JP2006206505A - Diabetic nephropathy preventing composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diabetic nephropathy preventing composition capable of being safely ingested by a person suspected of having diabetes and a diabetic patient for a long period from a stage prior to onset of the diabetes or an initial stage of the onset thereof and capable of preventing or inhibiting progress of the diabetes into diabetic nephropathy which is a complication thereof. <P>SOLUTION: This diabetic nephropathy preventing composition contains a low molecular weight chitosan and/or a salt thereof as active ingredients. The low molecular weight chitosan preferably has a number-average molecular weight of 10,000-30,000. The diabetic nephropathy preventing composition is preferably used for preventing diabetic nephropathy induced by nonobese non-insulin-dependent diabetic mellitus or preventing diabetic nephropathy induced by obese non-insulin-dependent diabetic mellitus. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a composition for preventing diabetic nephropathy that suppresses the prevention or progression of renal disease that accompanies diabetes, that is, diabetic nephropathy.

  According to the 2002 diabetes survey report (announced by the Ministry of Health, Labor and Welfare in June 2004), 12.8% of men and 6.5% of women are strongly suspected of having about half of them in Japan. People are currently receiving some kind of treatment. Moreover, it is said that 10.0% of men and 11.0% of women cannot deny the possibility of diabetes.

  And about half of the people who are treated for diabetes cause some complications caused by diabetes, including 15.6% for neuropathy, 13.1% for retinopathy, and 15.2 for nephropathy. %, Foot gangrene 1.6%. In addition, 3.1% of people who are not currently treated cause nephropathy. Furthermore, looking at the proportion of people who are suspected of having diabetes who have nephropathy by age, 0% at 20-39 years, 11% at 40-59 years, 33% at 60 years and older, It has been shown that the older the age, the higher the probability of becoming nephropathy.

  Diabetic nephropathy is a general term for kidney damage caused by diabetes. It usually refers to kidney damage caused by diabetic microangiopathy. At the same time as the blood vessels harden and the blood vessels narrow, the filtration action decreases, proteinuria is produced, urine is difficult to come out, waste products accumulate in the body, uremia, uremic neuralgia, nephrotic syndrome, etc. It is a disease that causes renal failure and eventually requires artificial dialysis. Diabetic nephropathy is a serious life-threatening complication for diabetic patients, and became the leading cause of dialysis in 1998. The number of dialysis patients is increasing at a rate of 10,000 per year.

  On the other hand, chitosan having a polysaccharide structure in which glucosamine is linked in a straight chain with β1-4 bonds is an insoluble dietary fiber prepared by deacetylating chitin obtained from crustacean shells such as crabs and shrimps It is known to have various physiological activities such as serum lipid improving action, blood pressure regulating action, intestinal regulating action, cholesterol lowering action and the like. For this reason, chitosan is already widely used as a food additive and in the field of functional foods.

  For example, in the following Patent Document 1, the present inventors are an antidiabetic agent effect promoter for enhancing the effect when used in combination with an antidiabetic agent, and include low molecular weight chitosan and / or a salt thereof. The present invention proposes an agent for promoting the effect of treating diabetes, which is characterized by comprising a composition for oral intake containing as an active ingredient.

In addition, in the following Non-Patent Documents 1 and 2, by administering orally administering low molecular weight chitosan obtained by lowering the molecular weight of chitosan to diabetic mice that are non-obesity type and obese type diabetes models that are not dependent on insulin. It has been reported that an increase in blood sugar level is suppressed.
JP 2004-67575 A Biol. Pharm. Bull. 23 (12) 1458-1464 (2000) Biol. Pharm. Bull. 25 (2) 188-192 (2002)

  Diabetic nephropathy is generally considered to develop about 10 years after the onset of diabetes. However, since there is no subjective symptom, nephropathy has progressed considerably when the subjective symptom appears, and is not dependent on insulin. In this case, it is necessary to pay attention to blood glucose and blood pressure control by daily meals and to conduct early detection by conducting microalbuminuria tests regularly, since the time of onset is not clearly known.

