JP6533412B2 - FoxO3a phosphorylation inhibition reagent - Google Patents

Description

The present invention relates to a FoxO3a phosphorylation inhibition reagent .

  FoxO is a transcription factor that has a DNA binding domain. One of FoxO, FoxO3a, is a protein of about 650 amino acids, which binds to a specific sequence in the promoter region of a gene and induces gene expression.

  FoxO3a promotes the expression of many genes associated with stress response, metabolic control, cell cycle, apoptosis and DNA repair. In addition, FoxO3a contributes to the action of enhancing resistance to various stresses, and is important for prolonging life and suppressing aging diseases.

  When phosphorylated, FoxO3a decreases its transcriptional activity. In particular, FoxO3a is negatively regulated by the PI3K-Akt pathway. When PI3K-Akt pathway is activated, Akt phosphorylates FoxO3a and promotes the nuclear export of FoxO3a. The extranuclear FoxO3a loses its function as a transcription factor.

  Therefore, by inhibiting the phosphorylation of FoxO3a, FoxO3a can be activated as a result, and an effect of enhancing resistance to various stresses can be obtained. By enhancing the resistance to stress, it is expected to suppress life extension and aging diseases.

  FoxO3a is known to be activated by TGFβ. For example, according to Patent Document 1, when TGFβ is allowed to act on leukemia stem cells, the proportion of cells in which FoxO3a is localized in the cell nucleus increases. On the other hand, when a TGFβ inhibitor is allowed to act on the leukemia stem cells, the proportion of cells in which FoxO3a is localized in the cell nucleus is reduced.

JP, 2010-281656, A

  TGFβ is a cytokine that regulates cell proliferation and differentiation and promotes cell death. Since TGFβ is a protein, there is room for improvement in formulation complexity and stability for use in FoxO3a activation as compared to small molecule compounds. In addition, TGFβ is known for its various actions and is involved in various diseases such as fibrotic diseases. For this reason, the use of TGFβ has effects other than activation of FoxO3a, ie there is a concern for selectivity. From these facts, it is difficult to say that TGFβ is easy to use to activate FoxO3a.

The present invention has been made in view of the above-mentioned circumstances, and an object thereof is to provide a novel reagent for inhibiting FoxO3a phosphorylation which is easy to use.

FoxO3a phosphorylation inhibitory agent according to the perspective of the present invention,
It contains carnosic acid or piciferin acid .

According to the present invention, novel FoxO3a phosphorylation inhibition reagents are provided that are easier to use.

FIG. 5 shows the amount of FoxO3a and phosphorylated FoxO3a in cells cultured in the presence of carnosic acid. (A) is a figure which shows the result of a western blotting method. (B) is a figure which shows the relative value of the ratio of phosphorylated FoxO3a with respect to FoxO3a. FIG. 5 shows the amount of FoxO3a and phosphorylated FoxO3a in cells cultured in the presence of piciferin acid. (A) is a figure which shows the result of a western blotting method. (B) is a figure which shows the relative value of the ratio of phosphorylated FoxO3a with respect to FoxO3a. FIG. 5 shows the amount of phosphorylated FoxO3a in the liver of mice administered with carnosic acid. (A) is a figure which shows the result of a western blotting method. (B) is a figure which shows the relative value of the quantity of phosphorylated FoxO3a. It is a figure which shows the amount of creatinine in the serum per body weight of the mouse | mouth which administered the carnosic acid. FIG. 1 shows images of stained kidney tissue sections. (A) is an image of a kidney tissue section of a carnosic acid-administered mouse. (B) is an image of a kidney tissue section of a control mouse.

An embodiment according to the present invention will be described.
Embodiment 1
First, the first embodiment will be described in detail. The FoxO3a phosphorylation inhibitor according to the present embodiment inhibits the phosphorylation of FoxO3a. The FoxO3a phosphorylation inhibitor comprises phenolic diterpenes. Phenolic diterpenes are compounds having the following structure:

The phenolic diterpene is preferably a phenolic diterpene carboxylic acid in which R ₃ is CO ₂ H. The phenolic diterpene carboxylic acid is preferably picifellic acid (hereinafter referred to as "PA") in which R ₁ and R ₂ are CH ₃ , R ₃ is CO ₂ H, and R ₄ is H, and particularly preferred. Is a carnosic acid (hereinafter referred to as “CA”) in which R ₁ and R ₂ are CH ₃ , R ₃ is CO ₂ H, and R ₄ is OH.

