JP6801894B2 - Agent for reducing blood uremic substances - Google Patents

Description

The present invention comprises one or more compounds selected from the group consisting of tyrosine and its pharmacologically acceptable salts and esters (hereinafter, these may be collectively referred to as "tyrosines"). The present invention relates to a reducing agent for a uremic substance in blood and a preventive or ameliorating agent for uremia contained as an active ingredient.

The kidney is an organ that regulates homeostasis in the body. As one of the renal functions, it has an action of taking in waste products and toxins (uremic substances) in blood, sending them out into urine, and excreting them out of the body. When such renal function is reduced, uremic substances that should be excreted by the kidney are likely to be accumulated in the body, causing symptoms such as nausea and loss of appetite, as well as damage to cells in the kidney and further deterioration of renal function. It is also known that those with impaired renal function are at higher risk of suffering from stroke or myocardial infarction than healthy subjects. Therefore, prevention or treatment of symptoms and diseases (uremia) caused by the accumulation of such uremic substances not only treats symptoms such as nausea and loss of appetite, and exacerbation of renal disorders, but also causes stroke and It is also necessary from the viewpoint of preventing heart disease.

Dialysis is considered to be the most effective treatment for uremia. In fact, if uremic substances are excreted from the body by dialysis therapy, even if renal function is abolished, the death of the patient due to the uremic substances can be avoided. However, long-term dialysis has created new problems with complications such as anemia, renal osteodystrophy, renal cancer, epicarditis, aluminum osteopathy, amyloidosis, crystalline arthritis, and polycystic kidney disease. .. In addition, there are medical economic problems such as a decrease in the quality of life (QOL) of patients who frequently go to dialysis facilities and the occurrence of high costs.

On the other hand, as a preparation having a function of adsorbing a uremic substance, a kind of activated carbon, spherical adsorption charcoal Kuremedin (registered trademark: Kureha Chemical Co., Ltd.) is known (Patent Documents 1 and 2). Kremezin is a black spherical particle obtained by oxidizing and reducing spherical microporous carbon derived from petroleum hydrocarbons at high temperature, and adsorbs harmful substances existing in the digestive tract without being absorbed in the body. It is used for the purpose of removing (reducing) blood uremic substances and suppressing deterioration of renal function by being excreted together with feces. In addition, carbon nanotubes (Patent Document 3), uremic substance-specific recognition polymers (Patent Document 4), and the like are known as agents for reducing blood uremic substances having a function of adsorbing uremic substances. However, the relationship between tyrosine and the reducing action of blood uremic substances has not been known so far.

Special Publication No. 62-29368 Special Publication No. 63-60009 Japanese Unexamined Patent Publication No. 2006-8602 Japanese Unexamined Patent Publication No. 2004-131406

An object of the present invention is an agent for reducing a blood uremia substance, which has an effect of effectively reducing (reducing) the concentration of a blood uremia substance, and a uremia having an effect of effectively improving uremia. To provide a preventive or ameliorating agent for.

The present inventor is continuing diligent research to solve the above problems. In the process, it was found that tyrosine has an effect of effectively reducing the concentration of a blood uremic substance and an effect of effectively improving uremia, and has completed the present invention. ..

That is, the present invention is as follows.
[1] An agent for reducing a blood uremic substance, which comprises one or more compounds selected from the group consisting of tyrosine and a pharmacologically acceptable salt and ester thereof as an active ingredient. ..
[2] The reducing agent according to the above [1], which is characterized by oral administration.
[3] The reducing agent according to the above [1] or [2], wherein the compound is Lm-tyrosine or 2-aza-DL-tyrosine.
[4] One or more uremic substances selected from p-cresol sulfate, phenyl sulfate, indoxyl sulfate, 1-methyladenosine, creatinine, trimethylamine-N-oxide, p-cresol, phenol, and indole. The reducing agent according to any one of the above [1] to [3], which is characterized by being the substance of.
[5] An agent for preventing or ameliorating uremia, which comprises, as an active ingredient, one or more compounds selected from the group consisting of tyrosine and a pharmacologically acceptable salt and ester thereof.
[6] The preventive or ameliorating agent according to the above [5], which is characterized by oral administration.
[7] The prophylactic or ameliorating agent according to the above [5] or [6], wherein the compound is Lm-tyrosine or 2-aza-DL-tyrosine.
[8] One or more types of uremia selected from p-cresol sulfate, phenyl sulfate, indoxyl sulfate, 1-methyladenosine, creatinine, trimethylamine-N-oxide, p-cresol, phenol, and indole. The preventive or ameliorating agent according to any one of the above [5] to [7], which is characterized by a symptom or disease caused by a substance.

