JP6077880B2 - TNF-α production inhibitor - Google Patents

Description

  The present invention relates to a TNF-α production inhibitor.

  TNF-α was isolated as a factor that induces hemorrhagic necrosis on tumors implanted in mice. Subsequent research has shown that it is involved in infection protection and anti-tumor effects by inducing apoptosis, enhancing inflammation mediators and antibody production, but overexpression leads to the development of diseases such as rheumatoid arthritis and psoriasis. became. In particular, rheumatoid arthritis, which is a chronic inflammatory disease, has clinical symptoms such as joint destruction, and TNF-α is identified as one of the cytokines that play a central role in the pathogenesis of rheumatoid arthritis along with IL-6. ing. In Japan, biological preparations targeting TNF-α are also used, and etanercept, a fusion protein of sTNFR and immunoglobulin G, and infliximab and adalimumab, which are anti-TNF-α monoclonal antibodies, are indicated. Yes. However, these drugs having a TNF-α inhibitory action have many side effects, and it is warned that the risk of infection and carcinogenesis increases. In addition, adipose tissue secretes inflammatory cytokines, and TNF-α inhibits glucose uptake into cells and decreases sensitivity to insulin, and further promotes fatty acid production in adipocytes and hepatocytes, mainly TNFR1 It is known to cause anti-glycerinemia via.

In recent years, a search for a substance that suppresses the production of TNF-α in a living body has been advanced.
Patent Document 1 (JP 2012-207043 A) discloses a TNF-α inhibitor containing nigerooligosaccharide as an active ingredient. Patent Document 2 (Japanese Patent Application Laid-Open No. 2011-251950) describes that sulfobasin B isolated from a kind of marine bacterium, Chryserobacterium sp., Has a TNF-α production inhibitory action. ing. Patent Document 3 (Japanese Patent Application Laid-Open No. 2008-106014) describes that naturally-derived compounds such as chalcone and hydroxychalcone have TNF-α inhibitory action. Patent Document 5 (Japanese Patent Application Laid-Open No. 2011-153139) discloses a drug having an inhibitory action on TNF-α containing a vitamin E derivative as an active ingredient.

  In addition, it is known that macrophages incorporating vitamin E (tocopherol) reduce the amount of TNF-α secreted by stimulation with LPS or the like (Non-patent Document 1).

JP 2012-207043 A JP 2011-251950 A JP 2008-106014 A JP 2007-197392 A JP 2011-153139 A

Sakamoto, W. et al. Biochim. Biophys. Acta, in press, 1998

  It is an object of the present invention to provide an agent that suppresses TNF-α production of macrophages.

  The present inventors have been studying a method for suppressing macrophage stimulation-induced TNF-α production. As a result, the inventors have found a strong ability to suppress TNF-α production by the combined use of an acylated sterol glycoside and tocopherol. Completed the invention.

The main configuration of the present invention is as follows.
(1) TNF- containing β-sitosterol glucoside palmitate and β-sitosterol glucoside palmitate as active ingredients, and containing 10 to 10 times the amount of tocopherol with respect to 1 part by mass of β-sitosterol glucoside palmitate α production inhibitor.

  The present invention provides a new TNF-α production inhibitor.

The graph which shows the test result which confirmed that the beta-sitosterol glucoside palmitate (beta) -sitosterol glucoside palmitate, (alpha) -tocopherol, and the combination agent thereof suppressed the secretion of TNF- (alpha) of the macrophage stimulated with palmitic acid. is there.

  The sterol skeleton of the acylated sterol glycoside used in the present invention is preferably β-sitosterol. The sugar to which the acylated sterol glycoside binds is preferably D-glucose. Furthermore, the acyl group of the acylated sterol glycoside is preferably a palmitic acid group.

  The specific compound of the acylated sterol glycoside is most preferably β-sitosterol glucoside palmitate.

The TNF-α production inhibitor of the present invention is preferably formulated as an acylated sterol glycoside so that an adult can take 1 mg to 1000 mg per day.
Furthermore, the TNF-α production inhibitor of the present invention can enhance its TNF-α inhibitory effect by blending α-tocopherol or an α-tocopherol derivative. α-tocopherol is preferably blended in an equivalent amount to 10 times the amount of acylated sterol glycoside. As long as the α-tocopherol derivative has an action as vitamin E, any compound is acceptable. As a typical α-tocopherol derivative, sodium vitamin E phosphate (TPNa: trade name) can be exemplified.
The safety of acylated sterol glycosides and α-tocopherol is well known, and it is known that there are no problems with extremely low toxicity substances.