  Currently, for the treatment of diabetic nephropathy, exercise therapy, diet therapy, oral hypoglycemic agent, and blood glucose control with insulin are performed. Further, when symptoms progress, antiplatelet agents, angiotensin inhibitors, diuretics, oral adsorbents and the like are used.

  However, long-term administration of sulfonylurea (SU) agents, biguanides, α-glucosidase inhibitors, thiazolines, phenylalanine derivatives, etc., used for the purpose of lowering blood sugar, causes hypoglycemia, gastrointestinal disorders, liver disorders In addition, there are side effects such as skin disorders, and other drugs are problematic because various side effects are strongly concerned.

  For diabetic nephropathy, growth hypertrophy factors such as TGF-β, angiotensin II, platelet-derived growth factor-B (PDGF-β), growth hormone (GH), insulin-like growth factor-I (IGF-I), etc. It is thought that factors such as endothelial cell type NO synthase, ornithine decarboxylase are involved, but the clinical application is the angiotensin converting enzyme inhibitor related to angiotensin II and AT1 receptor antagonist Only medicine. These are so-called blood pressure lowering drugs, and development of effective drugs targeting the kidney is expected.

  Therefore, the object of the present invention is to prevent a person suspected of having diabetes and a diabetic patient to safely take a long-term from the onset of diabetes or from the early stage of the onset of diabetes and prevent progression to diabetic nephropathy, which is a complication. Another object is to provide a composition for preventing diabetic nephropathy that can be suppressed.

  In order to achieve the above object, the composition for preventing diabetic nephropathy of the present invention is characterized by containing low-molecular chitosan and / or a salt thereof as an active ingredient.

  In the composition for preventing diabetic nephropathy of the present invention, the low molecular chitosan preferably has an average molecular weight of 10,000 to 30,000.

  The composition for preventing diabetic nephropathy of the present invention contains low molecular weight chitosan and / or a salt thereof as an active ingredient, so that there is no worry of side effects even when ingested for a long period of time, and the onset of diabetic nephropathy is effective. Can be prevented or suppressed.

  The composition for preventing diabetic nephropathy of the present invention is used for the prevention of diabetic nephropathy induced by non-obese insulin-independent diabetes or the prevention of diabetic nephropathy induced by obese insulin-independent diabetes. Preferably used.

  The composition for preventing diabetic nephropathy of the present invention can be safely and continuously taken by a person suspected of having diabetes and a person suffering from diabetes. The onset can be effectively prevented or suppressed for both diabetic nephropathy induced from resistant type 2 diabetes.

  The low molecular chitosan which is an active ingredient of the diabetic nephropathy preventing composition of the present invention is a hydrolyzate of chitosan prepared by a deacetylation reaction by chitin alkali treatment, and preferably has an average molecular weight of 10,000 to 30. , And more preferably those having an average molecular weight of 15,000 to 25,000. For example, low molecular weight chitosan having an average molecular weight of 20,000 is a mixture of chitosan degradation products distributed in the molecular weight range of 1,000 to 100,000.

  The low molecular chitosan may be a free form or a salt. The low molecular weight chitosan salts may be pharmaceutically acceptable, and specifically include hydrochloride, lactate, acetate, sulfate, citrate, ascorbate, malate, succinate. Examples thereof include acid salts, adipates, gluconates, and tartrate salts.

  Low molecular weight chitosan can be prepared directly by low-molecular-weight chitosan by alkali treatment of chitin, but it is difficult to control. Therefore, it is preferable to produce commercially available chitosan by acid degradation or enzymatic degradation to adjust molecular weight. . For example, after chitosan is heated with an acid such as hydrochloric acid, acetic acid, formic acid, etc., the acid is removed or neutralized and desalted, and powdered by crystallization, etc., chitosan is dissolved in dilute acid, chitosanase, glucosaminidase, Examples thereof include a method in which a chitosan degrading enzyme such as hemicellulase is allowed to act. The chitosan hydrolyzate (low molecular weight chitosan) thus obtained can be used as it is, but may be used after being purified by a method such as column chromatography or solvent fractionation. In addition, what is necessary is just to set hydrolysis conditions suitably so that the chitosan hydrolyzate (low molecular weight chitosan) of said average molecular weight range may be obtained.