In addition to the above CA and PA, as phenolic diterpenes, for example, compounds in which R ₁ and R ₂ are CH ₃ , R ₃ is CH ₂ OH, and R ₄ is H (piciferol), R ₁ and R ₂ are Compounds in which each of CH ₃ and R ₃ is CHO and R ₄ is H (Piciferal), compounds in which R ₁ and R ₂ are each CH ₃ , R ₃ is CO ₂ CH ₃ , and R ₄ is H, R ₁ A compound in which R ₂ is CH ₃ , R ₂ is CH ₂ OH, R ₃ is CH ₃ , and R ₄ is H, R ₁ is CH ₃ , R ₂ is CO ₂ H, R ₃ is CH ₃ , and R ₄ is H And compounds wherein R ₁ is CO ₂ H, R ₂ is CH ₃ , R ₃ is CH ₃ , and R ₄ is H.

  The phenolic diterpenes may be commercially available or may be extracted from natural products. For example, phenolic diterpenes can be extracted by methanol extraction from the leaves of Sawara (Chamaecyparis pisifera), rosemary (Rosmarinus officinalis) and sage (Salvia officinalis). In particular, PA is contained in the leaves of sawara. On the other hand, CA is contained in rosemary and sage.

  Various phenolic diterpenes can be isolated by chromatography on a silica gel support from extracts obtained from methanol from sawara leaves, rosemary or sage. Specifically, for example, in the isolation of PA, sawara leaves are methanol extracted for 24 hours, and the extract is concentrated. Next, the concentrate is subjected to column chromatography on a silica gel carrier using hexane-ethyl acetate as a solvent to obtain PA. The phenolic diterpene is not limited to extraction, and may be synthesized according to the structural formula. For example, CA can be synthesized by introducing a hydroxyl group into PA by oxidation at an ortho position or the like.

  The FoxO3a phosphorylation inhibitor according to the present embodiment suppresses the phosphorylation of FoxO3a in cultured cells as shown in Example 1 below. Therefore, expression of FoxO3a-regulated gene can be maintained or enhanced even under the condition that the transcription activity of FoxO3a in cells is decreased. Therefore, it can be used for functional analysis of many FoxO3a-regulated genes associated with stress response, metabolic control, cell cycle, apoptosis, DNA repair and the like.

  As described above in detail, the FoxO3a phosphorylation inhibitor according to the present embodiment can activate FoxO3a because it inhibits the phosphorylation of FoxO3a. In addition, since the phenolic diterpene contained in the FoxO3a phosphorylation inhibitor according to the present embodiment is a compound, it is easy to formulate as compared with a protein and is excellent in stability, and hence easy to use.

  In addition, the above-mentioned FoxO3a phosphorylation inhibitor enhances the resistance to various stresses, reduces the risk for various diseases, prolongs the life, suppresses aging diseases and the like, and is effective in preventing the improvement .

  Furthermore, in the present embodiment, the phenolic diterpene may be a phenolic diterpene carboxylic acid, particularly CA or PA. Since CA and PA are obtained from naturally occurring plants as described above, they can be used safely for humans.

  Also, in another embodiment, there is provided a phenolic diterpene for use as a FoxO3a phosphorylation inhibitor. Yet another embodiment is the use of a phenolic diterpene for the preparation of a FoxO3a phosphorylation inhibitor.

Second Embodiment
Next, the second embodiment will be described. The medicine concerning this embodiment contains the FoxO3a phosphorylation inhibitor concerning the above-mentioned Embodiment 1. The said medicine contains the said FoxO3a phosphorylation inhibitor as an active ingredient. The said medicine is manufactured by a well-known method.

