KR101841589B1 - Pharmaceutical composition for the prevention and treatment of fibrosis comprising of lobeglitazone - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for prevention and treatment of fibrosis including lobeglitazone as an active component. When using the lobeglitazone of the present invention as the active component, kidney fibrosis is suppressed by inhibiting an expression of mRNA or protein of p-Smad3, α-SMA, PAI-1 and type 1 collagen of the kidney, and non-diabetic kidney fibrosis can be effectively prevented and treated, thereby being usefully used in relevant pharmaceutical or medical industries.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for prevention or treatment of fibrosis comprising robeglitazone as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing or treating fibrosis comprising robeglitazone as an active ingredient.

Renal fibrosis is a common pathologic finding in end stage renal failure, the last stage of multiple renal disease [Kanwar YS, Annu Rev Pathol, 2011, 6: 395-423]. The most important mechanism of renal fibrosis is the accumulation of extracellular matrix (ECM) through the activation of the signaling pathway of Transforming Growth Factor β (TGF-β) Although many studies for the prevention or treatment of fibrosis have been continuing, there are no drugs that can effectively prevent or treat renal fibrosis. Thiazolidinediones (TZD) is a PPARγ agonist that increases the activity of PPARγ (peroxisome proliferator-activated receptor-gamma). It improves glucose metabolism abnormality caused by insulin resistance in diabetic patients through improvement of insulin sensitivity, It is an oral diabetic drug that is involved in metabolism and improves dyslipidemia. It has already been widely used worldwide [Kim H, Diabetologia 2004, 47: 2215 ~ 2225]. In addition to the glucose and lipid metabolism mentioned above, PPARγ is known to regulate various biological responses such as cell proliferation and apoptosis, inflammatory response, immune response, oxidative stress response (Guan Y, Drug News Perspect 2002, 15: 147-154), (M. Ricote, Nature 1998; 391 79e82), (G. Derosa, Curr. Mol. Pharmacol. 2012; 5 272e281), (Guo B, Diabetes 2004; 53: 200-208) .

Lobeglitazone is the most recently developed TZD family of drugs and has been proven to demonstrate the homeostatic effects of glucose and lipid metabolism comparable to the TZD family through PPARγ activation [ Kim, PLoS One 2014; 9), (SM Jin, Diabetes Obes.

Although there is a need for further studies on the effect of diabetic drugs on diabetic vascular complications, there is no known study except for the prevention of arterial sclerosis by inhibiting vascular endothelial cell proliferation [Soo Lim, Atherosclerosis 2015, 243, 107e119].

Accordingly, the inventors of the present invention have investigated a new therapeutic agent effective for the prevention and treatment of renal fibrosis, and found that the use of robeglitazone effectively prevents and treats non-diabetic renal fibrosis, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating fibrosis comprising lobeglitazone as an active ingredient.

It is also an object of the present invention to provide a food composition for preventing or improving fibrosis comprising robeglitazone as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating fibrosis comprising lobeglitazone as an active ingredient.

The present invention also provides a food composition for preventing or ameliorating fibrosis comprising robeglitazone as an active ingredient.

When the robeglitazone of the present invention is used as an active ingredient, fibrosis of the kidney is suppressed through a mechanism of inhibiting the expression of mRNA or protein of p-Smad3, -SMA, PAI-1 and type 1 collagen in the kidney, Can effectively prevent and treat non-diabetic renal fibrosis, and can be usefully used in related pharmaceutical or medical industries.

