JP2021017432A - Anti-inflammatory drug - Google Patents

Abstract

To provide a novel anti-inflammatory drug with high safety.SOLUTION: The anti-inflammatory drug contains an ester of a monoterpene alcohol and a branched-chain amino acid, or a salt thereof, as an active ingredient. The monoterpene alcohol contained in the anti-inflammatory drug may be menthol. The ester contained in the anti-inflammatory drug may be a compound represented by the formula (2) in the figure, where R is a C3-6 branched alkyl group.SELECTED DRAWING: Figure 1

Description

The present invention relates to anti-inflammatory agents.

Examples of pharmaceutical additives used in applications such as refreshing agents, fragrances, and stabilizers include monoterpene alcohols. In particular, menthol, which is a major volatile monoterpene alcohol contained in peppermint, is added to many analgesic and anti-inflammatory external preparations for the purpose of giving a feeling of coldness and refreshing feeling to the skin.

On the other hand, it has been reported that the anti-inflammatory analgesic effect is enhanced by adding menthol to an anti-inflammatory analgesic containing a phenylacetic acid derivative, which is one of the non-steroidal anti-inflammatory drugs, as an active ingredient (for example, patent). Reference 1). It has also been reported that menthol itself exhibits a local anesthetic effect (see, for example, Non-Patent Document 1) and an anti-inflammatory effect in the intestine (see, for example, Non-Patent Document 2).

In addition, monoterpene alcohol represented by menthol is known to have extremely low toxicity and irritation. From these facts, the application of monoterpene alcohol is expected for the development of safer anti-inflammatory agents.

Japanese Unexamined Patent Publication No. 2003-286161

Galeotti et al. , Planta Med. 2001; 67; 174 Bastaki et al. , Am. J. Translation. Res. 2018; 10; 4210

The present invention has been proposed in view of the above, and an object of the present invention is to provide a novel anti-inflammatory agent having high safety.

As a result of diligent research on the above object, the present inventors have found that an ester of a monoterpene alcohol and a branched-chain amino acid exhibits a high anti-inflammatory effect, and have completed the present invention. More specifically, the present invention provides the following.

<1> An anti-inflammatory agent containing an ester of monoterpene alcohol and a branched chain amino acid or a salt thereof as an active ingredient.

<2> The anti-inflammatory agent according to <1>, wherein the monoterpene alcohol is menthol.

［式中、Ｒは炭素数３〜６の分岐アルキル基を示す。］ <3> The anti-inflammatory agent according to <1> or <2>, wherein the branched chain amino acid is a compound represented by the following formula (1).

[In the formula, R represents a branched alkyl group having 3 to 6 carbon atoms. ]

［式中、Ｒは炭素数３〜６の分岐アルキル基を示す。］ <4> The anti-inflammatory agent according to any one of <1> to <3>, wherein the ester is a compound represented by the following formula (2).

[In the formula, R represents a branched alkyl group having 3 to 6 carbon atoms. ]

According to the present invention, it is possible to provide a novel anti-inflammatory agent having high safety.

It is a figure which shows the mRNA amount of TNF-α and IL-1β in the lipopolysaccharide-stimulated RAW264.7 cells to which menthol and different kinds of menthol amino acid esters were contacted. It is a figure which shows the mRNA amount of TNF-α in the lipopolysaccharide-stimulated RAW264.7 cells contacted with different concentrations of menthol, menthol valine ester and menthol isoleucine ester. FIG. 5 shows cell viability in RAW264.7 cells contacted with different concentrations of menthol, menthol valine ester and menthol isoleucine ester.

Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

The anti-inflammatory agent according to the present embodiment contains an ester of monoterpene alcohol and a branched chain amino acid or a salt thereof as an active ingredient.