  The dosage forms of the above-mentioned pharmaceuticals include, but are not limited to, solutions, tablets, capsules, injections, rectal suppositories, vaginal suppositories, nasal absorbents, transdermal absorbents, pulmonary absorbents and oral absorbents etc. is there. The medicine may be, for example, a combined preparation mixed with a pharmacologically acceptable carrier. The pharmacologically acceptable carrier is various organic carrier substances or inorganic carrier substances used as pharmaceutical materials. In addition, the medicine may contain pharmacologically acceptable additives such as sucrose, buffer and the like. Additives such as preservatives, antioxidants, coloring agents, and sweeteners can also be used, if necessary.

  Although the administration method of the above-mentioned medicine is optional, preferably it is oral administration, subcutaneous injection or intravenous injection. The dosage of the medicine is appropriately adjusted according to the patient's body weight, condition of the patient such as pathological condition, but it is 1 mg to 1 g, preferably 50 to 500 mg per person, as a daily dosage. The medicament may be administered over an extended period of time, such as 1 to 36 months, preferably 12 to 24 months. Of course, the administration method such as the administration frequency and the administration interval can be appropriately adjusted while observing the condition of the patient.

  The above drug activates FoxO3a by inhibiting the phosphorylation of FoxO3a. Since FoxO3a promotes the expression of genes associated with oxidative stress resistance, the drug is a disease caused by oxidative stress, in particular, hepatic diseases such as fatty liver, cirrhotic liver and hepatitis, atherosclerosis, Parkinson's disease , Angina pectoris, myocardial infarction, Alzheimer's disease, schizophrenia, bipolar disorder and chronic fatigue syndrome. In addition, as shown in Example 2 below, the above-mentioned medicine extends the life span to suppress the aging disease and the like in order to enhance the survival rate of the aging promoting mouse.

  Moreover, the said medicine suppresses the phosphorylation of FoxO3 in a kidney, as shown in the following Example 3. For this reason, the above-mentioned medicine is preferably used for treatment or prevention of kidney disease. For example, in diabetic model animals, it is known that the phosphorylation of FoxO3 in the kidney is enhanced, and as a result, the expression level of anti-oxidative stress enzyme under the control of FoxO3 decreases and the oxidative stress increases. It is thought to cause renal function decline. For this reason, the above-mentioned medicine is effective in diabetic nephropathy. Renal diseases include, in addition to diabetic nephropathy, for example, acute nephritis, chronic nephritis, nephrotic syndrome, acute renal failure and chronic renal failure.

  As described above in detail, since the above-mentioned medicine can activate FoxO3a, it can treat and prevent various diseases, in particular, oxidative stress related diseases and aging diseases. In addition, since the medicine has a renal protective action, it can treat or prevent a renal disease.

  Another embodiment is a method of inhibiting the phosphorylation of FoxO3a by administering the above-mentioned medicine to a patient, a method of activating FoxO3a, or a method of treating an oxidative stress related disease or a renal disease. Yet another embodiment is the use of a FoxO3a phosphorylation inhibitor as described above for the manufacture of a medicament for inhibiting FoxO3a phosphorylation or a medicament for activating FoxO3a. Another embodiment is the use of the above FoxO3a phosphorylation inhibitor for the manufacture of a medicament for treating oxidative stress related diseases or renal diseases. In still another embodiment, the medicine may be a therapeutic agent for renal disease or a prophylactic agent for renal disease.

Third Embodiment
Furthermore, Embodiment 3 will be described. The additive for FoxO3a phosphorylation inhibition according to the present embodiment contains a phenolic diterpene. As shown in Example 2 below, the above-mentioned phenolic diterpenes, even when used orally, inhibit the phosphorylation of FoxO3a, and therefore their action can be obtained even when used as an additive.

  The additive for inhibiting FoxO3a phosphorylation is added to, for example, food. The food includes nutritive drinks and functional foods. Specifically, the above-mentioned additive for inhibiting FoxO3a phosphorylation is soft drinks, beverages such as black tea and green tea, candy, cookies, tablets, sweets such as chewing gum and jelly, cereals such as noodles, bread, cooked rice and biscuits It may be contained in processed products, kneaded products such as sausage, ham and kamaboko, dairy products such as butter and yoghurt, and seasonings such as sprinkles. When the above-mentioned FoxO3a phosphorylation inhibitor additive is added to a food, the food may contain other additives such as sweeteners, flavors and colorants.

