JP2018058807A - HGF inducer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide hepatocyte growth factor inducers consisting of a combination of a substance which promotes hepatocyte growth factor gene expression in transcription step, and a substance which promotes hepatocyte growth factor protein synthesis in translation step, as well as having remarkably high activity compared with the prior art, because an inducer which induces hepatocyte growth factor production in vivo is desired in order to utilize for treatment/prophylactic agents to various diseases, cosmetic products, functional foods (supplements), and the like.SOLUTION: The present invention is a hepatocyte growth factor inducer containing as active ingredients a hepatocyte growth factor transcription promoting agent which promotes the transcription of hepatocyte growth factor genes, and a hepatocyte growth factor translation promoting agent which promotes the translation of hepatocyte growth factor protein.SELECTED DRAWING: Figure 1

Description

The present invention relates to an HGF inducer comprising, as active ingredients, a hepatocyte growth factor transcription promoter that promotes transcription of a hepatocyte growth factor gene and a hepatocyte growth factor translation promoter that promotes translation of a hepatocyte growth factor protein. About.

Hepatocyte growth factor (hereinafter also referred to as “HGF”) is a protein discovered using growth of mature liver parenchymal cells as an index. It has become clear that proliferative effects are also exerted on cell lines and some mesenchymal cells. It is also known that HGF exhibits not only cell proliferation activity but also various activities such as cell migration promotion, morphogenesis promotion, cell death suppression, and angiogenesis action (Non-patent Document 1). Due to its pharmacological action, HGF is a liver disease treatment agent, renal disease treatment agent, epithelial cell proliferation promoter, lung disorder treatment agent, gastric / duodenal injury treatment agent, cranial nerve disorder treatment agent, immunosuppressive side effect inhibitor, collagen degradation accelerator, Development as a cartilage disorder therapeutic agent, an arterial disease therapeutic agent, a wound therapeutic agent, a neuropathy ameliorating agent, a hair growth promoter and the like (see Patent Documents 1 to 5) is expected.

HGF is mainly produced by mesenchymal cells, but its production increases in response to organ damage. For example, when an injury occurs in the liver, the production of HGF increases in liver non-parenchymal cells. At this time, the expression of HGF also increases in other organs such as the intact lung, spleen, and kidney, and the blood HGF level increases. This indicates that there are factors in the blood that induce HGF expression in response to organ damage. Such factors are collectively referred to as indulin (Non-patent Document 2).

Indulin is not a single substance. For example, cytokines such as interleukin-1, FGF (fibroblast growth factor), cAMP inducers, prostaglandins and the like have been identified as indulin. These factors increase HGF production by increasing transcription of HGF mRNA. Further, it has been found that glycosaminoglycans such as heparin, heparan sulfate and chondroitin sulfate work at a translation level that promotes synthesis of HGF protein from HGF mRNA (Patent Document 6, Non-Patent Document 3).

It is known that substances exhibiting HGF-inducing activity are also contained in natural products. Examples thereof include forskolin (Non-patent Document 4) in Coleus forskohlii, fucoidan derived from Gagome Kombu (Patent Document 7), chicory acid from Koei Sung (Patent Document 8), and the like.

JP-A-4-18028 JP-A-4-49246 JP-A-6-172207 JP 7-89869 A JP-A-6-329441 JP 2002-3384 A JP 2001-226392 A JP 2010-043013 A

Nakamura, T., “The discovery of Hepatocyte Growth Factor (HGF) and its significance for cell biology, life sciences and clinical medicine.” Proc. J. Acad, Ser. B, 86, 588-610, (2010). Matsumoto, K., “Identification and characterization of" injurin ", an inducer of expression of the gene for hepatocyte growth factor.“ Proc. Natl. Acad. Sci. USA, 89, 3800-3804, (1992). Matsumoto, K., “Heparin as an inducer of hepatocyte growth factor.” J. Biochem., 114, 820-826, (1993). Matsunaga, T., “Expression of hepatocyte growth factor is up-regulated through activation of a cAMP-mediated pathway.” Exp. Cell Res., 210, 326-335, (1994).

There is a need for an inducer that induces the production of hepatocyte growth factor in vivo for use in therapeutic and preventive drugs for various diseases, beauty products, cosmetics, functional foods (supplements), and the like.

Therefore, the present invention comprises a combination of a substance that promotes the expression of hepatocyte growth factor gene at the transcription stage and a substance that promotes the synthesis of hepatocyte growth factor protein at the translation stage. Provided is a hepatocyte growth factor inducer having significantly high activity. That is, the present invention comprises a hepatocyte growth factor transcription promoter that promotes transcription of a hepatocyte growth factor gene and a hepatocyte growth factor translation promoter that promotes translation of a hepatocyte growth factor protein as active ingredients. It is a cell growth factor inducer.