Figure 1 shows H & E and Sirius levels in kidney fibers of the group administered with robeglitazone (UUO + lobeglitazone), untreated group (UUO) or control (CON) in a model of kidney fibrosis induced via unilateral urinary bladder closure (UUO) red staining results.
Figure 2 shows the degree of tubular atrophy and the degree of Sirius staining (UUO + lobeglitazone), untreated group (UUO) or control group (CON) administered with robeglitazone in a model of kidney fibrosis induced through unilateral urinary tract obstruction Fig.
Figure 3 shows the immunohistochemical staining of renal fibers in the kidney fibers of mice treated with robeglitazone (UUO + lobeglitazone), untreated (UUO) or control (CON) in a model of kidney fibrosis induced via unilateral urinary tract closure (UUO) And the results are shown in FIG.
FIG. 4 is a graph showing the effect of α-SMA on renal fibers of the group administered with robeglitazone (UUO + lobeglitazone), untreated group (UUO) or control (CON) in a model of renal fibrosis mice induced through unilateral urinary obstruction (UUO) , And PAI-1, type 1 collagen mRNA.
Figure 5 shows the effect of p-Smad3 on kidney fibers of the group administered with robeglitazone (UUO + lobeglitazone), untreated group (UUO) or control (CON) in a model of renal fibrosis mice induced through unilateral urinary tract obstruction (UUO) , α-SMA, PAI-1, and type 1 collagen.

The present invention provides a pharmaceutical composition for preventing or treating fibrosis comprising lobeglitazone as an active ingredient.

In the present invention, "Lobeglitazone" is a thiazolidinediones (TZD) -based drug, which activates peroxisome proliferator-activated receptor gamma (PPARgamma) Is a commercially available oral diabetic drug that has been proven to exhibit the homeostasis effect of metabolism.

In the present invention, "fibrosis" means that excessive fibrous connective tissue is formed in an organ or tissue during reparative or reactive processes.

The fibrosis is one or more fibrosing diseases selected from the group consisting of renal fibrosis, cystic fibrosis, myelo fibrosis, lung fibrosis and liver fibrosis, Preferably, but not limited to, renal fibrosis.

The renal fibrosis is non-diabetic and the renal fibrosis is caused by ureteral obstruction, ischemia-reperfusion injury, nephrotoxic agents damage, radiocontrast media, Acute kidney injury due to acute kidney injury due to tubulointerstitial disease and acute kidney damage due to glomerulus disease, and the nephrotoxicity The medicament may be at least one selected from the group consisting of cisplatin, gentamicin, non-steroidal anti-inflammatory drug (NSAID), and ifosfamide, preferably at least one selected from the group consisting of renal fibrosis But is not limited thereto.

Non-diabetic renal fibrosis can occur due to the above-described various causes, and the cause is different from diabetic renal fibrosis caused by hyperglycemia because common causes do not lead to hyperglycemia. The major mechanism of non-diabetic renal fibrosis is activation of the TGF-β / Smad signaling system by oxidative stress reaction or inflammatory reaction, which occurs irrespective of hyperglycemia, And the prevention and / or treatment of kidney complications through the use of the drug. Therefore, the cause and mechanism of non-diabetic renal fibrosis and diabetic renal fibrosis are different from each other, so that the composition and medicines related to diabetic renal fibrosis in the past can not be considered to have the same effect on non-diabetic renal fibrosis.

In the present invention, "unilateral ureteral obstruction" refers to unilateral or bilateral occlusion, which obstructs the flow of the ureter blocked by the ureter site connected to the kidney by the bladder.

In the present invention, "ischemia-reperfusion injury" is one of the important factors for inhibiting renal function in acute renal failure or kidney transplantation.

In the present invention, "Acute Kidney Injury" refers to a sudden loss of kidney function. When there is not enough blood to the kidney, when the kidney itself becomes diseased, The most common cause of urinary incontinence is urinary incontinence. This means that the kidneys are not functioning properly and that waste products are not being excreted and accumulating in the body.

The pharmaceutical composition according to the present invention may be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers for use in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method have.

In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin And the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweetening agents, fragrances and preservatives . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Propellants, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of suppositories, witepsol, tween 61, cacao paper, laurin, and glycerogelatin can be used.