  Each compound of the present invention and a pharmacologically acceptable salt thereof may be natural products isolated and purified from plants or the like, or may be synthesized by a known synthesis method.

  When the drug of the present invention is administered into the body, it is preferably administered orally, and it can also be used as a health food having a TNF-α inhibitory action by being added to and blended with foods and drinks or supplements.

  The TNF-α production inhibitor of the present invention can be administered to animals and humans as it is or as a pharmaceutical composition together with a conventional pharmaceutical preparation carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected according to need. For example, oral preparations such as tablets, capsules, granules, fine granules, powders, and injections And parenterals such as suppositories.

  In the present invention, oral preparations such as tablets, capsules, granules, fine granules, and powders are produced according to a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, and the like. . The compounding amount of the compound of the present invention in these preparations is not particularly limited and can be appropriately designed. In addition to the compound of the present invention, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be appropriately used for this type of preparation.

  Examples of the binder include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, sodium methylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol and the like. Examples of the disintegrant include starch, hydroxypropyl starch, carboxymethylcellulose sodium, carboxymethylcellulose calcium, carboxymethylcellulose, and low-substituted hydroxypropylcellulose. Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like. Examples of lubricants include talc, waxes, hydrogenated vegetable oils, sucrose fatty acid esters, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like. Examples of the fluidity promoter include light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like. The compounds of the present invention can also be administered as suspensions, emulsions, syrups, and elixirs, and these various dosage forms may contain flavoring agents and colorants.

  Moreover, other active ingredients can be added as needed within the range not impairing the effects of the present invention. For example, a substance that adjusts the immune response of the living body can be added to regulate the immune function.

Hereinafter, the present invention will be described in more detail with reference to examples and test examples.
Test 1. Test for confirming TNF-α production suppression effect of α-tocopherol and β-sitosterol glucoside palmitate Using the samples having the concentrations shown in Table 1, the production suppression effect of TNF-α induced by palmitic acid was tested.

(1) Preparation of sample, reagent and medium
[GW9508 (Cayman Chemical Company)]
Prepared to 20 mM with DMSO. This was diluted to the desired concentration by dilution with DMSO. GW9508 is a compound that has been confirmed to have a TNF-α inhibitory effect, and its chemical name is 4- (3-phenoxybenzylamino) phenylpropionic acid.
[Indomethacine (Sigma-Aldrich)]
Prepared to 10 mM with DMSO. This was diluted to the desired concentration by dilution with DMSO. This compound has been confirmed to have a TNF-α inhibitory effect.
[Α-Tocopherol (Sigma-Aldrich)]
Prepared to 4 mg / mL with DMSO. This was diluted twice with DMSO to prepare a dilution series.
[Β-Sitosterol glucoside palmitate (Funakoshi)
Prepared to 20 mM with DMSO. This was diluted twice with DMSO to prepare a dilution series.

(2) Cell lysis buffer (RIPA)
A buffer solution having the following composition was used.
50 mM Tris-HCl, pH 8.0 (Wako), 150 mM NaCl (Wako), 1% (v / v) NP-40 Alternative (CALBIOCHEM), 0.1% (w / v) SDS (Wako) , 0.5% (w / v) Na-deoxycholate (Wako)
+ Protease Inhibitor Cocktail (nacalai tesque)

<Growth medium> DMEM-10% FBS-1% P / S
DMEM (Gibco, Inc.) containing 4.5 g / L glucose, 110 mg / mL sodium pyruvate, 15 m / L phenol red, 445 mL, fetal calf serum (FBS, 171012, NICHIREI BIOSCIENCES INC) 50 mL, penicillin-streptomycin 5 mL of the solution (Sigma) was added and mixed by pipetting.

<Assay medium> DMEM-0.5% FBS-1% P / S
4.5 g / L glucose, 110 mg / mL sodium pyruvate, 15 m / L phenol red containing DMEM (Gibco) 495 mL and penicillin-streptomycin solution (Sigma) 5 mL, mixed, 39.8 mL Was dispensed into centrifuge tubes, fetal bovine serum (FBS, 171012, NICHIREI BIOSCIENCES INC) 0.2 mL was added, and the mixture was prepared by mixing.