  The content of the above-mentioned additive for inhibiting FoxO3a phosphorylation as an active ingredient in food is usually 0.0001 to 99% by weight, preferably 1 to 90% by weight. The intake amount of the above-mentioned additive for inhibiting FoxO3a phosphorylation as an active ingredient may be in the range of 1 mg to 100 g, 10 mg to 100 g, or 100 mg to 50 g per adult per day.

  Since the additive for inhibiting FoxO3a phosphorylation according to the present embodiment contains the above-described phenolic diterpene that inhibits the phosphorylation of FoxO3a, it can impart an antioxidant stress action, an anti-aging action and a nephroprotective action to food etc. it can.

  As described above in detail, the additive for FoxO3a phosphorylation inhibition according to the present embodiment contains a phenolic diterpene that inhibits the phosphorylation of FoxO3a. For this reason, by ingesting the said additive agent for FoxO3a phosphorylation inhibition with a foodstuff etc., the antioxidant stress effect | action, anti-aging effect, and the renal protection effect by activating FoxO3a can be obtained.

  In another embodiment, there is provided a functional food comprising the above-mentioned additive for inhibiting FoxO3a phosphorylation. The functional food is preferably eaten for the prevention and amelioration of oxidative stress related diseases, aging diseases and kidney diseases. In this way, phenolic diterpenes that inhibit the phosphorylation of FoxO3a can be easily taken via the diet. In addition, the said additive for FoxO3a phosphorylation inhibition is contained as an active ingredient in the foodstuff provided with the package in which the effects, such as an oxidative stress related disease, an aging disease, or a kidney disease, were indicated, such as prevention, suppression, and improvement. May be

  The present invention will be described more specifically by the following examples, but the present invention is not limited by the examples.

(Example 1: Phosphorylation inhibition experiment of FoxO3a using SH-SY5Y cells)
Human neurofibroblastoma cells, SH-SY5Y cells (European Collection of Cell Culture; obtained from DA Pharma Biomedical Co., Ltd.), were cultured in a 6 cm dish until they became nearly confluent. As a culture medium, a DMEM medium (manufactured by GIBCO) containing 10% fetal bovine serum (FBS) and an antibiotic (50 unit / ml penicillin and 50 μg / ml streptomycin (manufactured by GIBCO)) was used.

  Change the medium to DMEM medium (GIBCO) containing 1% fetal bovine serum (FBS) and antibiotics (50 unit / ml penicillin and 50 μg / ml streptomycin (GIBCO)) and dissolve in dimethyl sulfoxide (DMSO) The added CA or PA was added to the medium. As a control, the same amount of DMSO was added. After 24 hours, proteins were extracted using M-Per reagent (PIERCE, manufactured by Thermo Scientific Co., Ltd.), and proteins in the supernatant were recovered by centrifugation (14,000 rpm) for 10 minutes.

  Total FoxO3a and phosphorylated FoxO3a contained in the obtained protein were quantified by Western blotting using a FoxO3a antibody (# 12829, manufactured by Cell Signaling Technology) and a phosphorylated FoxO3a antibody (ab154786, manufactured by abcam). For Western blotting, Western Breeze Chemiluminescent Kit, anti-rabbit (manufactured by Life technologies) was used.

(result)
FIG. 1 shows the amount of FoxO3a and phosphorylated FoxO3a when 5 μM of CA is added. CA suppressed the phosphorylation of FoxO3a (see FIG. 1 (a)). Calculation of the ratio of phosphorylated FoxO3a to total FoxO3a based on the concentration of bands showed that CA significantly suppressed the phosphorylation of FoxO3a by about 40% compared to the control group (n = 3 ) (See FIG. 1 (b)).

  FIG. 2 shows the amount of FoxO3a and phosphorylated FoxO3a when 5 μM or 10 μM PA is added. The phosphorylation of FoxO3a was suppressed by 10 μM of PA (see FIG. 2 (a)). In the ratio of phosphorylated FoxO3a to FoxO3a, it was shown that 10 μM of PA tends to suppress phosphorylation of FoxO3a to about 60% (n = 3) (see FIG. 2 (b)).

