JP4348254B2 - Food and drugs for inhibiting the progression of cirrhosis - Google Patents

Description

  The present invention relates to a food and drug for inhibiting the progression of cirrhosis, comprising a peptide rich in L-glutamine.

  Cirrhosis is a major problem among liver diseases. Currently, there are about 4 million chronic hepatitis patients (type B: about 1.5 million, type C: about 2.5 million) in Japan. Chronic hepatitis shifts to cirrhosis, but the transition rate is 20% for hepatitis B and 60% for hepatitis C. As a result, in Japan, 2370 people, including 647 from hepatitis B and 1723 from hepatitis C, are reported to have cirrhosis every year in 100,000 people. In addition, the annual number of deaths due to cirrhosis reaches 17,000. Therefore, there is a demand for a method for suppressing the progression from hepatitis to cirrhosis, but there is no appropriate method at present.

  Cirrhosis is a condition in which the liver is fibrotic and hardened. Here, liver fibrosis is a pathological condition in which an extracellular matrix (ECM) such as collagen is excessively accumulated as a result of wound healing in response to tissue injury in the liver.

  Normally, collagen in a living body maintains a certain balance while repeating production and degradation, but under pathological conditions, the balance is lost and fibrosis occurs. Liver fibrosis occurs mainly due to inflammation caused by hepatitis virus and alcohol, and becomes prominent especially when inflammation becomes chronic. When hepatocytes are repeatedly damaged by hepatitis or alcohol, fibrosis progresses beyond the limit plate in the portal vein area, and the lobular structure is destroyed. Hepatocytes regenerate and a structure called regenerative nodules is created, and eventually the state of fibrosis called cirrhosis progresses.

  The main ECM-producing cells in the liver are hepatic stellate cells, and humoral factors that increase due to liver damage act in a complex manner, activating the stellate cells and causing liver fibrosis. Astrocytes are known as vitamin A storage cells (Ito cells) in the liver and belong to the myofibroblast family. In the process of liver fibrosis, stellate cells are activated by cytokines from Kupffer cells and infiltrating cells and transformed into activated cells to produce prominent ECM such as collagen. Furthermore, matrix metalloprotease (TMP), TGF-β (transforming growth factor-β), PDGF (platelet-derived growth factor), etc. It produces growth factors such as HGF (hepatocyte growth factor) and plays a central role in liver fibrosis. Activated stellate cells also increase the expression of various cytokine receptors and become highly sensitive to cytokines.

Collagen production and degradation are complexly regulated by various cytokines and matrix degrading enzymes and their inhibitors. MMP is involved in the degradation and metabolism of collagen, but as fibrosis progresses, the proportion of active form (MMP acting as a protease) of MMP-2 (responsible for degradation of basement membrane components) decreases, and collagen turn It is known that over is suppressed.

In WO03 / 074071, it is known that a glutamine peptide obtained by enzymatically hydrolyzing wheat gluten has an effect of improving liver diseases. However, its detailed actions are not well known.
WO03 / 074071

  Even if chronic hepatitis can be maintained in a good state, it can be expected that the prognosis and quality of life will be greatly improved. Thus, an object of the present invention is to provide a food and a drug that suppress the progression from chronic hepatitis to cirrhosis.

As a result of intensive studies on the effects of glutamine peptides in the case of chronic inflammation in the liver (chronic hepatitis), the inventor has found that glutamine peptides have the effect of suppressing the progression from chronic hepatitis to cirrhosis. . More specifically, a glutamine peptide produced by the method described in WO03 / 074071 was administered to an animal model, and its biological action was examined in detail. As a result, the administration of glutamine peptide is often used as an indicator of liver diseases such as severe hepatitis, GOT (Glutamic Oxaloacetic Transaminase) and GPT (Glutamic) in serum.
Even when there is no change in Pyruvic Transaminase), it was discovered that the progression of cirrhosis was suppressed, and the present invention was completed.

Therefore, the present invention is characterized by comprising a peptide composed of an L-amino acid constituting a natural protein, having an L-glutamine content of 15 to 60% by mass and an average molecular weight of 200 to 100,000. The present invention relates to a food for suppressing the progression of cirrhosis. The peptide preferably has an L-glutamine content of 20 to 40% by mass and an average molecular weight of 500 to 20,000, and particularly preferably a peptide produced from wheat flour gluten.

  In the following description, a peptide having a high glutamine content according to the present invention is referred to as a “glutamine peptide”. Further, in the present specification, the terms “peptide” and “glutamine peptide” mean a peptide composed of L-amino acids constituting a natural protein. Therefore, the term “L-amino acid” or “amino acid” means an L-amino acid that constitutes a natural protein.