In addition, as another embodiment of the present invention, a blood uremic substance is administered by administering one or more compounds selected from tyrosine to a patient who needs reduction of the blood uremic substance. One or more compounds selected from tyrosine for use as a method for reducing blood uremic substances or as a reducing agent for blood uremic substances, and tyrosine for use in reducing blood uremic substances. Examples include the use of one or more compounds selected from the class and one or more compounds selected from the tyrosine species for producing a reducing agent for blood uremic substances. ..

Further, as another embodiment of the present invention, urinary toxicosis can be prevented or prevented by administering one or more compounds selected from tyrosine to a patient who needs reduction of blood urinary toxicant substances. In the method of improvement (treatment), one or more compounds selected from tyrosine for use as a preventive or ameliorating (therapeutic) agent for urinary poisoning, and prevention or improvement (treatment) of urinary poisoning. One or more compounds selected from tyrosine for use, and one or more selected from tyrosine for producing preventive or ameliorating (therapeutic) agents for urinary poisoning The use of compounds can be mentioned.

According to the present invention, an increase (increase) in blood uremic substance concentration can be reduced (suppressed) or prevented, and the uremia substance concentration in blood can be adjusted to a normal value, thus preventing or improving uremia. Useful for (treatment).

In FIGS. 1A to 1C, plasma samples were prepared from obese diabetes model mice (db / db mice) before, after, and after withdrawal of L-meta (m) -tyrosine, and three types of uremia in plasma were prepared. It is a figure which shows the result of having measured the concentration of the uremia substance (phenyl sulfate [FIG. 1A], para [p] -cresyl sulfate [FIG. 1B], and indoxyl sulfate [FIG. 1C]). FIG. 1D is a diagram showing the results of preparing urine samples from db / db mice before, after, and after withdrawal of Lm-tyrosine and measuring the concentration of albumin in urine. The albumin / creatinine correction value on the vertical axis indicates the relative value of the urinary albumin concentration to the urinary creatinine concentration. Plasma samples were prepared from db / db mice (n = 10) before and after administration of Lm-tyrosine, and two types of uremic substances in plasma (phenyl sulfate [Fig. 2A] and indoxyl sulfate [Fig.] It is a figure which shows the result of having measured the concentration of 2B]). "*" In the figure indicates that there is a statistically significant difference (p <0.05) by Tukey's test. 3A and 3C show plasma samples prepared from db / db mice before and after administration of 2-aza-DL-tyrosine and two uremic substances in plasma (creatinine [FIG. 3A] and 1-methyl). It is a figure which shows the result of having measured the concentration of adenosine [FIG. 3C]). 3B and D show urine samples prepared from db / db mice before and after administration of 2-aza-DL-tyrosine and two uremic substances in urine (creatinine [FIG. 3B] and 1-methyl). It is a figure which shows the result of having measured the concentration of adenosine [FIG. 3D]). In FIGS. 4A and 4B, plasma samples were prepared from db / db mice before and after administration of 2-aza-DL-tyrosine, and two types of uremic substances (indoxyl sulfate [Fig. 4A] and sulfuric acid] in plasma were prepared. It is a figure which shows the result of having measured the concentration of phenyl [FIG. 4B]). FIG. 4C is a diagram showing the results of preparing urine samples from db / db mice before and after administration of 2-aza-DL-tyrosine and measuring the concentration of a uremic substance (phenyl sulfate) in urine. .. Plasma samples were prepared from db / db mice (n = 4) before and after administration of 2-aza-DL-tyrosine, and three types of uremic substances (phenyl sulfate [Fig. 5A], indoxyl sulfate) in plasma were prepared. FIG. 5B is a diagram showing the results of measuring the concentrations of [FIG. 5B] and creatinine [FIG. 5C]). "*" In the figure indicates that there is a statistically significant difference (p <0.05) by Tukey's test. FIG. 5D is a diagram showing the results of preparing urine samples from db / db mice before and after administration of 2-aza-DL-tyrosine and measuring the concentration of albumin in urine. The albumin / creatinine correction value on the vertical axis indicates the relative value of the urinary albumin concentration to the urinary creatinine concentration. 4 types of groups (normal breeding group [“NC” in the figure], 2-aza-DL-tyrosine administration normal breeding group [“NA” in the figure], adenine diet breeding group [“CC” in the figure], And 2-aza-DL-tyrosine-administered adenine dietary group [“CA” in the figure]), serum samples were prepared, and four types of uremic substances (phenyl sulfate [Fig. 6A], indoxyl sulfate] in the serum were prepared. FIG. 6B] shows the results of measuring the concentrations of trimethylamine-N-oxide [TMAO] [FIG. 6C] and creatinine [FIG. 6D]). "*" In the figure indicates that there is a statistically significant difference (p <0.05) by Tukey's test. Kidneys were collected from two groups (Adenin dietary group [“CC” in the figure] and 2-aza-DL-tyrosine-administered Adenine dietary group [“CA” in the figure]), and Masson's trichrome. It is a figure which shows the microscopic image (FIG. 7A) which tissue was stained by the staining (MTS) method, and the result (FIG. 7B) which measured the ratio (%) of the renal tubule to the whole kidney. "*" In the figure indicates that there is a statistically significant difference (p <0.05) by Tukey's test.