The pharmaceutical composition according to the present invention can be administered to the subject in various routes. All modes of administration may be expected, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The therapeutically effective dose of the pharmaceutical composition of the present invention will vary depending upon the species, body weight, age and individual condition of the subject, the disorder or disease being treated, or their severity. The daily dose of the composition of the present invention is 0.0002 to 1000 mg / kg, based on robeglitazone active ingredient, and can be administered 1 to 10 times per day, and the dose is in any sense limited to the scope of the present invention It is not.

In one embodiment of the present invention, the expression of p-Smad3, a-SMA, PAI-1 and type 1 collagen is inhibited when robeglitazone is administered to a model of a renal fibrosed mouse through unilateral urinary tract obstruction (UUO) Respectively. Thus, it has been confirmed that robeglitazone significantly inhibits non-diabetic renal fibrosis caused by urinary tract obstruction and thus can be effectively used for the prevention or treatment of renal fibrosis associated therewith.

In addition, the present invention provides a therapeutic agent for fibrosis comprising robeglitazone as an active ingredient, wherein the administration of a therapeutically effective amount of robeglitazone of the present invention to a subject suffering from or susceptible to fibrosis effectively inhibits fibrosis Prevention or treatment.

The present invention also provides a food composition for preventing or ameliorating fibrosis comprising robeglitazone as an active ingredient.

The robeglitazone of the present invention can be used as a health functional food, a food additive or a dietary supplement.

When robeglitazone is used as a food additive, it can be suitably used according to a conventional method such as adding it directly or mixing it with another food or food ingredient.

In addition, the mixing amount of the robeglitazone may be suitably changed according to the purpose of use (prevention, health or therapeutic treatment), preferably 0.01 to 95% by weight based on the total weight of the food composition, More preferably 1 to 80% by weight. If the content is less than 0.01% by weight, the efficiency of taking may be poor. If the content is more than 95% by weight, formulation may be difficult.

As a specific example, when food or beverage is prepared, it is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material of robeglitazone of the present invention. However, when it is intended for health and hygiene purposes or for the purpose of controlling health, it can be added in an amount below the above range, and there is no problem in terms of safety. Therefore, the active ingredient can be used in an amount exceeding the above range have.

There is no particular limitation on the kind of the food, but examples of the food to which the robeglitazone of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, Dairy products including beverages, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes and the like.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavors or natural carbohydrates such as ordinary beverages. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweetening agents such as dextrin and cyclodextrin; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

The food composition of the present invention can be used for various foods, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusters, stabilizers, preservatives, glycerin, Carbonation agents used in beverages, etc., and may include, but is not limited to, natural fruit juices, fruit juice beverages, and flesh for the manufacture of vegetable beverages. These components may be used independently or in combination. The proportion of the above additives is not particularly limited, but is preferably within a range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

For long-term consumption intended for health and hygiene purposes or health control purposes, the food composition of the present invention has no problem in terms of safety and can be taken for a long period of time.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Experimental Example  One. One side  Unilateral ureteral  obstruction, UUO ) In the kidney fibrosis mouse model Robe glitazone  administration

The present invention utilizes a model of kidney fibrosis mice induced through unilateral ureteral obstruction (UUO). Unilateral closure of the urinary tract was performed by anesthetizing the mice with pentobarbital at a concentration of 50 mg / kg, and then tying the unilateral urinary tract at two points and cutting between them. In the mouse model, robeglitazone was administered by gavage at a dose of 1 mg / kg daily for 3 days before unilateral closure of the urothelial system, and was administered daily for up to 7 days after the procedure. A mouse model without robeglitazone was used as a non-administration group and a wild-type mouse was used as a control group. In order to compare the degree of kidney fibrosis between the group administered with robeglitazone and the group administered with no rosiglitazone, each kidney tissue after sacrifice of the mice in each group was confirmed by the following series of experiments.

Experimental Example  2. H & E staining or Sirius staining and immunohistochemical staining

H & E staining, Sirius staining, or immunohistochemical staining was performed to confirm renal tissue changes of the robeglitazone-treated group, the non-treated group, or the control group of Experimental Example 1 described above.