  The composition for preventing diabetic nephropathy of the present invention includes, in addition to the above active ingredients, excipients, stabilizers, preservatives, preservatives, brighteners, thickeners, coloring agents, minerals, vitamins, and sugars. In addition, additives such as fragrances, fats and oils, amino acids and the like can be appropriately included.

  The product form of the diabetic nephropathy preventing composition of the present invention is not particularly limited as long as it is a form suitable for oral administration, and examples thereof include tablets, powders, granules, solutions, capsules and the like.

The effective intake of the composition for preventing diabetic nephropathy of the present invention is preferably 1.0 to 5000 mg, more preferably 50 to 500 mg in terms of low molecular weight chitosan and / or its salt per adult day. The safety of low-molecular chitosan has already been confirmed, and as a result of an acute toxicity test by oral administration in rats, LD ₅₀ is 5 g or more per kg of body weight.

  Further, the diabetic nephropathy preventing composition of the present invention can be ingested by being mixed with food and drink such as nourishing tonic drinks, juices, coffees, mineral water, miso soup, seasonings, sauces and dressings. . The amount of the composition for preventing diabetic nephropathy in the food or drink may be set based on the above effective intake, but is usually 0.01 to 20 mass in terms of low molecular weight chitosan and / or its salt. % Is preferable, and 0.1 to 3% by mass is more preferable.

  The composition for preventing diabetic nephropathy of the present invention reduces the amount of urinary albumin excreted by oral administration at an early stage of diabetes, more preferably before the onset of diabetes, and the histopathological change (proximal) of renal tissue. It effectively suppresses the onset of diabetic nephropathy by inhibiting tubule meganucleation, extent of distal tubule protrusion, glomerular mesangial cell area, tubulointerstitial fibrosis, glomerular hypertrophy, etc. Can be prevented or suppressed.

  In addition, safety has already been confirmed for taking in combination with other anti-diabetic drugs and low-molecular-weight chitosan, and the composition for preventing diabetic nephropathy of the present invention is used for the treatment of diabetic nephropathy. May be taken at the same time as a therapeutic drug for diabetes, or may be taken individually.

  3.0 kg of chitosan was dispersed in 300 L of water, and lactic acid was added and dissolved with stirring (pH 5.0). To this solution was added 1500 U of chitosanase (trade name “chitosanase-U”, manufactured by Meiji Seika Co., Ltd.) (the titer was indicated by the manufacturer), and the decomposition reaction was performed at 45 ° C. This reaction solution was filtered with a filter press to remove insoluble matters, and then subjected to ultrafiltration membrane treatment. In the ultrafiltration membrane treatment, the non-permeate side liquid treated with the trade name “Microza ACV-3010” (manufactured by Asahi Kasei Kogyo Co., Ltd., molecular weight cut off 13,000) is recovered, and then the trade name “Microza AHV-3010”. (Asahi Kasei Kogyo Co., Ltd., molecular weight cut off: 50,000), and the liquid on the permeate side was recovered. This permeate was freeze-dried to obtain 570 g of low molecular chitosan. When the molecular weight of this low molecular chitosan was measured, the molecular weight distribution was in the range of 15,000 to 30,000, and the average molecular weight was about 20,000. The test described later was performed using this low-molecular chitosan.

In the study, as a disease model animal, 1) Streptozotocin (STZ) induction characterized by having impaired insulin secretion in response to glucose stimulation, which is considered to closely resemble human non-obese non-insulin dependent diabetes SPF male ICR mice (8 weeks old, purchased from Japan SCL), which are progressive diabetic mice, and 2) severe obesity and hyperinsulinemia that are considered to closely resemble human non-insulin-dependent diabetes Two types of diabetic mouse models, SPF male KK-A ^y mice (6 weeks old, purchased from CLEA Japan), which are hereditary obese diabetic mice characterized by insulin resistance, were used. In addition, after both mice have been bred in advance, symptoms such as polydipsia, polyuria, and increase in urinary albumin are observed with an increase in blood glucose, and damage to the kidney is seen from pathological analysis of the kidney Have confirmed.