The agent for reducing blood uremic substances of the present invention contains one or more compounds selected from tyrosine, whose use is limited to "to reduce blood uremic substances", as an active ingredient. It is an agent contained (hereinafter, may be referred to as "Reducing Agent"), and the preventive or ameliorating agent for uremia of the present invention has a limited use for "to prevent or ameliorate uremia". An agent containing one or more compounds selected from tyrosine as an active ingredient (hereinafter, may be referred to as "preventive / ameliorating agent") (these agents are collectively referred to as "the agent". There is a case). This drug has specific functions such as health supplements, foods with health claims, supplements, etc., in addition to pharmaceuticals (preparations for reducing blood urinary toxic substances, preparations for prevention or improvement [treatment] of urinary toxicosis). It also includes pharmaceutical-like (food) compositions and functional foods that are eaten for the purpose of maintaining health.

In the present invention, the "uremic substance (also referred to as" uremic toxin ")" is a substance (waste products, toxins, etc.) excreted by the normal kidney, and is excreted for some reason such as decreased renal function. It means a substance that increases (accumulates) in the blood and causes symptoms or diseases of uremia when its function deteriorates. "Uremic substances" are usually classified into three types: water-soluble low-molecular-weight substances (500 daltons or less), medium-molecular-weight substances (more than 500 daltons), and protein-binding substances (literature "Vanholder, R. , et al: Kidney Int., 63 (5): 1934-1943, 2003 ").

Specific examples of the water-soluble low molecular weight substance include 1-methyladenosine, 1-methylguanosine, 1-methylinosine, and ADMA (Asymmetric dimethylarginine). α-keto-δ-guanidinovaleric acid, α-N-acetylarginine, Arabitol, Argininic acid, benzyl alcohol (Benzylalcohol) ), Β-guanidinopropionic acid, β-lipotropin, Creatine, Creatinine, Cytidine, Dimethylglycine, Erythritol, γ -Guanidinobutyric acid, Guanidine, Guanidinoacetic acid, Guanidonosuccinic acid, Hypoxanthine, Malondialdehyde, Mannitol, Methylguanitol (Methylguanidine), myo-inositol ^{(myoinositol), N 2, N} 2 - dimethyl-guanosine ^{(N 2, N 2 -dimethylguanosine)} , N 4 - acetyl cytidine ^{(N 4 -acetylcytidine), N 6} - methyl adenosine (N ⁶ -methyladenosine ), ^{N 6} - threonyl carbamoyl adenosine (N ⁶ -threonylcarbamoyladenosine), orotic acid (orotic acid), orotidine (orotidine), oxalate (oxalate), phenylacetylglutamine (phenylacetylglutamine), pseudouridine (pseudouridine), SDMA (Symmetric Dimethylarginine), sol Sorbitol, Taurocyamine, Threitol, Thymine, Uracil, Urea, Uric acid, Uridine, Xanthine, Xanthosine ), Trimethylamine-N-oxide (TMAO), 4-ethylphenylsulfate and the like.

Specific examples of the medium-molecular-weight substance include adrenomedullin, atrial natriuretic peptide (ANP), β _2- microglobulin (β _2- microglobulin), and β-endolphin (β-). endorphin, Cholecystokinin, CC16 (Clara cell protein), Complement factor D, Cystatin C, Degranulation inhibiting protein 1, Delta sleep-inducing peptide (Delta-sleep inducing peptide), Endothelin, Hyaluronic acid, Interleukin-1β, Interleukin-6, κ-Ig light chain (κ-Ig light) Chain), λ-Ig light chain, λ-Ig light chain, Leptin, Methionine-enkephalin, Neuropeptide, Parathyroid hormone, Retinol- Binding protein), tumor necrosis factor (TNF) -α (Tumor necrosis factor-α) and the like can be mentioned.

Specific examples of the protein-binding substance include 2-methoxyresorcinol, 3-deoxyglucosone, and CMPF (3-Carboxy-4-methyl-5-propyl-2-furanpropionate). ), Fructoselysine, Glyoxal, Hippuric acid, Homocysteine, Hydroquinone, Indole-3-acetic acid, Indoxyl sulfate or its precursor (Indole), Kinurenine, Kynurenic acid, Leptin, Melatonin, Methylglyoxal, N-carboxymethyllysine (N-( carboxymethyl) lysine), p-Cresyl sulfate or its precursor (p-Cresol), Pentosidine, phenyl sulfate (Phenyl sulfate) or its precursor (phenol [Phenol]) ), P-OH-horseuric acid (P-OHhippuric axid), putrescine, quinolinic acid, Retinol-binding protein, spermidine, spermine, etc. Can be done.