  By containing the glutamine peptide of the present invention in a food or drink, the glutamine peptide can be taken more easily in daily life. Furthermore, the peptide can also be administered as a drug for inhibiting the progression of cirrhosis.

According to the present invention, it comprises a peptide composed of an L-amino acid constituting a natural protein, having an L-glutamine content of 15 to 60% by mass and an average molecular weight of 200 to 100,000. A cirrhosis progress-suppressing food and a cirrhosis progress-suppressing drug are provided. The glutamine peptide used in the present invention is excellent in safety and suitable for long-term administration. For this reason, it is extremely useful as a food and drug for suppressing the progression of cirrhosis.

The glutamine peptide of the present invention, for example, can be produced by the method described in WO03 / 074071. Further, glutamine peptides derived from wheat gluten are already commercially available (for example, “Glutamine Peptide GP-1” manufactured by Nisshin Pharma Inc., “Glutamine Peptide GPA” manufactured by DMV, etc.). These glutamine peptides may be used as they are or may be subjected to treatments such as hydrolysis and fractionation depending on circumstances.

  Such glutamine peptides, for example, bread-based foods such as bread, udon noodles, cookies, rice crackers, teas, soups, sprinkles, butter, jams, spreads, liquid foods, juices, jelly-like beverages, etc. The food for suppressing the progression of cirrhosis is produced. As such a food, it is desirable that the daily dose can be controlled as a health food or a functional food. The daily intake for an adult is usually 4 to 80 g, preferably 6 to 60 g as a glutamine peptide.

  The cirrhosis progression-suppressing drug containing the glutamine peptide of the present invention can be made into oral preparations such as tablets, granules, powders, capsules, syrups, dry syrups, and liquids by using a glutamine peptide by a conventional method. In this case, the daily dose for adults is the same as that for food.

  Examples of blood biochemical analysis values used as an index for diagnosis of cirrhosis include hyaluronic acid content, ammonia content, and thymol turbidity.

  Hyaluronic acid is a high-molecular-weight mucopolysaccharide in which N-acetylglucosamine and D-glucuronic acid are polymerized, and is widely distributed in connective tissues in vivo together with other glycosaminoglycans (umbilical cord, joint fluid, vitreous) Is particularly abundant) and is responsible for joint lubrication, biological defense against bacterial invasion, and regulation of electrolytes and water. The produced and released hyaluronic acid is transferred to the lymphatic tissue along the bloodstream, and further transferred to the liver for metabolism. On the other hand, when the fibrosis of the liver proceeds, the production of hyaluronic acid such as Ito cells and fibroblasts in the liver increases. In addition, hepatic sinusoidal endothelial cells, which are supposed to decompose about 90% or more of blood hyaluronic acid, lose their function with fibrosis, and the concentration of hyaluronic acid in the blood increases. Clinically, it is highly useful for differentiating between cirrhosis and non-cirrhosis, and is also useful for predicting IFN treatment effect in chronic active hepatitis C.

Ammonia (NH ₃ ) is produced by deamination from amino acids during protein metabolism, and is used for urea synthesis in the liver, but most of the blood NH ₃ is derived from the digestive tract (small intestinal mucosa and colonic bacteria). Is done. NH ₃ detoxification depends on the urea cycle in hepatocytes, and urea is excreted in the urine from the kidney. Therefore, reduction of the reduction due to the urea cycle activities of liver function, in the case of portal blood by increasing and portal collateral branches NH ₃ production in the intestine, such as inflow to large circulation, the blood NH ₃ concentration High price.

  The thymol turbiduty test (TTT) is a colloidal reaction generally performed together with the zinc sulfate turbidity test (ZNN), and has been used for a long time in the diagnosis and follow-up of liver diseases. However, although there is a drawback that it changes due to fluctuations in protein ratio, it is still used as an index to know the degree of chronic hepatitis and chronic liver disease. TTT abnormalities are more frequent when IgG and IgM are increased (hepatitis A), albumin is decreased, lipids and lipoproteins are increased, and in the case of cholestasis, the increase in glycoproteins in the blood is particularly modified (suppressed). )I do.

  Both GOT and GPT are typical aminotransferases using pyridoxal phosphate (PALP) as a coenzyme. It is most commonly used to know the degree of disorder such as hepatobiliary disease, heart disease, muscle disease, hemolytic disease, clinical course, etc., and is often used as a measure for determining the progress of the pathology of cirrhosis. This time, the present inventors, when ingesting a peptide with a high glutamine content to an animal individual who has cirrhosis, even if there is no change to be seen in GOT and GPT by ingesting a peptide with a high glutamine content, It has been found that data serving as another index can be improved, and a peptide having a high glutamine content is used as a food and drug for inhibiting the progression of cirrhosis.