<Monotelpene alcohol>
The monoterpene alcohol constituting the ester is not particularly limited, and for example, menthol, thymol, carvacrol, terpineol, linalool, hinokithiol, geraniol, nerol, terpinen-4-ol, citronellol, lavandulol, borneol, peryl alcohol and the like. Can be mentioned. Further, when the monoterpene alcohol constituting the ester has an isomer, the type of the isomer is not limited.

As described above, the monoterpene alcohol constituting the ester is preferably menthol from the viewpoint of having an anti-inflammatory effect by itself. It is known that a large number of isomers are present in menthol, but from the viewpoint of availability, the monoterpene alcohol constituting the ester is more preferably l-menthol or d-menthol. -Menthol is more preferred.

<Branched chain amino acids>
The branched chain amino acids that make up the ester are represented by the following formula (1). In the following formula (1), R represents a branched alkyl group having 3 to 6 carbon atoms. The branched alkyl group is not particularly limited, and for example, isopropyl group, isobutyl group, sec-butyl group, tert-butyl group, isopentyl group, neopentyl group, sec-pentyl group, tert-pentyl group, 2-methylbutyl group, 1- Ethylpropyl group, 1,2-dimethylpropyl group, isohexyl group, neohexyl group, sec-hexyl group, tert-hexyl group, 2-methylpentyl group, 3-methylpentyl group, 2,2-dimethylbutyl group, 2- Ethylbutyl group and the like can be mentioned. In one embodiment, the branched alkyl group is an isopropyl group or a sec-butyl group. Further, the type of isomer of the branched chain amino acid constituting the ester is not limited, and may be any of D-form, L-form, and DL-form. From the viewpoint of economy and availability, the branched chain amino acid constituting the ester is preferably L-form.

Specific examples of the branched chain amino acid represented by the above formula (1) include essential amino acids such as valine, isoleucine, and leucine. Of these, the branched chain amino acid constituting the ester is preferably valine or isoleucine.

<Ester>
The ester contained in the anti-inflammatory agent is represented by the following formula (2). In the following formula (2), R represents a branched alkyl group having 3 to 6 carbon atoms. The type of ester isomer is not limited.

Specific examples of the ester represented by the above formula (2) include those in which R in the above formula (2) is an isopropyl group, those having a sec-butyl group, and the like.

The ester may be in the form of a pharmaceutically acceptable salt. For example, the ester may be in the form of a salt with an inorganic acid such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc., acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citrate. It may be in the form of a salt with an organic acid such as an acid, succinic acid, methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid.

<Anti-inflammatory agent>
The anti-inflammatory agent according to this embodiment can be used for the prevention or treatment of inflammation. "Inflammation" refers to various reaction mechanisms caused by various stimuli, injuries, infections, and the like. Whether or not inflammation is induced can be identified using known inflammatory markers. As an inflammation marker, for example, TNF-α, IL-1β, iNOS, INF-γ, COX-2, NF-κB and the like are known. In addition to preventing the onset of the disease, "prevention" also includes delaying the onset of the disease. In addition, "treatment" includes not only eliminating or alleviating the symptoms of the disease, but also suppressing the degree of progression of the symptoms.

The above-mentioned "inflammation" includes general inflammatory diseases, for example, various dermatomyositis including atopic dermatitis, dermatomyositis, polymyositis, allergy, systemic lupus erythematosus, lupus erythematosus, after-stomatitis, retina. Eye diseases such as inflammation, gastric inflammation, hepatitis, bronchitis, esophagitis, enteritis, pancreatitis, lupus erythematosus, nephritis, floor rubbing, lupus, chronic thyroiditis, various chronic inflammatory diseases such as multiple sclerosis, septicemia , Shock, radiation damage, various acute inflammatory diseases such as rejection of organ transplantation, systemic edema and localized edema, etc.