The uremic substance in the present invention is selected from p-cresol sulfate, phenyl sulfate, indoxyl sulfate, 1-methyladenosine, creatinine, TMAO, and precursors thereof (p-cresol, phenol, and indole) 1. Alternatively, two or more kinds of substances are preferable.

In the present invention, "reduction of blood uremic substance" means blood by suppressing the production (production) of the uremic substance or its precursor and promoting the excretion of the uremic substance or its precursor to the outside of the body. It means that the concentration of uremic substances contained in the substance is reduced (decreased).

In the present invention, tyrosine means a compound represented by the following general formula (I) and pharmacologically acceptable salts and esters thereof.

(In the formula, any one of X _{1 to} X ₅ represents C-OH, and the remaining four independently represent C-H or N.)

The compound represented by the general formula (I) has optical activity and may be any of DL form (racemic form), D form and L form, and all of them are included in the tyrosine of the present invention.

The following examples can be given as the positions of −OH of the compound of the above formula (I).

In the compounds represented by the above three formulas, the compound (tyrosine) in which all four ring atoms except the ring atom having a substituent are CH and one of the four ring atoms represents N, and the rest. The compounds (azatyrosine) in which all three are all CH are exemplified below.

As is clear from the above chemical formula, in the case of tyrosine, any of ortho (o) -tyrosine, meta (m) -tyrosine, and para (p) -tyrosine can be used. Specific examples of the tyrosine of the present invention include Lo-o-tyrosine, Lm-tyrosine, Lp-tyrosine, DL-o-tyrosine, DL-m-tyrosine, DL-p-tyrosine, and D-. o-tyrosine, Dm-tyrosine, Dp-tyrosine, 2-aza-L-tyrosine, 3-aza-L-tyrosine, 2-aza-DL-tyrosine, 3-aza-DL-tyrosine, 2- Examples thereof include aza-D-tyrosine and 3-aza-D-tyrosine, with Lm-tyrosine and / or 2-aza-DL-tyrosine being preferred.

In addition to the above, the following compounds are exemplified as compounds represented by the general formula (I).

The "pharmaceutically acceptable salt of the compound represented by the general formula (I)" of the present invention is not particularly limited, and is, for example, a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, or a nitrate salt. , Sulfates, phosphates and other inorganic acid salts; oxalate, malonate, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetic acid Organic salts such as salts, acetates, methanesulfonates, p-toluenesulfonates, trifluoromethanesulfonates; alkali metal salts such as sodium salts and potassium salts; alkaline earth metals such as calcium salts and magnesium salts Salts; ammonium salts; amino acid salts such as asparagate, glutamate, lysine, arginine, histidine salts and the like can be mentioned.

The "pharmaceutically acceptable ester of the compound represented by the general formula (I)" of the present invention is hydrolyzed in the body such as in the stomach or blood to produce a compound represented by the general formula (I). It is not particularly limited as long as it is, for example, an ester of tyrosine and ethanol, an ester of tyrosine and n-propanol, an ester of tyrosine and i-propanol, an ester of tyrosine and ethylene glycol, and an ester of tyrosine and glycerin. And so on.

The agent can be used as a pharmaceutically acceptable usual carrier, binder, stabilizer, excipient, diluent, pH buffer, disintegrant, isotonic agent, additive, coating agent, as required. Examples thereof include those to which a compounding component such as a solubilizer, a lubricant, a gliding agent, a solubilizing agent, a lubricant, a flavoring agent, a sweetening agent, a solvent, a gelling agent, and a nutritional agent is further added. Specific examples of such compounding ingredients include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, and polyalkylene glycol. Polyvinyl alcohol and glycerin can be exemplified.