Specifically, kidney tissue was fixed in 4% paraformaldehyde overnight, and then hardened in paraffin to make blocks. After cutting to a thickness of 4 μm, xylene was deparaffinized and tissue staining was performed. H & E staining was performed in hematoxylin for 3 to 4 minutes to perform nuclear staining, followed by decolorization by addition of acid alcohol, followed by neutralization in ammonia solution. After that, it was reacted with eosin for staining of cytoplasm and the like. To perform Sirius red staining, kidney tissue sections were stained with saturated PICR for 18 h in a solution containing 0.1% Sirius red F3BA and then washed in 0.01 N HCl for 2 min. Immunohistochemical staining was performed with 0.01 M citric acid buffer and allowed to react with 3% H ₂ O ₂ to remove the endogenous peroxidase activity so that the antibody could penetrate well into the tissue . Subsequently, p-smad3, a-SMA, PAI-1 and type 1 collagen antibody were reacted overnight, then the secondary antibody was reacted for 1 hour and nuclear staining was performed with Mayer hematoxilin Respectively. All tissue sections were stained, dehydrated, covered with coverslips using permount solution, and sealed.

Experimental Example  3. RT- PCR  Of the kidney mRNA  Measurement of changes in Western Blot  Perform

To confirm the renal tissue changes of the robeglitazone-treated group, the untreated group, or the control group of Experimental Example 1, RT-PCR or Western blotting was performed to measure the mRNA or protein of Smad3,? -SMA, PAI-1 and type 1 collagen Was confirmed.

Specifically, to perform RT-PCR for mRNA analysis, RNA was extracted from the cells using a trizol solution. cDNA synthesis was performed using the cDNA synthesis kit and the relative expression levels of α-SMA, PAI-1 and type 1 collagen mRNA were analyzed using the SYBR Green PCR Master Mix Kit using the StepOnePlus Real-Time PCR System . To confirm the expression level of Smad3, α-SMA, PAI-1 and type 1 collagen proteins in renal tissues of each experimental group, cells were lysed using RIPA buffer and centrifuged at 15,000 × g for 10 min. After 30 μg of protein was electrophoresed on SDS-PAGE, polyvinylidene fluo-ride membranes were transferred using an electric device. After blocking with 5% skimmed milk, the primary antibody was reacted overnight, and the secondary antibody was reacted for 1 hour to perform Western blotting. Expression of each protein was compared using chemiluminescence Western blot search system.

Example  1. Through H & E staining and Sirius staining Robe glitazone  Confirming inhibition of renal fibrosis in the administration group

Renal fibrosis in the kidney fibrosis mice model induced by unilateral urinary obstruction (UUO) in Experimental Example 1 was compared with that in the group administered with robeglitazone (UUO + lobeglitazone), the untreated group (UUO) or the control group (CON) For confirmation, the kidney tissues of the above groups were confirmed by H & E staining and Sirius staining method of Experimental Example 2. In addition, the tubular atrophy of the kidney and the degree of staining of Sirius were confirmed, and the effect of inhibiting renal fibrosis by administration of robeglitazone was confirmed. The results are shown in Figs. 1 and 2. Fig.

As shown in Fig. 1, the group administered with robeglitazone (UUO + lobeglitazone) to a model of kidney fibrosis induced through unilateral urinary tract obstruction (UUO) showed a decrease in renal fibrosis And it was confirmed to be similar to the control group.

As shown in FIG. 2, the untreated group (UUO) showed high renal tubular atrophy and high degree of Sirius staining, indicating that renal fibrosis progressed. However, in a model of renal fibrosis via unilateral URT, (UUO + lobeglitazone) inhibited kidney fibrosis because of low tubular atrophy and Sirius staining.