The anti-inflammatory agent according to the present embodiment may contain components other than the ester or a salt thereof. For example, the anti-inflammatory agent may contain a conventional organic or inorganic carrier as a formulation material. This carrier can be used as an excipient, a lubricant, a binder, a disintegrant, etc. in a solid preparation, and as a solvent, a solubilizing agent, a suspending agent, an tonicity agent, a buffer, etc. in a liquid preparation. It is compounded. In addition, the anti-inflammatory agent may contain a preparation additive such as a preservative, an antioxidant, a drug solubilizer, a transdermal absorption promoter, a colorant, a sweetener, and a fragrance.

The dosage form of the anti-inflammatory agent according to this embodiment is not particularly limited. Dosage forms of anti-inflammatory agents include oral preparations such as tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions and films; injections, infusions, external preparations, suppositories, etc. Parenteral agents such as pellets, nasal agents, pulmonary agents (inhalants), eye drops; and the like.

The anti-inflammatory agent according to the present embodiment can be produced by any method adopted in the field of pharmaceuticals or a method with appropriate improvements.

The dose of the anti-inflammatory agent according to the present embodiment is appropriately determined according to the administration target, administration route, target disease, symptom and the like. Further, the number of administrations, the administration interval, and the like are not particularly limited, and the administration may be a single administration or a plurality of administrations.

The anti-inflammatory agent according to the present embodiment may be administered in combination with other agents depending on the purpose of administration and the like. The type and amount of the drug to be used in combination with the anti-inflammatory agent are appropriately selected based on the effect to be obtained and the like, and they may be administered together with the anti-inflammatory agent or may be administered separately.

The administration target of the anti-inflammatory agent according to the present embodiment is not particularly limited, and mammals and the like can be preferably mentioned. The mammal may be either a human or a non-human animal (mouse, rat, cow, pig, dog, cat, etc.).

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

(Synthesis of menthol amino acid ester)
The synthesis of esters of l-menthol and L-amino acids (also referred to herein as "menthol amino acid esters") is described in the article by Harada et al. (Harada et al., Bull. Chem. Soc. Jpn .; 1964; 37). It was carried out according to the method described in 191). First, 1 mol equivalent of L-amino acid, 1.3 mol equivalent of p-toluenesulfonic acid monohydrate, and 1.5 mol equivalent of l-menthol are suspended in toluene, and Dean is equipped with a reflux cooler. -The Stark apparatus was used to heat at 110 ° C. for 3 days. After diluting with ethyl acetate, it was washed with saturated aqueous sodium hydrogen carbonate solution, then washed with saturated brine, and dried over sodium sulfate. Then, filtration and concentration under reduced pressure were carried out, and the residue was purified by silica gel chromatography to obtain a menthol amino acid ester.

(Test Example 1)
In the following tests, a RAW264.7 cell line (mouse macrophage-like cell line) was used. For culturing the RAW264.7 cell line, 0.85% physiological saline containing FCS (Fujifilm Wako Pure Chemical Industries, Ltd.) with a final concentration of 10%, 100 U / mL penicillin, and 100 μg / mL streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd.) ) Was added to DMEM medium (SIGMA). The RAW264.7 cell line was seeded on a 12-well-plate (IWAKI) so as to be 5.0 × 10 ⁵ cells / well, and cultured at 37 ° C. in a 5% CO ₂ incubator for 24 hours.

Menthol and different types of menthol amino acid esters dissolved in aqueous solution containing 0.1 mM MES and 0.1% DMSO were added to the medium to a final concentration of 100 μM and incubated for 24 hours. Next, lipopolysaccharide (LPS) was added to the medium to a final concentration of 1 μg / mL, and the mixture was incubated for 1 hour or 6 hours to stimulate inflammation. Then, the mRNA expression levels of TNF-α and IL-1β were measured. In addition, TNF-α and IL-1β are inflammatory marker genes as described above.