Uremia, which is a disease to be prevented or ameliorated by the preventive / improving agent, includes the above-mentioned uremic substances (preferably p-crecil sulfate, phenyl sulfate, indoxyl sulfate, 1-methyladenosine, creatinine, and precursors thereof. The symptoms or diseases are not particularly limited as long as they are caused by one or more substances selected from the body [p-cresol, phenol, and indole], and such symptoms or diseases include, for example, loss of appetite and nausea. , Vomiting, mouth odor, stomatitis, enteritis, etc.; gastrointestinal abnormalities; selflessness, indifference, poor memory, depression, tilting, coma, polyneuritis, (developmental) coordination disorder, loss of appetite, etc. Abnormalities; Cardiovascular abnormalities such as arteriosclerosis, anemia, erythroid hematopoietic disorders, hypertension, ischemic heart disease, peritonitis, myocarditis, (blood) coagulation abnormalities, heart failure, cardiovascular disorders; pigmentation, itching (feeling) , Subcutaneous bleeding, skin atrophy, infection, atopy, hair loss and other skin abnormalities (diseases); albuminuria, acute renal failure, acute uremic nephropathy, renal anemia, chronic renal failure, uremic interstitial disorder, acute nephritis, chronic Renal dysfunction such as nephritis, nephropathy, diabetic nephropathy, arteriosclerotic nephropathy, renal osteodystrophy (eg, renal osteodystrophy), flooding; stroke; myocardial infarction; cancer; autism; immunity Insufficiency; bone disorders; hyperparathyroidism; insulin resistance; malnutrition; inflammation; quake; etc. In addition, the above-mentioned urinary toxicosis is a symptom associated with LPS (Lipopolysaccharide) such as IgA nephropathy, cystic kidney, liver dysfunction (for example, fulminant hepatitis, fatty liver, NASH, NAFLD) and cancer (for example, bile carcinogenesis). Or diseases; inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; arteriosclerosis-related diseases; autoimmune diseases such as lupus, dermatitis and rheumatism; obesity; metabolic syndrome; diabetes; autism; Parkinson's disease; Alzheimer's disease Disease; sarcopenia is also included.

The administration form of the present drug includes oral administration in which the dosage form is powder, granules, tablets, capsules, syrup, suspension, etc., or injection or spraying in the dosage form such as solution, emulsion, suspension, etc. Parenteral administration, which is administered intranasally, can be mentioned as a dosage form, and oral administration is preferable.

The dose of the present drug is appropriately determined according to age, body weight, gender, symptoms, sensitivity to the drug, etc., and is, for example, in the range of 0.1 μg to 200 mg / kg (body weight) / day. In this example described later, according to an experiment using a model mouse, a dose of Lm-tyrosine of 10 to 50 mg / kg / day and 2-aza-DL- of 3 to 5 mg / kg (body weight) / day The dose of tyrosine, that is, the dose of tyrosine of 3 to 50 mg / kg / day is specifically shown. Such doses are doses to humans based on the Human Equivalent Dose (HED) 12.3 in mice (see document "Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers"). When converted to, it is 0.24 to 4.1 mg / kg / day. Therefore, the dose of the present drug is preferably 0.01 to 100 mg / kg / day, more preferably 0.03 to 60 mg / kg / day, further preferably 0.06 to 30 mg / kg / day, and 0. .1 to 10 mg / kg / day is even more preferable, and 0.2 to 6.0 mg / kg / day is most preferable. The drug may be administered once or in multiple doses (for example, 2 to 4 doses) per day.

The administration target of this drug is not particularly limited, but usually, the concentration of the uremic substance in the blood is due to a person who needs to reduce the blood uremic substance, specifically, a decrease in renal function or the like. Those who have increased (increased) or who have accumulated uremic substances in their bodies.

The reducing agent may contain a component for reducing blood uremic substances other than tyrosine, but tyrosine alone exerts an excellent effect of reducing blood uremic substances. Those containing no components other than tyrosine that reduce blood uremic substances (for example, proteins, DNA, RNA, plant-derived extracts, polymers) are preferable. In addition, the preventive / ameliorating agent may contain a component for preventing or improving uremia other than tyrosine, but tyrosine alone exerts an excellent preventive or ameliorating effect on uremia. , Tyrosine and other components that prevent or improve uremia (eg, proteins, DNA, RNA, plant-derived extracts, polymers) are not contained.

Tyrosine can be produced by any known method such as chemical synthesis, production by microorganisms, production by enzyme, etc., but commercially available products can also be used. For example, when Lm-tyrosine is used as the tyrosine, commercially available products such as those manufactured by Santa Cruz (catalog #; sc-358267) and Watanabe Chemical Industry Co., Ltd. (Code number; H00219) can be mentioned, and DL- When m-tyrosine is used, commercially available products such as those manufactured by Santa Cruz (catalog #; sc-207493) and Sigma-Aldrich (product number; T3629) can be used. When 2-aza-L-tyrosine is used as the tyrosine, it can be produced by the method described in Document "J. Org. Chem. 1996, 61, 813-815". When 2-aza-DL-tyrosine is used as the tyrosine, it can be produced according to the method described in the document "Biochemistry 2001, 40, 14862-14868".

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

In order to confirm that tyrosine has the effect of reducing blood uremic substances, an analysis was performed using obese diabetic model mice (db / db mice) that develop diabetic nephropathy.