Test Example 1 (Test on urinary albumin excretion in STZ-induced progressive diabetic mice)
8 week old SPF male ICR mice fasted for 20 hours were distributed into 3 groups (10 mice each), and STZ was injected intraperitoneally at a single dose of 100 mg / kg. In addition, STF-untreated SPF male ICR mice (8 weeks old) were used as a normal control group (10 groups).

  From the 2nd week to the 23rd week after the STZ treatment, 2 groups of the STZ treatment groups were administered drinking a solution of low molecular chitosan dissolved in distilled water (concentration 0.2 mass%, 0.8 mass%). The low-molecular chitosan administration group and the remaining 1 group (diabetes control group) were administered with distilled water. The normal control group was administered with distilled water.

  Then, 22 weeks after the STZ treatment, each group of individual mice was placed in a metabolic cage, urine was collected for 24 hours, and urinary albumin excretion per 24 hours was measured. The result is shown in FIG.

  From Fig. 1, the 0.2% by mass and 0.8% by mass low-molecular-weight chitosan-administered groups significantly suppressed the increase in urinary albumin excretion per day (p <0.05) compared to the diabetic control group. (0.2 mass% low molecular chitosan administration group: 12 ± 1 μg, 0.8 mass% low molecular chitosan administration group: 13 ± 5 μg, diabetes control group: 369 ± 305 μg).

Test Example 2 (Studies on meganucleation of proximal tubules and protrusion of distal tubules in STZ-induced progressive diabetic mice)
From the mice in Test Example 1, 24 weeks after STZ treatment, each group of individual mice was placed in a metabolic cage, the kidneys were removed, and the histopathological changes in the kidneys were evaluated. The kidney was sliced to about 2 mm after excision, fixed in a 10% neutral buffered formalin solution all day and night, and then immersed in 0.01% phosphate buffer (pH 7.2) for 48 hours. Next, each was immersed in 70, 80, 90, and 95% alcohol sequentially for 3 hours, further immersed in 99.5% alcohol all day and night, and then immersed in absolute alcohol for 1 hour to completely dehydrate. The tissue was then placed in benzene I, II, III and paraffin saturated benzene for 30 minutes, respectively, to remove the alcohol. Then, the tissue was infiltrated with paraffin, and the tissue was embedded in paraffin. After preparing the tissue block, a tissue section having a thickness of about 1 μm was prepared using a large sliding microtome (trade name “LS-113”, manufactured by Yamato).

  The obtained tissue specimen is observed with an optical microscope. Histopathological changes (proximal tubule nucleation and extent of distal tubule protrusion) are classified into 4 stages (-: no change, +: slight change) , ++: change, +++: significant change). FIG. 2 shows the result of the degree of megakaryosis of the proximal tubule, and FIG. 3 shows the result of the degree of protrusion of the distal tubule.

  2 and 3, it can be seen that in the low-molecular chitosan-administered group, renal histopathological changes such as megakaryosis of the proximal tubule and protrusion of the distal tubule are suppressed compared to the diabetic control group. .

Test Example 3 (Test on the area of glomerular mesangial cells in STZ-induced progressive diabetic mice)
The glomeruli were observed by PAS staining of the kidney tissue specimen removed in Test Example 2. That is, the tissue sections were immersed in xylene and anhydrous xylene for 20 minutes to remove paraffin, and were sequentially attached to 99.5, 90, and 80% alcohol to remove xylene and rehydrate the tissue. Then, it was immersed in a 0.5% periodic acid aqueous solution for 10 minutes and washed several times with purified water. Next, it was immersed in a Schiff reagent for 40 minutes, and then immersed in sulfite water three times for 3 minutes each and washed with tap water. Next, it was immersed in a hematoxylene solution for 10 seconds and washed again with tap water. Finally, it was dehydrated with 80, 90, 99.5% and anhydrous alcohol, and clarified with xylene and xylene and sealed. This is observed quantitatively by observing 15 glomeruli in each tissue specimen with an image analyzer (trade name “Image analyzer V1,” manufactured by TOYOBO), and measuring the area of PAS-positive basement membrane and mesangial cells. The degree of change was evaluated. The result is shown in FIG.