  Hereinafter, the effects of glutamine peptide on the progression of cirrhosis will be described in detail with reference to examples.

Summary of results from biological tests To examine the effect on progression from hepatitis to cirrhosis, a chronic hepatitis model with rat carbon tetrachloride was used. In this model, administration of carbon tetrachloride subcutaneously twice a week causes chronic inflammation in the liver, but it is known that transition from hepatitis to cirrhosis will occur if continued for more than 20 weeks. It has been.

  Three weeks after the start of administration of carbon tetrachloride to rats, the control group diet was switched to that containing glutamine peptides (4% and 8%), and carbon tetrachloride administration was continued for 25 weeks, with no glutamine peptide given. Compared to the group. As a result, although there was almost no difference in serum GOT and GPT, ammonia, thymol turbidity test, and hyaluronic acid showed significantly lower values in the glutamine peptide administration group than in the control group. Further, the condition of the removed liver was harder in the control group, and the surface was not smooth and covered with countless nodules, whereas it was milder in the glutamine peptide administration group. In addition, liver atrophy was milder in the glutamine peptide administration group, and the liver weight was larger than that in the control group. In the control group, ascites was observed in more than half of the individuals, whereas in the glutamine peptide administration group, ascites was only observed in 0 or 1 of 6 cases.

When the activity of MMP was examined by gelatin zymography, a larger amount of active MMP-2 was observed in the glutamine peptide administration group than in the control group, suggesting that collagen metabolism was activated. Moreover, as a result of measuring collagen in the liver as a soluble fraction and hydroxyproline (Hyp) in the insoluble fraction, the glutamine peptide-administered group had a higher Hyp in the soluble fraction than the control group. It is suggested that the metabolism of is activated. These biological tests are described in detail below.

Glutamine peptide administration test in cirrhosis model rats
(1) Test method Seventeen 7-week-old SD male rats were used in the experiment. A liquid (1 ml / kg / dose) containing an equal amount of carbon tetrachloride and olive oil was subcutaneously administered to each rat twice a week (Tuesday and Friday) for 25 weeks.
Rats were fed a 20% casein diet for the first 3 weeks, after which a control group (n = 5), 4% glutamine peptide group (n = 6), 8% so that there was no difference in body weight between groups. It was divided into glutamine peptide groups (n = 6). The control group continued to receive a 20% casein diet, and the 4% glutamine peptide group received a test diet (4% GP diet) in which 4% of casein was replaced with glutamine peptide GP-1 (Nisshin Pharma Co., Ltd.) The 8% glutamine peptide group was fed a test meal (8% GP meal) in which 8% of casein was replaced with GP-1. Details of the feed used in the test are shown in Table 1.

The glutamine peptide used was produced according to the method described in WO03 / 074071. The average molecular weight and glutamine content of the glutamine peptide measured by the method described in WO03 / 074071 are 10,000 and 32% by mass, respectively.
During the test period, rats received food and water ad libitum. The animals were fasted from the day before the last day of the test and sacrificed under ether anesthesia on the day of collection, and blood, tissues, etc. were subjected to analysis.

(2) Table 2 shows the weight and the weight at the end of the anatomical finding test.

  The body weight was slightly larger in the glutamine peptide administration group but was not statistically significant. The presence or absence of ascites was checked at the time of dissection. Ascites was observed in 3 out of 5 cases in the control group, but ascites was confirmed in 4% glutamine peptide group in 0 out of 6 cases and 8% glutamine. In the peptide group, only 1 out of 6 cases was found.

  As for the liver, the control group had a rough surface and a large number of nodules, and was generally fibrotic, whereas the glutamine peptide-administered group was generally larger than the control group and was hardened. The degree was milder than the control group. As shown in Table 2, it was also statistically confirmed that the liver weight of the glutamine peptide administration group was larger than that of the control group (Student t test, p <0.05).

(3) Blood biochemical analysis GOT and GPT are JSCC standardization methods (Hiroshi Miura, Japanese Clinical, 53-Zan-266, 1995), Ammonia is Okada-Fujita Modified Method (Michio Inari, Japanese Clinical, 53-Zan- 478, 1995), Thymol turbidity test (TTT) is the recommended method of the Gastroenterological Society Liver Function Research Group (Takeyuki Kadana et al., Japanese Clinical, 53-163-1995), hyaluronic acid is the latex agglutination immunoturbidimetric method (Shimmura) Reit, Medicine and Pharmacology, 44 (6): 1141-1146, 2000).
The analysis results are shown in Table 3.