[Lm-tyrosine]
[Method 1]
Blood samples and urine samples were collected from the tail vein of 10 9-week-old db / db mice (BKS. Cg Leprdb m / Jcl male, Claire Japan) on a normal diet and individually bred (pre-administration samples). After that, the amount of water consumed every 24 hours was measured, the average amount of water consumed per day was calculated, and the dose of Lm-tyrosine (manufactured by Astatech) was 50 mg / kg (body weight) / day. It was dissolved in water and administered for 10 days from 10 weeks of age. After administration for 10 days, breeding in normal water was carried out again for 10 days. Blood samples and urine samples (samples after administration) were collected on the 10th day of administration, and blood samples and urine samples (samples after drug suspension) were collected on the 10th day after discontinuation of administration.

After preparing plasma from blood samples before and after administration and after drug suspension according to a standard method, liquid chromatograph-tandem mass spectrometer (LC-MS / MS) (LC: NANOSPACE SI-2 [SHISEIDO], MS / MS: TSQ Quantum Ultra [manufactured by Thermo Fisher Scientific]) was used to measure the concentrations of phenyl sulfate, p-cresyl sulfate, and indoxyl sulfate in plasma (FIGS. 1A to 1C, Tables 1 to 3). In addition, the urinary albumin concentration was measured by the Turbidimetric Immuno Assay Kit (Albumin-mouse manufactured by Levis) using urine samples before and after administration and after drug suspension (Fig. 1D, Table 4).

[Result 1]
As a result of measuring the blood phenyl sulfate concentration in db / db mice after administration of Lm-tyrosine, it decreased to 3.5 to 50.1% before administration (FIG. 1A, "after administration" in Table 1). And "before administration"). Similarly, the blood p-cresyl sulfate concentration in db / db mice after administration of Lm-tyrosine decreased from 25.0 to 80.0% before administration (Fig. 1B, Table 2, "after administration"). (Comparison with "before administration"), and the concentration of blood indoxyl sulfate in db / db mice after administration of Lm-tyrosine decreased to 26.1 to 68.3% before administration (Fig.). 1C, Table 3, "Post-administration" and "Before administration" comparison), even after drug suspension, it decreased to 17.4 to 70.6% before administration (Fig. 1C, Table 3, "Pre-administration"). Comparison between "post-drug" and "pre-dose"). Furthermore, as a result of measuring the urinary albumin concentration in db / db mice after administration of Lm-tyrosine, it decreased to 10.7 to 79.2% before administration (FIG. 1D, "after administration" in Table 4). (Comparison with "before administration"), even after withdrawal, it decreased to 21.0 to 58.4% before administration (Fig. 1D, Table 4 "after withdrawal" and "before administration". Comparison).
These results show that tyrosine such as Lm-tyrosine reduces uremic substances (phenyl sulfate, p-cresyl sulfate, indoxyl sulfate, etc.) accumulated in the blood due to decreased renal function, resulting in diabetic nephropathy. It is shown that it has an effect of improving renal dysfunction such as, and that it is effective in improving symptoms and diseases caused by uremic substances, that is, uremia.

The table shows the phenyl sulfate concentration (μM) in plasma. The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

The table shows the p-cresyl sulfate concentration (μg / mL) in plasma. The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

The table shows the concentration of indoxyl sulfate in plasma (μg / mL). The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

The table shows the relative value of urinary albumin concentration to urinary creatinine concentration. The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

[Method 2]
The dose of Lm-tyrosine was changed from 50 mg / kg (body weight) / day to 10 mg / kg (body weight) / day, and the administration period of Lm-tyrosine was changed from 10 days to 7 days. The concentrations of phenyl sulfate and indoxyl sulfate in plasma in db / db mice were measured according to the method described in [Method 1].

[Result 2]
The blood phenyl sulfate concentration of 4.02 μM in db / db mice was significantly reduced to 1.33 μM by administration of Lm-tyrosine (Fig. 2A). In addition, the blood indoxyl sulfate concentration of 5.28 μg / mL in db / db mice was significantly reduced to 3.70 μg / mL by administration of Lm-tyrosine (Fig. 2B). As is clear from this result, it is shown that tyrosine such as Lm-tyrosine has an effect of reducing blood uremic substances.

[2-Aza-DL-Tyrosine]
[Method 3]
Blood samples and urine samples were collected from the tail vein of 4 normal diet / individual-reared 9-10 week-old db / db mice (BKS. Cg Leprdb m / Jcl male, Claire Japan) (pre-administration sample). After that, the amount of water consumed every 24 hours was measured, the average amount of water consumed per day was calculated, and 2-aza-DL-tyrosine was dissolved in the water of the water supply bottle so that the dose was 5 mg / kg (body weight) / day. It was administered for 10 days from the age of 10 weeks. Blood samples and urine samples (samples after administration) were collected on the 10th day of administration. 2-Aza-DL-tyrosine was produced according to the method described in the document "Biochemistry 2001, 40, 14862-14868".