From FIG. 4, the low-molecular-weight chitosan-administered group had a significantly lower (p <0.01) increase in the area of the mesangial region compared to the diabetic control group, and was suppressed to the same level as the normal control group. (0.2% by mass low molecular chitosan administration group: 15 ± 1 × 10 ⁻⁴ mm ² , 0.8% by mass low molecular chitosan administration group: 14 ± 0.4 × 10 ^{− 4} mm ² , diabetes control group: 19 ± 1 × 10 ⁻⁴ mm ² ).

Test Example 4 (Test for urinary albumin excretion in hereditary obese diabetic KK-A ^y mice)
As normal mice, 6 week-old SPF male ICR mice (12 mice per group) were bred with a standard diet (trade name “MF”, manufactured by Oriental Yeast Co., Ltd.) (normal group). On the other hand, 6-week-old SPF male KK-A ^y mice as diabetic mice were divided into 2 groups (12 mice in each group), and 1 group was bred with a high fat-containing diet (standard diet + 10% by mass lard) (control group). ) The other group was a group to which a high fat-containing feed and a 1.8% by mass low molecular chitosan aqueous solution were administered (low molecular chitosan administration group).

  At 0, 3, 6, 9, 12, 18, 24, and 30 weeks after the start of the experiment, each group of animals was placed in a metabolic cage, and the amount of water consumed and the amount of urine per 24 hours and the amount of urinary albumin excreted were measured. The result of urinary albumin excretion is shown in FIG.

  From FIG. 5, the amount of urinary albumin excreted in the normal group was 0.01 to 0.04 mL / 24 h throughout the experimental period, whereas the control group and the low-molecular chitosan-administered group showed an increase until the 12th week. Then, it decreased with a decrease in urine volume. The low-molecular chitosan-administered group showed lower values at 3, 6, 12, and 24 weeks than the control group's urinary albumin level, but no significant difference was observed.

Test Example 5 (Test on the amount of thiobarbituric acid (TBA) reactive substance in hereditary obese diabetes KK-A ^y mice)
In Test Example 4, at the 12th and 30th weeks, half of the mice in each group were laparotomized under ether anesthesia and both kidneys were removed. After measuring the wet weight of the kidney, one kidney was homogenized and the amount of thiobarbituric acid (TBA) reactant was measured as an index of lipid peroxidation. The result is shown in FIG.

  From FIG. 6, the amount of TBA reactive substance in the renal tissue at 12 weeks was about three times that of the normal group in both the control group and the low molecular chitosan administration group, but the low molecular chitosan administration group was the control group at 30 weeks. It is significantly lower (p <0.01) than the normal group (p <0.01), indicating that the oxidative stress on the kidney is reduced (week 30: normal group: 1.31 ± 0.3, control group: 5.08 ±) 0.4, low molecular chitosan administration group: 2.04 ± 0.2).

Test Example 6 (Test for fibrosis of tubulointerstitium in hereditary obese diabetes KK-A ^y mice)
One kidney extracted in Test Example 5 was fixed in 10% neutral buffered formalin solution, embedded in paraffin, and then sliced. The obtained sections were stained by Masson's trichrome according to a conventional method, and the stained fibrosis area was measured using image analysis software (trade name “Win ROOF”, manufactured by Mitani Corporation). The result is shown in FIG.

  From FIG. 7, in the control group, more advanced fibrosis was observed at the 30th week, whereas in the low-molecular chitosan-administered group, the progression of fibrosis was suppressed to the same level as in the normal group. It can be seen that histopathological changes are suppressed (30th week: normal group: 4.59 ± 0.33, control group: 14.5 ± 0.69, low-molecular chitosan administration group: 5.13 ± 0.10).

Test Example 7 (Test on glomerular area and glomerular mesangial area of hereditary obese diabetes KK-A ^y mouse)
The kidney tissue sections obtained in Test Example 6 were stained with PAS according to a conventional method. The glomerular area and glomerular mesangial area were measured from the PAS-stained specimen using image analysis software (trade name “Win ROOF”, manufactured by Mitani Corp.). The results are shown in FIGS.