In another present invention, the hepatocyte growth factor transcription promoter is forskolin, and the hepatocyte growth factor translation promoter is chondroitin sulfate or dermatan sulfate. In yet another aspect of the present invention, the hepatocyte growth factor translation promoter is a polysaccharide, and the polysaccharide has a repeating structure of glucuronic acid disulfate and acetylgalactosamine 6 sulfate.

Furthermore, another aspect of the present invention is the hepatocyte growth factor inducer, wherein the hepatocyte growth factor transcription promoter is derived from Coleus forskohlii and the hepatocyte growth factor translation promoter is derived from shark cartilage.

According to the present invention, by combining an HGF transcription promoter that promotes transcription of a hepatocyte growth factor (HGF) gene and an HGF translation promoter that promotes translation of a hepatocyte growth factor (HGF) protein, It is possible to maximize the production of HGF in the cells to be administered. Compared to the single action of each of the conventional HGF transcription promoter and HGF translation promoter, the present invention demonstrates the additive synergistic effect of HGF transcription promoter and HGF translation promoter, and the strongest HGF induction. It is expected that agents can be created.

As will be described later, when forskolin and shark cartilage-derived chondroitin sulfate D are added at the same time, the amount of HGF produced reaches 100 to 150 times that of the negative control. It has been clarified that the HGF-inducing effect in the presence of an HGF translational promoter that promotes the translation of the protein of this protein is at a high level that has never been observed before.

  The powerful HGF inducer of the present invention can be easily imagined from the demonstration of the prevention and treatment effects of various acute or chronic organ diseases that have been clarified so far by using recombinant HGF protein or HGF gene medicine. In addition, it can be a pharmaceutical, health food or supplement aimed at preventing or treating many intractable diseases.

It is a figure which shows the expression induction of HGF mRNA by the hepatocyte growth factor transcription promoter contained in this invention. It is a figure which shows the HGF induction activity by the hepatocyte growth factor transcription promoter contained in this invention. It is a figure which shows the HGF induction activity by the hepatocyte growth factor translation promoter contained in this invention. It is a figure which shows HGF induction activity by the Example of this invention. It is a figure which shows the increase in the mouse | mouth blood HGF amount by the Example of this invention.

Hereinafter, the hepatocyte growth factor inducer of the present invention will be described in detail based on the mode for carrying out the invention.

The hepatocyte growth factor inducer of the present invention is an effective combination of a hepatocyte growth factor transcription promoter that promotes transcription of a hepatocyte growth factor gene and a hepatocyte growth factor translation promoter that promotes translation of a hepatocyte growth factor protein. Ingredients. By inducing the production of hepatocyte growth factor at both the transcription stage of the hepatocyte growth factor gene and the translation stage of the protein of the hepatocyte growth factor, a remarkable effect of inducing hepatocyte growth factor not found in the prior art is achieved. Can be obtained.

The hepatocyte growth factor transcription promoter has an action of promoting the synthesis of mRNA using the base sequence of DNA encoding hepatocyte growth factor as a template in vivo and increasing the amount of the mRNA. Specific examples of the hepatocyte growth factor transcription promoter include forskolin. Forskolin is a kind of terpene compound, which is classified into diterpenes composed of four isoprenes, and is contained in plants such as Plectranthus barbatus.

In addition, hepatocyte growth factor translation promoters promote hepatocyte growth factor protein synthesis based on mRNA synthesized in vivo using the DNA base sequence encoding hepatocyte growth factor as a template. Has the effect of increasing the amount of factor protein. As the hepatocyte growth factor translation promoter, polysaccharides are preferable, and even polysaccharides are more preferably glucosaminoglycans. Specific examples of the hepatocyte growth factor translation promoter include chondroitin sulfate or dermatan sulfate.

Chondroitin sulfate is a glucosaminoglycan having a repeating structure of acetylgalactosamine and glucuronic acid. Those having a sulfate group added to the 4-position of acetylgalactosamine are called chondroitin 4-sulfate or chondroitin sulfate A, and those having a sulfate group added to the 6-position of acetylgalactosamine are called chondroitin 6-sulfate or chondroitin sulfate C. Chondroitin sulfate A and chondroitin sulfate C are known as components of cartilage.

Glucosaminoglycan having a repeating structure of glucuronic acid disulfate and acetylgalactosamine 6 sulfate is called chondroitin sulfate D and is known as a component of shark cartilage.

Furthermore, dermatan sulfate is a glucosaminoglycan having a repeating structure of iduronic acid disulfate and acetylgalactosamine tetrasulfate, and is sometimes called chondroitin sulfate B. Chondroitin sulfate B is known as a skin component.