Plasma was prepared from blood samples before and after administration according to a conventional method, and then the concentrations of creatinine, 1-methyladenosine, indoxyl sulfate, and phenyl sulfate in the plasma were measured using LC-MS / MS (Fig. 3 and). 4, Tables 5-11). In addition, after diluting the urine sample before and after administration 10-fold, the concentrations of creatinine, 1-methyladenosine, and phenyl sulfate in urine were measured using LC-MS / MS.

[Result 3]
As a result of measuring the blood creatinine concentration in db / db mice after administration of 2-aza-DL-tyrosine, it was reduced to 66.5-82.2% before administration (Fig. 3A, Table 5). Similarly, the urinary creatinine concentration in db / db mice after administration of 2-aza-DL-tyrosine decreased to 18.8-90.4% before administration (Fig. 3B, Table 6, P = 0.006). ). In addition, the blood 1-methyladenosine concentration in db / db mice after administration of 2-aza-DL-tyrosine decreased to 45.4 to 82.3% before administration (Fig. 3C, Table 7), and in urine. The 1-methyladenosine concentration decreased from 65.4 to 87.8% before administration (Fig. 3D, Table 8, P = 0.043). In addition, the blood indoxyl sulfate concentration in db / db mice after administration of 2-aza-DL-tyrosine decreased from 59.0 to 83.5% before administration (FIG. 4A, Table 9, P = 0. 032). In addition, the blood phenyl sulfate concentration in db / db mice after administration of 2-aza-DL-tyrosine decreased to 19.1 to 45.8% before administration (Fig. 4B, Table 10), and urinary phenyl sulfate. The concentration decreased from 16.9 to 97.3% before administration (Fig. 4C, Table 11).
These results show that tyrosine such as 2-aza-DL-tyrosine reduces uremic substances (creatinine, 1-methyladenosine, indoxyl sulfate, phenyl sulfate, etc.) accumulated in the blood due to decreased renal function. It has been shown to be effective in improving uremia.

The table shows the plasma creatinine concentration (μM). The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

The table shows the creatinine concentration (μM) in urine. The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

The table shows the concentration of 1-methyladenosine (ng / mL) in plasma. The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

The table shows the concentration of 1-methyladenosine (ng / mL) in urine. The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

The table shows the concentration of indoxyl sulfate in plasma (μg / mL). The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

The table shows the phenyl sulfate concentration (μM) in plasma. The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

The table shows the urinary phenyl sulfate concentration (μM). The numerical values in parentheses indicate relative values (%) when the value of "before administration" is taken as 100%.

[Method 4]
The dose of 2-aza-DL-tyrosine was changed from 5 mg / kg (body weight) / day to 3 mg / kg (body weight) / day, and the administration period of 2-aza-DL-tyrosine was changed from 10 days to 14 days. The concentration of phenyl sulfate, indoxyl sulfate, and creatinine in plasma in db / db mice was measured according to the method described in [Method 3] above, and urinary albumin was measured according to the method described in [Method 1] above. The concentration was measured.

[Result 4]
The blood phenyl sulfate concentration of 4.15 μM in db / db mice was significantly reduced to 1.64 μM by administration of 2-aza-DL-tyrosine (Fig. 5A). In addition, the blood indoxyl sulfate concentration of 3.66 μg / mL in db / db mice was significantly reduced to 2.46 μg / mL by administration of 2-aza-DL-tyrosine (Fig. 5B). In addition, the blood creatinine concentration of 6.25 μg / mL in db / db mice was significantly reduced to 4.43 μg / mL by administration of 2-aza-DL-tyrosine (Fig. 5C). Furthermore, the urinary albumin concentration in db / db mice increased when 2-aza-DL-tyrosine was not administered, whereas such increase was suppressed by administration of 2-aza-DL-tyrosine (2). FIG. 5D).
As is clear from these results, tyrosine such as 2-aza-DL-tyrosine has an effect of reducing blood uremic substances and significantly lowers blood creatinine level, which is an index of renal function. It is expected to have a renal protective effect because it lowers it and suppresses the increase in urinary albumin level.

In order to confirm that tyrosine has the effect of reducing blood uremic substances, a model mouse that developed chronic renal failure by feeding Adenin (hereinafter referred to as "Adenin dietary renal failure model mouse"). ) Was administered 2-aza-DL-tyrosine, and the concentration of blood uremic substances was analyzed. The adenine-phagocytic model mouse is a mouse in which fed adenine crystallizes as insoluble 2,8-dihydroxyadenine in the renal tubules to cause renal damage (Cozzolino, M. et al. Kidney Int). . 64, 441-450 (2003), Tamagi, K. et al. Nephrol. Dial. Transplant 21, 651-659 (2006)).