  From FIG. 8, the glomerular area of the low-molecular chitosan-administered group is about 1.3 times that of the normal group, but the glomerular hypertrophy is significantly suppressed (p <0.01) compared to the control group. It can be seen that the histopathological changes of the kidney are suppressed (Week 12: Normal group: 3415 ± 71.9, Control group: 5543 ± 90.0, Low-molecular chitosan administration group: 4599 ± 77.2, Week 30: Normal group : 3806 ± 91.1, control group 6464 ± 142.3, low-molecular chitosan administration group: 4832 ± 139.6).

  FIG. 9 also shows that the mesangial area ratio to the glomerulus is significantly suppressed in the low-molecular chitosan-administered group, and the histopathological change of the kidney is suppressed (12th week: normal group). : 11.2 ± 0.32, control group: 23.2 ± 0.49, low molecular chitosan group: 21.6 ± 0.38, 30th week… normal group: 10.0 ± 0.50, control group 27.9 ± 0.54, low molecular chitosan group: 22.5 ± 0.52).

From the above test results, it has been clarified that continuous administration of low molecular chitosan can suppress impaired renal function in STZ-induced progressive diabetic mice and hereditary obese diabetic KK-A ^y mice, characterized by impaired insulin secretion. It has been shown to effectively suppress the development of diabetic nephropathy induced by type 2 diabetes characterized by insulin resistance in type 2 diabetes and obesity.

It is a figure which shows the result of having measured the urinary albumin excretion amount of the mouse | mouth of each group in the 22nd week at the time of administering low molecular chitosan to STZ induction progressive diabetes mouse | mouth. It is a figure which shows the result of having observed the degree of the meganucleation of the proximal tubule of the renal tissue sample extracted from the mouse | mouth of each group in the 22nd week at the time of administering low molecular chitosan to STZ induction progressive diabetes mouse | mouth. It is a figure which shows the result of having observed the extent of the protrusion of the distal tubule of the kidney tissue sample extracted from the mouse | mouth of each group in the 22nd week at the time of administering low molecular chitosan to STZ induction progressive diabetes mouse | mouth. It is a figure which shows the result of having measured the glomerular mesangial area of the kidney tissue sample extracted from each group mouse | mouth in the 24th week when low molecular chitosan is administered to STZ induction progressive diabetes mouse | mouth. It is a figure showing the change of the fluctuation | variation of the urinary albumin excretion amount of each group mouse | mouth at the time of administering low molecular chitosan to the hereditary obesity diabetes KK- ^Ay mouse | mouth. It is a figure which shows the result of having measured the amount of TBA reaction substance in the renal tissue of each group mouse | mouth in the 12th week and 30th week when low molecular chitosan is administered to the hereditary obesity diabetes KK- ^Ay mouse | mouth. It is a figure which shows the result of having observed and compared the fibrillation of the tubulointerstitium of each group mouse | mouth in the 12th week and 30th week when low molecular chitosan is administered to the hereditary obesity diabetes KK- ^Ay mouse | mouth. It is a figure which shows the result of having measured the glomerulus area of each group mouse | mouth in the 12th week and the 30th week at the time of administering low molecular weight chitosan to the hereditary obesity diabetes KK- ^Ay mouse | mouth. It is a figure which shows the result of having measured the glomerular mesangial area ratio with respect to the glomerular area of each group mouse | mouth in the 12th week and the 30th week at the time of administering low molecular chitosan to the hereditary obesity diabetes KK- ^Ay mouse | mouth.

The composition for preventing diabetic nephropathy of the present invention can be suitably used for prevention or suppression of the onset / progression of kidney damage caused by diabetes.

Claims (4)

  A composition for preventing diabetic nephropathy, comprising low-molecular chitosan and / or a salt thereof as an active ingredient.   The composition for preventing diabetic nephropathy according to claim 1, wherein the low molecular chitosan has an average molecular weight of 10,000 to 30,000.   The composition for preventing diabetic nephropathy according to claim 1 or 2, which is used for prevention of diabetic nephropathy induced by non-obese insulin-independent diabetes.   The composition for preventing diabetic nephropathy according to claim 1 or 2, which is used for the prevention of diabetic nephropathy induced by obese insulin-independent diabetes.