In the present invention, it is preferable that chondroitin sulfate D obtained from shark cartilage is an active ingredient among hepatocyte growth factor translation promoters. This is because chondroitin sulfate D obtained from shark cartilage has higher activity of promoting the translation of hepatocyte growth factor than other chondroitin sulfates and dermatan sulfate.

In the present invention, a hepatocyte growth factor transcription promoter contained as an active ingredient promotes the synthesis of mRNA using a DNA base sequence encoding hepatocyte growth factor as a template. Furthermore, the synthesis of a hepatocyte growth factor translation promoter contained as an active ingredient promotes the synthesis of hepatocyte growth factor protein based on the mRNA whose synthesis has been promoted, so that significant hepatocytes not found in the prior art A growth factor-inducing effect can be obtained.

The hepatocyte growth factor-inducing agent of the present invention can be ingested orally by mixing the extract with a pharmaceutically or food hygienically acceptable carrier that is optionally blended by a known method. It can be easily prepared as a medicine or food. The food includes health foods, functional foods and supplements. It is also possible to prepare a medicine to be taken nasally or transdermally, a medicine to be administered by injection, or a cosmetic product to be used transdermally.

The hepatocyte growth factor inducer of the present invention may be formulated using a pharmaceutically or food hygienically acceptable carrier. Examples of the preparation include a solid preparation and a liquid preparation. As a carrier acceptable for pharmaceutical or food hygiene, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations and solvents in liquid preparations. , Solubilizers, suspending agents (emulsifiers, thickeners) and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners, and fragrances can be used. The hepatocyte growth factor inducer of the present invention can be prepared as a therapeutic agent, a prophylactic agent, a cosmetic product, a cosmetic product, or a functional food.

The therapeutic agent, prophylactic agent, cosmetic product or functional food containing the hepatocyte growth factor inducer of the present invention is preferably taken orally. More preferably after meal, before meal or between meals. The intake amount and the number of intakes of the present invention vary depending on age, weight, dosage form, and the like. Furthermore, the therapeutic agent or prophylactic agent of the present invention may be administered nasally or transdermally, and may be administered intravenously, abdominally, intradermally, subcutaneously, muscle, etc. using a syringe. The cosmetic product of the present invention may be administered transdermally.

The hepatocyte growth factor inducer of the present invention induces the production of hepatocyte growth factor in vivo. Therefore, it exerts an effect on various diseases that are treated and / or prevented by production of hepatocyte growth factor. Specifically, liver diseases (hepatitis, cirrhosis, liver failure, other liver disorders), kidney diseases (glomerulonephritis, kidney failure, renal anemia, diabetic nephropathy, other kidney disorders), skin diseases, Eye disease (corneal ulcer, etc.), lung disease (pneumonia, emphysema, pulmonary tuberculosis, chronic obstructive pulmonary disease, pneumoconiosis, pulmonary fibrosis, other lung disorders), gastroduodenal disease (gastritis, gastric ulcer, duodenal ulcer, other gastroduodenum) Disorder), disorders in cancer diseases and cancer treatment, ischemic or arterial diseases of the heart or extremities, blood diseases (anemia, thrombocytopenia, blood flow disorders, etc.), bone diseases (osteoporosis, osteodysplasia, osteoarthritis) , Other bone disorders), or central diseases (such as abnormal neurodifferentiation). The therapeutic agent, preventive agent, beauty product, or functional food of the present invention can be used for the treatment and / or prevention of these diseases.

As a subject for administration or ingestion of a therapeutic agent, prophylactic agent, cosmetic product or functional food containing the hepatocyte growth factor inducer of the present invention, humans are preferable, but mice, rats, rabbits, dogs, cats, cows, It may be administered or ingested to mammals such as monkeys and pigs, pets belonging to birds, reptiles, amphibians and fish, livestock / cultured species, laboratory animals, and the like.

The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

The amount of HGF mRNA when forskolin or chondroitin sulfate was used as a hepatocyte growth factor transcription promoter was measured. SF4-1 cells, which are human skin fibroblasts, were seeded on a 6-well plate at a cell density of 1.8 × 10 ⁵ cells / well and cultured for 1 day in DMEM medium containing 10% FCS. Next, the medium of the cells was removed by aspiration, and the medium was replaced with a DMEM medium containing 1% FCS, and the culture was continued. At this time, 10 μM forskolin and chondroitin sulfate D derived from shark cartilage were added at a concentration of 5 mg / ml. After 16 hours, the cells were collected and mRNA was extracted. CDNA was synthesized using Oligo (dT) and RT-PCR was performed. Electrophoresis was performed with 1.5% agarose, and the fluorescence intensity of the band after Et-Br staining was measured. As shown in FIG. 1, the amount of HGF mRNA was increased by the addition of forskolin, but the amount of HGF mRNA was not changed by the addition of chondroitin sulfate D. Therefore, it was found that forskolin is preferably used as the hepatocyte growth factor transcription promoter.