[Method 5]
Analysis was performed using adenine dietary renal failure model mice according to the following procedures [1] to [4].
[1] Male C57BL / 6 mice (purchased from Japan CLEA Co., Ltd.) were bred on normal feed (CE-2, manufactured by Japan CLEA Co., Ltd.) until 6 weeks of age.
[2] At 7 weeks of age, the group was divided into a normal breeding group bred with a normal feed and an adenine diet breeding group bred with a normal feed containing 0.2% adenine (manufactured by Wako).
[3] In the adenine dietary rearing group, after 8 weeks of adenine dietary rearing (15 weeks of age), the feed was returned to the normal diet, and two groups (adenine dietary rearing group [n = 5] and 2-aza-DL- Tyrosine-administered adenine diet-fed group [n = 5]), normal water was orally administered to the adenine diet-fed group, and 2-aza-DL-tyrosine-administered adenine diet-fed group was 2-aza-DL. Water containing -tyrosine was orally administered so that the dose of 2-aza-DL-tyrosine was 5 (mg / kg (body weight) / day).
On the other hand, the normal breeding group consists of two groups (normal breeding group [n = 5] and 2-aza-DL-tyrosine-administered normal breeding group [n = 5]) after 8 weeks of normal breeding (15 weeks of age). Oral administration of normal water to the normal breeding group and administration of 2-aza-DL-tyrosine to the normal breeding group, water containing 2-aza-DL-tyrosine and 2-aza-DL-tyrosine. Was orally administered so that the dose of was 5 (mg / kg (body weight) / day).
[4] After oral administration of the above [3] for 10 days, a blood sample and the entire kidney were collected on the 11th day, serum was prepared from the blood sample according to a conventional method, and then LC-MS / MS was used. The concentrations of four uremic substances (phenyl sulfate, indoxyl sulfate, TMAO, and creatinine) in the serum were measured (Fig. 6). In addition, the collagen fibers of the kidney were tissue-stained using the MTS method (Fig. 7).

[Result 5]
First, control mice in which the concentrations of four types of uremic substances (phenyl sulfate, indoxyl sulfate, TMAO, and creatinine) in the blood of adenine dietary renal failure model mice (“CC” in FIG. 6) were bred on a normal diet. It was confirmed that the increase was compared with (“NC” in FIG. 6). Next, it was shown that when 2-aza-DL-tyrosine was administered to adenine dietary renal failure model mice (“CA” in FIG. 6), the elevated concentrations of the above four uremic substances decreased. Specifically, the phenyl sulfate concentration significantly decreased from 14.45 μM to 10.92 μM, the indoxyl sulfate concentration significantly decreased from 15.09 μM to 11.55 μM, and the TMAO concentration decreased from 27.75 μM. It decreased to 23.47 μM, and the creatinine concentration decreased significantly from 39.13 μM to 30.71 μM. Furthermore, the proportion of renal tubules of 2-aza-DL-tyrosine-administered Adenine-dietary renal failure model mice was determined by the 2-aza-DL-tyrosine-naive Adenine-dietary renal failure model mice (“CC” in FIG. 7). It was shown to increase compared to.
These results show that tyrosine such as 2-aza-DL-tyrosine has the effect of reducing uremic substances (phenyl sulfate, indoxyl sulfate, TMAO, and creatinine) accumulated in the blood due to decreased renal function. It is shown that it has the effect of improving renal dysfunction such as renal failure. Therefore, tyrosine is considered to be effective in improving symptoms and diseases caused by uremic substances, that is, uremic disease.

The present invention contributes to the prevention or amelioration (treatment) of symptoms or diseases caused by the accumulation (increase) of uremic substances in blood.

Claims (6)

An agent for reducing a blood uremic substance, which comprises Lm-tyrosine or 2-aza-DL-tyrosine as an active ingredient . The reducing agent according to claim 1, which is orally administered. The uremic substance is one or more substances selected from p-cresol sulfate, phenyl sulfate, indoxyl sulfate, 1-methyladenosine, creatinine, trimethylamine-N-oxide, p-cresol, phenol, and indole. The reducing agent according to claim 1 or 2 , wherein the reducing agent is present. A preventive or ameliorating agent for uremia, which comprises Lm-tyrosine or 2-aza-DL-tyrosine as an active ingredient . The preventive or ameliorating agent according to claim 4 , which is orally administered. Uremia is caused by one or more substances selected from p-cresyl sulfate, phenyl sulfate, indoxyl sulfate, 1-methyladenosine, creatinine, trimethylamine-N-oxide, p-cresol, phenol, and indole. The preventive or ameliorating agent according to claim 4 or 5 , which is characterized by having a symptom or disease.