Example  2. Through immunohistochemical staining Robe glitazone  Confirming inhibition of renal fibrosis in the administration group

p-smad3, a-SMA, PAI-1 and type 1 collagen are fibrosis-related factors, and the degree of fibrosis can be confirmed by their expression.

(UUO + lobeglitazone), untreated (UUO) or control (CON) kidney tissues in a mouse model of renal fibrosis induced by unilateral urinary obstruction (UUO) Chemical staining was performed to confirm the expression level of p-smad3, a-SMA, PAI-1 and type 1 collagen, and kidney fibrosis was confirmed by administration of robeglitazone. The results are shown in Fig.

As shown in FIG. 3, renal fibrosis progresses in the tissues of the untreated group (UUO) by confirming that the positive staining area and expression of p-Smad3, α-SMA, PAI-1 and type 1 collagen are significantly high (UUO + lobeglitazone) in the kidney fibrosis mouse model induced by unilateral urinary obstruction (UUO) showed that the degree of positive staining and the expression level of UUO + lobeglitazone inhibited kidney fibrosis Respectively.

Example  3. p- Smad3 , α- SMA , PAI Of type 1 and type 1 collagen mRNA  Or through protein expression Robe glitazone  Confirming inhibition of renal fibrosis in the administration group

p-smad3, a-SMA, PAI-1 and type 1 collagen are fibrosis-related factors, and the degree of fibrosis can be confirmed by their expression.

The renal tissues of the kidney fibrosis mice induced through unilateral urinary obstruction (UUO) in Experimental Example 1 were injected into the renal tissues of the group administered with robeglitazone (UUO + lobeglitazone), the untreated group (UUO) or the control group (CON) RT-PCR and Western blotting of Example 3 were performed to confirm the mRNA or protein expression level of p-smad3, a-SMA, PAI-1 and type 1 collagen, and the degree of renal fibrosis upon administration of robeglitazone was confirmed . The results are shown in Figs.

As shown in Figs. 4 and 5, the expression of mRNA or protein of p-Smad3, a-SMA, PAI-1 and type 1 collagen was significantly higher in tissues of the untreated group (UUO) Respectively. On the other hand, mice treated with robeglitazone (UUO + lobeglitazone) in a model of renal fibrosis induced via unilateral Urethral Obstruction (UUO) showed mRNA or protein of p-Smad3, a-SMA, PAI-1 and type 1 collagen Was significantly lower than that of the control group, confirming that renal fibrosis was effectively inhibited.

Through the above results, it was shown that treatment of robeglitazone with a model of kidney fibrosis mice induced through unilateral urinary obstruction (UUO) inhibited the expression of p-Smad3, a-SMA, PAI-1 and type 1 collagen Respectively. Thus, it has been confirmed that robeglitazone significantly inhibits renal fibrosis caused by urinary tract obstruction and thus can be effectively used for the prevention or treatment of renal fibrosis associated therewith.

Claims (7)

A pharmaceutical composition for preventing or treating renal fibrosis comprising lobeglitazone as an active ingredient. delete The method according to claim 1,
A pharmaceutical composition for preventing or treating renal fibrosis, wherein the renal fibrosis is non-diabetic. The method of claim 3,
The renal fibrosis may be caused by at least one of ureteral obstruction, ischemia-reperfusion injury, nephrotoxic agents damage, radiocontrast media damage, tubulointerstitial inflammation, And acute kidney damage due to acute kidney injury and glomerulus disease caused by a disease caused by a disease or a disorder of the kidney. 5. The method of claim 4,
The nephrotoxic drug is at least one selected from the group consisting of cisplatin, gentamicin, non-steroidal anti-inflammatory drug (NSAID), and ifosfamide. A pharmaceutical composition for therapeutic use. The method according to claim 1,
Wherein said robeglitazone inhibits the expression of mRNA or protein of p-Smad3,? -SMA, PAI-1 and type 1 collagen in the kidney. A food composition for preventing or improving renal fibrosis comprising robeglitazone as an active ingredient.

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