(Example 2: Oral administration experiment to aging accelerated mice)
Administration of CA was started after preliminary breeding of 3 weeks old senescence accelerated mice (Sensence accelerated mouse: SAMP 8) for 1 week. CA was dissolved in distilled water so as to be 6, 50 or 60 μM, and was freely ingested as drinking water. The control group received distilled water as well. Hereinafter, the aging promoting mouse is simply referred to as "mouse". There were no clear differences in body weight, food consumption and water consumption during the feeding period between the CA administration group and the control group. The water intake at 40 weeks of age was 3.77 g / animal / day in the control group, 3.68 g / animal / day in the 6 μM CA administration group, and 4.20 g / animal / day in the 60 μM CA administration group. there were.

  Administration of CA was continued, and surviving mice at 10 months of age were counted to calculate the survival rate. For mice receiving 50 μM of CA for 11 months, livers were collected, and phosphorylated FoxO3a in the liver was quantified using Western blotting as described above.

(result)
Table 1 shows the survival rates of mice at 10 months of age. The survival rate of the control group which did not receive CA was 50 to 80%. On the other hand, mice receiving CA had higher survival rates than the control group, and at 60 μM, survival rates of mice receiving CA were 100%. This suggests that intake of CA reduces the risk of disease by suppressing the phosphorylation of FoxO3a.

  FIG. 3 shows the amount of phosphorylated FoxO3a in livers from 12 month old mice and control groups receiving 50 μM of CA for 11 months. The amount of phosphorylated FoxO3a was reduced in the CA administration group as compared to the control group (see FIG. 3 (a)). When the amount of phosphorylated FoxO3a was compared based on the concentration of the band, the amount of phosphorylated FoxO3a was significantly reduced to about 60% in the CA administration group, assuming that the average value of the control group is 100% (control group) Is n = 7, CA administration group is n = 9) (see FIG. 3 (b)).

  From the above results, CA and PA were shown to suppress the phosphorylation of FoxO3a. No adverse events were observed when CA was administered to aging-promoting model mice for a long period of 11 months. Furthermore, it has been revealed that CA suppresses the phosphorylation of FoxO3a, thereby prolonging the lifespan of mice.

Example 3 Examination of Renal Protective Action of CA
In Example 2 above, blood was collected from mice to which 50 μM of CA was administered for 11 months to obtain serum. The amount of creatinine in serum was measured using Lampia Liquid CREA (manufactured by Kyoto Pharmaceutical Co., Ltd.). On the other hand, kidneys were collected from mice that received 50 μM of CA for 11 months, and the kidneys were fixed with 10% neutral formalin and then embedded in paraffin. Renal tissue sections were prepared and immunohistochemical staining was performed using a phosphorylated FoxO3a antibody (ab154786, manufactured by abcam).

(result)
The concentration of serum creatinine was calculated and divided by the weight of each mouse is shown in FIG. The values in the CA administration group were significantly lower than in the control group. Creatinine is a metabolite of creatine, which is abundant in muscle, and is usually removed from the blood in the glomeruli of the kidney and excreted in urine, so its concentration in blood is usually low. However, as kidney function declines, the concentration of creatinine in the blood increases. Therefore, it became clear that renal function is protected by CA administration.

  FIG. 5 shows microscopic images of stained kidney tissue sections. In the control group, phosphorylated FoxO3 was abundantly present in kidney tissues (see FIG. 5 (a)), but in the CA administration group, it was revealed that phosphorylated FoxO3 was less (FIG. 5 (b (b) )reference). This result indicates that phosphorylation of FoxO3 is suppressed in the CA administration group, and that CA acts protectively on kidney cells. From the results of FIGS. 4 and 5, it was shown that administration of CA protected renal function.

  Since it is known that non-phosphorylated FoxO3 acts as a transcription factor and is important for renal protection, CA that suppresses the phosphorylation of FoxO3 in kidney cells is useful as a renal protective agent.

  The embodiments described above are for explaining the present invention, and do not limit the scope of the present invention. That is, the scope of the present invention is indicated not by the embodiments but by the claims. And, various modifications made within the scope of the claims and the meaning of the invention are considered to be within the scope of the present invention.

The present invention is suitable for inhibiting the phosphorylation of FoxO3a .

Claims (1)

Including carnosic acid or piciferic acid ,
FoxO3a phosphorylation inhibition reagent .