The HGF-inducing activity of forskolin, which can be a hepatocyte growth factor transcription promoter, was measured. SF4-1 cells, which are human skin fibroblasts, were seeded in a 96-well plate at a cell density of 1.5 × 10 ⁴ cells / well. On the next day, the medium was replaced with DMEM medium containing 1% FCS, and forskolin was added to a concentration of 0 to 30 μM. Moreover, heparin known to have HGF-inducing activity was used as a positive control. After 48 hours, the culture supernatant was collected and the amount of secreted HGF was measured. HGF was quantified by ELISA. As shown in FIG. 2, the maximum HGF-inducing activity was shown by adding 10 μM forskolin.

As hepatocyte growth factor translation promoters, chondroitin sulfate A, chondroitin sulfate B (also called dermatan sulfate), chondroitin sulfate C, and chondroitin sulfate D were measured for HGF-inducing activity. SF4-1 cells, which are human skin fibroblasts, were seeded in a 96-well plate at a cell density of 1.5 × 10 ⁴ cells / well. On the next day, the medium was replaced with DMEM medium containing 1% FCS, and chondroitin sulfate was changed between 0 to 10 mg / ml and added. Moreover, heparin known to have HGF-inducing activity was used as a positive control. After 48 hours, the culture supernatant was collected and the amount of secreted HGF was measured. HGF was quantified by ELISA. As shown in FIG. 3, the addition of chondroitin sulfate A to D showed an increase in HGF-inducing activity in a concentration-dependent manner.

HGF-inducing activity was measured using forskolin, a hepatocyte growth factor transcription promoter, and chondroitin sulfate D derived from shark cartilage, a hepatocyte growth factor translation promoter, as active ingredients. SF4-1 cells, which are human skin fibroblasts, were seeded in a 96-well plate at a cell density of 1.5 × 10 ⁴ cells / well. On the next day, the medium was changed to a DMEM medium containing 1% FCS, and 10 μM forskolin and 5 mg / ml chondroitin sulfate were added. Moreover, heparin known to have HGF-inducing activity was used as a positive control. After 48 hours, the culture supernatant was collected and the amount of secreted HGF was measured. HGF was quantified by ELISA. As shown in FIG. 4, when forskolin or chondroitin sulfate D derived from shark cartilage was added alone, the amount of HGF produced was promoted 20 times as much as the addition of distilled water as a negative control. On the other hand, when forskolin and shark cartilage-derived chondroitin sulfate D were added simultaneously, the amount of HGF produced reached 100 to 150 times that of the negative control, and a remarkable synergistic effect was observed.

Forskolin, a hepatocyte growth factor transcription promoter, and chondroitin sulfate D (glucuronic acid disulfate and acetylgalactosamine 6 sulfate) derived from shark cartilage, a hepatocyte growth factor translation promoter, and dermatan sulfate A hepatocyte growth factor inducer containing) was administered to mice and the amount of HGF in plasma was measured. ICR mice (8 weeks old, female) were orally administered with 20 μg forskolin and 20 mg chondroitin sulfate. Plasma was collected after 30 hours and the HGF concentration was measured. As shown in FIG. 5, even when compared with an inducer containing only forskolin as an active ingredient or an inducer containing only chondroitin sulfate D as an active ingredient, the inducer of this example has a remarkable blood HGF level. An increase was observed, indicating that the HGF-inducing effect was remarkably high.

The hepatocyte growth factor inducer of the present invention can be used for the treatment and prevention of various diseases by inducing production of hepatocyte growth factor in the fields of medicine, food and beauty.

Claims (4)

A hepatocyte growth factor transcription promoter that promotes transcription of the hepatocyte growth factor gene;
An active ingredient is a hepatocyte growth factor translation promoter that promotes translation of hepatocyte growth factor protein,
Hepatocyte growth factor inducer The hepatocyte growth factor transcription promoter is forskolin;
The hepatocyte growth factor translation promoter is chondroitin sulfate or dermatan sulfate,
The hepatocyte growth factor inducer according to claim 1 The hepatocyte growth factor translation promoter is a polysaccharide;
The polysaccharide is
It has a repeating structure of glucuronic acid 2 sulfate and acetylgalactosamine 6 sulfate,
The hepatocyte growth factor inducer according to claim 1 or 2 The hepatocyte growth factor transcription promoter is derived from Coleus forskori,
The hepatocyte growth factor translation promoter is derived from shark cartilage,
The hepatocyte growth factor inducer according to any one of claims 1 to 3.