing the same severity of diabetes 4 weeks after administration of STZ or saline And 4 groups of post-saline hPS group (SAL / p-hPS) and non-diabetes physiological saline group (SAL / p-SAL) over the next 4 weeks Then, hPS or physiological saline was subcutaneously administered using an osmotic pressure minipump (FIG. 16A). Two groups (STZ / SAL and STZ / hPS) to which STZ was administered developed diabetes compared with the group given physiological saline, and showed significant weight loss and increased blood glucose level. At this time, a significant difference in body weight was observed between the two groups (STZ / SAL and STZ / hPS) administered with STZ, but there was no difference in blood glucose level (FIG. 16B). In the renal tissue collagen and hydroxyproline, blood creatinine, L-FABP, renal tissue PAS staining region and apoptotic cell region, the STZ / SAL group significantly increased compared to the STZ / hPS group (FIGS. 16C, D, FIG. 17). Compared with the control group and STZ / hPS group, the expression level of TGFβ1 mRNA was significantly increased and the expression level of podosin was significantly decreased in the STZ / SAL group (FIG. 16E). On the other hand, there was no difference in the TAT amount of the coagulation activation marker (FIG. 18).

  In another experiment, when diabetes was induced in STZ (WT / STZ) in hPS-TG mice (hPS / STZ) and wild-type mice that had undergone unilateral nephrectomy, the severity of diabetes based on blood glucose levels was equivalent. Therefore, the STZ dose for the former mouse was larger than the STZ dose for the latter mouse (FIG. 19A). As expected, there was no difference in blood glucose level between WT / STZ and hPS / STZ (FIG. 19B). On the other hand, glomerular mesangial enlargement and collagen deposition were significantly decreased in hPS / STZ compared to WT / STZ, despite blood glucose levels, plasma creatinine, L-FABP, and renal hydroxyproline at similar levels. (FIGS. 19B and 19C). It was also found that hPS can significantly suppress fibrosis caused by STZ (right side of FIG. 19D). Therefore, it is also effective for fibrosis caused by diabetes (eg, renal fibrosis, pulmonary fibrosis). Further, in the renal tissue, there was no significant difference in F4 / 80 + macrophage signal between the groups. There was no significant difference in clotting factor (TF) and fibrinolysis (PAI-1, t-PA) markers (FIG. 20).

As shown in the above embodiment, it was found that hPS suppresses the apoptosis of islet β cells and suppresses the development of diabetic complications.
<HPS suppresses exacerbation of diabetes by suppressing apoptosis>
hPS is a glycoprotein with a molecular weight of 69 kDA and exhibits biological activities such as anticoagulation / anti-inflammatory and anti-apoptotic activities. Specifically, suppression of cytokine (IL-6, TNFα) and chemokine (MCP-1) release from leukocytes, suppression of apoptosis due to macrophage phagocytosis, suppression of apoptosis via TAM receptor and Akt / PKB, etc. The biological activity is expressed through the mechanism of According to the present inventor, PS has been shown to suppress diabetes and diabetic complications based on the above mechanism and through experiments of diabetic mice induced by STZ. In particular, it is the first finding that hPS suppresses apoptosis of islet β cells caused by diabetes.

<Control of apoptosis by hPS>
β-cell apoptosis is known to be an important phenomenon, particularly in type 1 diabetes, but the relationship is unclear in type 2 diabetes. In type 2 diabetes, insulin resistance, high insulin concentrations, and β-cell hyperplasia are observed. Clinically, relative insulin deficiency is observed. This is believed to be due to the large amount of apoptosis in β cells. Apoptosis of β-cells is thought to occur due to the imbalance between apoptosis-inducing factor and anti-apoptotic factor, specifically, three types of apoptosis-inducing pathways activated by caspase 3 (Tyro3, Axl, Mer) The effects of exist. There are Akt / PKB pathway, NFκβ pathway, and pathways involving several proteins in the IAP protein and Bcl-2 family as pathways that suppress β-cell apoptosis due to diabetes. Among these, it is known that hPS suppresses the apoptotic pathway by TAM receptor / Akt / PKB signal, but there was no knowledge about IAP or Bcl-2 family proteins. In this study, hPS suppresses caspase 3 activation, increases phosphorylation of Akt / PKB and IκB, and increases the expression of anti-apoptotic factors (BIRC3 and Bcl-2 family proteins) in β cells, It has been shown to decrease the expression of apoptosis-inducing factor (Bax). Moreover, the anti-apoptotic effect by hPS was inhibited by the anti-Mer receptor antibody. (FIGS. 12A and B) These results indicate that hPS enhances the activation of Akt / PKB and NFκB signals and induces the expression of IAP and Bcl-2 proteins, mainly through the Mer of TAM receptors. Based on this mechanism, it has been shown to suppress apoptosis of β cells induced by diabetes.