  Although there was no difference in GOT and GPT, the values of ammonia, thymol turbidity method, and hyaluronic acid, which are indicators of cirrhosis, were lower in the glutamine peptide administration group.

(4) Matrix metalloprotease in the liver 100 mg of each rat liver was taken and 500 ml of PBS (137 mM NaCl, 2.68 mM KCl, 8.1 mM)
Homogenize in Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ , 5 mM EDTA, pH 7.2). The obtained homogenate was centrifuged (10,000 g × 5 min) and separated into a supernatant (soluble fraction) and a precipitate (residue). MMP-2 and MMP-9 were measured by analyzing a certain amount of the soluble fraction by gelatin zymography. Gelatin zymography is a method in which a sample is developed by SDS-PAGE using a gel containing gelatin, and then the gel is immersed in a neutral buffer containing a nonionic surfactant to remove SDS, thereby removing MMP-2 and In this method, MMP-9 is linaturated and gelatin in the gel is decomposed to detect an active band in which gelatin is decomposed after staining with CBB (Coomassie Brilliant Blue) as an unstained active band. According to this method, inactive MMPs are also converted to an active form during the linaturation process, and thus can be detected.

  The result of analysis by gelatin zymography is shown in FIG. From this result, it was confirmed that in the glutamine peptide administration group, the active form of MMP-2 in the liver increased and collagen degradation was enhanced. This indicates that the progression of liver fibrosis is suppressed by the active MMP.

(5) Collagen in the liver Take 100 mg of the liver of each rat and homogenize in 500 ml of PBS. The obtained homogenate was centrifuged (10,000 g × 5 min) and separated into a supernatant (soluble fraction) and a precipitate (residue). A certain amount of each fraction was mixed for each group, and then analyzed by a Pico-Tag amino acid analysis system (Nihon Waters) to analyze the content of hydroxyproline (Hyp). As a result, it was confirmed that the glutamine peptide administration group had a higher Hyp of the soluble fraction than the control group (FIG. 2).

Hyp is an amino acid that occupies 30% of collagen and is present specifically in collagen, and thus reflects the amount of collagen. From this, it was confirmed that the glutamine peptide administration group had a lot of soluble collagen, and collagen metabolism was activated.
From the above, it has been clarified that glutamine peptide suppresses the progression from hepatitis to cirrhosis by activating liver collagen metabolism and suppressing liver fibrosis.

(6) Examination of dose The above test was repeated by giving test meals containing different amounts of glutamine peptide in order to determine the dose of glutamine peptide for suppressing the progression from hepatitis to cirrhosis. As a result, when rats were used, it was found that the amount of glutamine peptide to be contained in the feed (20% protein) was 2 to 20%, preferably 3 to 15%.

  When the daily intake amount for adults is converted based on the results of the examination of this dose, the daily required amount of protein for adults is usually 80 g. Therefore, the glutamine peptide is 4 to 80 g, preferably 6 to 60 g. It becomes.

It is a figure which shows the result of gelatin zymography obtained as a result of the matrix metalloprotease test in a liver. In the figure, the image is inverted. It is a figure which shows the hydroxyproline content obtained as a result of the collagen test in a liver. In the figure, Con is a control group administered with a casein diet, GP4 is a group administered with a 4% glutamine peptide diet, and GP8 is a group administered with an 8% glutamine peptide diet.

Claims (6)

An agent for lowering hyaluronic acid, ammonia and / or thymol turbidity associated with cirrhosis, comprising a hydrolyzate of wheat gluten having an L-glutamine content of 20 to 40 % by mass and an average molecular weight of 500 to 20,000 . The agent according to claim 1, comprising an enzyme hydrolyzate of wheat gluten having an L-glutamine content of 32 % by mass and an average molecular weight of 10,000 . An agent for preventing and / or treating ascites associated with cirrhosis, comprising a hydrolyzate of wheat gluten having an L-glutamine content of 20 to 40 % by mass and an average molecular weight of 500 to 20,000 . The agent according to claim 3, comprising an enzyme hydrolyzate of wheat gluten having an L-glutamine content of 32 % by mass and an average molecular weight of 10,000 . A liver collagen metabolic activator associated with cirrhosis, comprising a hydrolyzate of wheat gluten having an L-glutamine content of 20 to 40 % by mass and an average molecular weight of 500 to 20,000 . The agent according to claim 5, comprising an enzyme hydrolyzate of wheat gluten having an L-glutamine content of 32 % by mass and an average molecular weight of 10,000 .

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