(3) Cell culture method 1) Seed cryopreserved cells (2 × 10 ⁶ cells) in a 150 mm dish (MS-10150, Sumitomo Bakelite Co., Ltd.). Cultured for days.
2) Cells grown in a 150 mm dish were seeded in a 48-well plate at a cell density of 2 × 10 ⁵ cells / well, and cultured in assay medium for 9 hours to completely adhere the cells.
3) The growth medium in the 48-well plate was carefully removed with a P200 micropipette.
4) Put 14 μL of each sample stock solution and 1386 μL of medium into a 1.5 mL microtube to prepare a sample-added medium (sample concentration is 1 / 100th of the added concentration, dimethyl sulfoxide (DMSO) concentration is 1%) .
5) After stirring well by vortexing, 400 μL was added to each well and allowed to stand for 14 hours.
6) After 14 hours, the medium was collected in a microtube.
7) Place 7 μL of 200 μM palmitic acid (PA), 7 μL of each sample stock solution (twice the pretreatment concentration), and 1386 μL of medium in a 1.5 mL microtube to prepare a PA + sample addition medium (sample and DMSO) The final concentration was adjusted to be the same as that in the pretreatment).
8) After stirring the prepared sample with a vortex mixer, 400 μL each was added to each well and cultured for 24 hours to induce secretion of TNF-α.
9) After 24 hours, the medium was collected and stored frozen at −80 ° C. until analysis.
10) After recovering the medium, the cells were washed once with PBS (−), and 100 μL of RIPA was added to each well and subjected to ultrasonic disruption.
11) The solubilized protein was pipetted into a microtube and stored frozen at −80 ° C. until analysis.

(4) Quantification of cell protein amount 1) The medium that had been cryopreserved at −80 ° C. was thawed in water and centrifuged at 4 ° C. and 20,400 × g for 10 minutes.
2) After centrifugation, the amount of protein in the supernatant was measured using Pierce BCA Protein Assay Kit (Thermo SCIENTIFIC) according to the attached protocol.

(5) Measurement of the amount of TNF-α by ELISA 1) The medium that had been cryopreserved at −80 ° C. was thawed in water and centrifuged at 1000 × g for 3 minutes.
-The amount of TNF-α in the supernatant was measured using “Quantikine Mouse TNF-α Immunoassay kit” (manufactured by R & D Systems) according to the protocol attached to the kit.

The measurement results are shown in FIG. 1 as relative values (%) where the average value of 3 wells was obtained for each sample and the TNF-α production amount stimulated by palmitic acid was taken as 100. The measurement results were evaluated by a significant difference test by Student's t test. “ASG” in the figure represents β-sitosterol glucoside palmitate.
Each compound suppressed TNF-α production of macrophages induced by palmitic acid stimulation.

Test 2. Test of combined effect of β-sitosterol glucoside palmitate and α-tocopherol The effect of using β-sitosterol glucoside palmitate and α-tocopherol in combination with β-sitosterol glucoside palmitate, which was confirmed to have a TNF-α inhibitory effect in Test 1 above, was confirmed.
A TNF-α suppression test was performed in the same manner as in Test Example 1. As test samples, 5 μg / mL of β-sitosterol glucoside palmitate was used in a combination group of 25 μM α-tocopherol, and 2.5 μg / mL of β-sitosterol glucoside palmitate was used in a combination group of 12.5 μM α-tocopherol. The α inhibitory effect was tested.
Α-tocopherol was prepared as follows.
α-Tocopherol (Sigma-Aldrich) was adjusted to a concentration of 20 mM with DMSO, and this stock solution was diluted with DMSO to prepare a desired concentration solution.

The results are shown in Figure 1.
In the β-sitosterol glucoside palmitate alone group, although a TNF-α production inhibitory effect was observed, it reached a peak at 10 μM, and the effect was limited.
In the α-tocopherol alone group, a TNF-α production inhibitory effect was observed in a concentration-dependent manner, and the production inhibitory effect was about 32% at 100 μM.
On the other hand, TNF-α is synergistically suppressed by using α-tocopherol in combination with β-sitosterol glucoside palmitate, and in the β-sitosterol glucoside palmitate 5 μg / mL and α-tocopherol 25 μM combination group, no palmitic acid is added. The result was equivalent to stimulation. This is an inhibitory effect equivalent to 100 μM or more of α-tocopherol.
Therefore, when α-tocopherol is used in combination with β-sitosterol glucoside palmitate, TNF-α produced by macrophages upon external stimulation may be completely suppressed.

  From the above test results, it was confirmed that the combined use of β-sitosterol glucoside palmitate and α-tocopherol significantly suppresses TNF-α production.

Claims (1)

  TNF-α production suppression containing β-sitosterol glucoside palmitate and β-sitosterol glucoside palmitate as active ingredients, and containing 10 to 10 times the amount of tocopherol with respect to 1 part by mass of β-sitosterol glucoside palmitate Agent.