<HPS suppresses insulin resistance>
Type 2 diabetes is characterized by excessive release of insulin and increased insulin resistance, accompanied by a decrease in blood glucose uptake into muscles and fat cells, and a decrease in glucose release from the liver. The Akt / PKB pathway is important for maintaining insulin sensitivity and blood glucose concentration homeostasis. Independent of insulin release from β-cells, activation of the Akt / PKB pathway is caused by glycogen, synthase, kinase 3 deactivation, glucose transporter expression increase, and 6-phosphofructo-2-kinase activation. Activation of the sugar system increases glucagon synthesis. Through these mechanisms, Akt / PKB enhances insulin sensitivity in surrounding tissues. Systemic administration of hPS improves insulin resistance by enhancing insulin sensitivity in db / db mice with reduced HOMA-IR values and enhancing insulin effects on adipocytes, skeletal muscle and hepatocytes showed that. (FIGS. 2 and 3) Thus, hPS was found to improve insulin sensitivity by activating the Akt / PKB pathway.

<HPS improves diabetic glomerulosclerosis>
Mammary protein kidney deposition, mesangial enlargement and tuberous sclerosis with glomerular basement membrane thickening are features of diabetic nephropathy. Growth factors such as TGF-β1 and chemokines stimulate the release of matrix proteins from myofibroblasts following renal cell glomerular apoptosis. According to this study, hPS has been shown to protect diabetic nephropathy separately from the protective function of β cells. Although the latter mechanism is not clear, it may be related to suppressing glomerular cell apoptosis.

<Clinical application>
As a result of this study, (1) the level of free PS was significantly decreased in patients with type 1 diabetes and type 2 diabetes, and (2) there was a significant inverse correlation between HbA1c and free PS in patients with type 2 diabetes. I found two points. Since free PS and HbA1c show a significant inverse correlation, it is considered that a low free PS concentration is associated with a clinical picture of diabetic patients. In other words, by adopting a strategy to increase the free PS concentration, there is a high possibility that the seriousness of diabetic patients can be eliminated or linked to treatment. Such measures include, for example, administering PS, administering an anti-C4bp antibody that blocks the binding between PS and C4bp, and administering a drug that increases the expression level of PS.
As described above, according to the present embodiment, a novel therapeutic drug for diabetes and / or a therapeutic drug for diabetic nephropathy can be provided.

In addition, about this embodiment, it can deform | transform as follows and can implement.
(1) In this embodiment, the biological activity was examined centering on hPS (human protein S), but it can be carried out using a protein having a homology of 96% or more and having a biological activity equivalent to that of PS. The homology can be calculated by NCBI <http://blast.ncbi.nlm.nih.gov/Blast.cgi>, for example. It is known that when the homology is high, it has a similar biological activity, and when the homology is low, it has another biological activity.
(2) In the present embodiment, PS and a protein having 96% or more homology with PS and having equivalent biological activity (hereinafter referred to as “PS etc.”) and a predetermined combination drug can be used. Examples of such a combination drug include at least one drug selected from the group consisting of sulfonylurea, glinide, biguanide, thiazolidinedione, glycosidase, DPP-4, incretin and sodium / glucose transporter 2. Specifically, (a) sulfonylurea (promoting insulin secretion) includes glimepiride, glipizide and glyburide; (b) glinide (promoting insulin secretion) includes nateglinide and repaglinide; (c) biguanide (liver sugar) (Formation reduction) as metformin, (d) thiazolidinedione (glitazone) (insulin resistance reduction) as pioglitazone and rosiglitazone, (e) glucosidase (sugar absorption inhibition) as acarbose and miglitol ( f) DPP-4 (dipeptidyl peptidase-4) inhibitors (increasing insulin secretion by sugar) are alogliptin, linagliptin, sitagliptin, saxagliptin, and (g) incretin preparation (increasing insulin secretion by sugar) is exenatide. , Liraglutide is (h) sodium-glucose transporter Examples of 2 (promoting glucose excretion in the urinary tract) include canagliflozin and dapagliflozin.
(3) According to this embodiment, PS and the like are diabetes and / or diabetic complications (for example, diabetic nephropathy, renal fibrosis, pulmonary fibrosis, stroke, myocardial infarction, heart failure, peripheral vascular disorder and renal disorder) Etc.).
(4) As for the administration form such as PS, intraperitoneal administration and subcutaneous administration were carried out, but other administration forms (for example, intramuscular, intravenous, intrabronchial, intrahepatic, intrauterine, intravaginal, intrarectal) The same effect can also be obtained using the oral cavity, sublingual, intranasal, transdermal, and the like. Further, the dose of PS or the like is not particularly limited, and is, for example, 0.1 mg / kg · day to 10 mg / kg · day (preferably 0.5 mg / kg · day to 5 mg / kg · day) per person. Although the hPS dose to mice used in the experiments described above is 2 mg / kg · day, it is strongly estimated as an amount that brings about an effect, but the final optimum dose range will be awaiting clinical trials.
(5) PS and the like are also effective for the prevention and treatment of fibrosis (for example, see the right side of FIG. 19D).
(6) By increasing free PS and the like that are not bound to C4bp in the blood, the effect of PS and the like can be enhanced, and the dose of PS and the like can be reduced (or the administration of PS and the like is eliminated). As such a strategy, for example, administration of an anti-C4bp antibody together with PS or the like or in place of PS or the like is exemplified. By this measure, similar to the above embodiment, an effect equivalent to improvement of diabetes and / or diabetic complications can be expected.
(7) Inhibition of pancreatic β-cell apoptosis can be expected by administering GAS6 (growth-arrest specific gene 6) in place of PS or in combination. Since GAS6 has a structure very similar to PS and the like and has biological activity similar to PS and the like, GAS6 can be treated for diabetes and / or diabetic complications using the knowledge of this embodiment. It is understood that it is used as a medicine.
As described above, by using proteins with biological activity such as PS for diabetes or diabetic complications, it is possible to prevent diabetes and diabetic complications, and to suppress progression and deterioration after onset. This has a great effect on reducing the burden on patients, reducing the burden on patients with onset, and extending lifespan.

Claims (5)

A therapeutic agent for diabetes and diabetic complications containing protein S, or protein having 96% or more homology with protein S, or a protein having equivalent biological activity. The dosage form is at least selected from the group consisting of subcutaneous, intramuscular, intravenous, intrabronchial, intraperitoneal, intrahepatic, intrauterine, intravaginal, intrarectal, buccal, sublingual, intranasal and transdermal The therapeutic agent according to claim 1, which is one. The therapeutic agent according to claim 1 or 2, further comprising at least one drug selected from the group consisting of sulfonylurea, glinide, biguanide, thiazolidinedione, glycosidase, DPP-4, incretin and sodium / glucose transporter 2. A therapeutic drug characterized by the combined use. The therapeutic agent according to any one of claims 1 to 3 is selected from the group consisting of diabetes, diabetic nephropathy, renal fibrosis, pulmonary fibrosis, stroke, myocardial infarction, heart failure, peripheral vascular disorder and renal disorder A therapeutic agent characterized by being administered to at least one of the above. The therapeutic agent according to any one of claims 1 to 4, wherein a dose of the therapeutic agent is 0.1 mg / day to 10 mg / day per kg of body weight.