Total RNA was extracted from the cells after LPS stimulation using SepazolRNA I SuperG (containing 8% quinolinol) (nacalai tesque). Then, cDNA was synthesized using River Tra Ace qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.). For the synthesized cDNA, the genes of TNF-α, IL-1β, and GAPDH as an endogenous control were amplified by a real-time PCR method using THUNDERBIRD SYBR qPCR Mix (Toyobo Co., Ltd.). For real-time PCR, initial denaturation was performed at 95 ° C. for 60 seconds using the CFX Connect Real-Time PCR detection system (Bio-Rad), followed by a denaturation reaction at 95 ° C. for 15 seconds and 60 ° C. for 30 seconds. The elongation reaction was carried out for 40 cycles. The mRNA expression levels of TNF-α and IL-1β were corrected by the mRNA expression levels of GAPDH. The sequence of the primers used is as follows.

The same experiment was performed three times to determine the mean and standard deviation. The results are shown in FIG. In addition, "*" in FIG. 1 indicates that it is statistically significant as compared with the example (cont (+ LPS)) in which the control solution was added and LPS stimulation was performed, and "ns" is Indicates that it is not statistically significant.

As shown in FIG. 1, it was confirmed that the addition of LPS increased the mRNA expression levels of TNF-α and IL-1β. Next, in addition to the addition of LPS, an ester of menthol and valine (also referred to herein as "menthol valine ester" or "ment-Val") and an ester of menthol and isoleucine (in the present specification, " The addition of "menthol isoleucine ester" or "ment-Ile") suppressed the increase in TNF-α mRNA expression. Further, in addition to the addition of LPS, when mentholvaline ester, menthol isoleucine ester, and ester of menthol and leucine (also referred to as "menthol leucine ester" or "ment-Leu" in the present specification) are added, The increase in the expression level of IL-1β mRNA was suppressed. On the other hand, the addition of menthol amino acid ester composed of amino acids of alanine, glycine and phenylalanine did not confirm the effect of suppressing the increase in mRNA expression level of these inflammatory marker genes. From these results, it was found that the ester of menthol and branched chain amino acid has an anti-inflammatory effect.

(Test Example 2)
In the following tests, cells cultured in the same manner as in Test Example 1 were used. A test solution was prepared by dissolving menthol, menthol valine ester, and menthol isoleucine ester in 100% ethanol and diluting with water so that the final concentration of ethanol was 0.5%. The test solution was added to the medium and incubated for 24 hours so that the concentrations of these compounds differed within the range of 1 to 500 μM. Next, LPS was added to the medium to a final concentration of 1 mg / mL and incubated for 1 hour to stimulate inflammation. Then, the mRNA expression level of TNF-α was measured. The amount of TNF-α mRNA was measured using the same method as in Test Example 1.

The same experiment was performed three times to determine the mean and standard deviation. The results are shown in FIG. In addition, "*" in FIG. 2 indicates that it is statistically significant as compared with the example in which the control solution was added and LPS stimulation was performed (indicated as 0 μM in each compound).

As shown in FIG. 2, it was confirmed that the addition of menthol, menthol valine ester, and menthol isoleucine ester in addition to the addition of LPS tended to suppress the increase in TNF-α mRNA expression level in a concentration-dependent manner. It was. Further, menthol had an IC ₅₀ value of 45.05 μM, whereas menthol valine ester had an IC ₅₀ value of 7.54 μM and menthol isoleucine ester had an IC ₅₀ value of 4.89 μM. From this, it was found that menthol valine ester and menthol isoleucine ester have a very high anti-inflammatory effect as compared with menthol.

(Test Example 3)
A cytotoxicity test was performed to confirm the cytotoxicity of the menthol amino acid ester.

In the following tests, cells cultured in the same manner as in Test Example 1 were used. Menthol, menthol valine ester, and menthol isoleucine ester dissolved in an aqueous solution containing 0.1 mM MES and 0.1% DMSO were added to the medium to a concentration of 1 μM, 10 μM, 100 μM, 500 μM, 1000 μM, and 10000 μM. And incubated for 24 hours. The recovered cells were then exposed to propidium iodide (PI) at a final concentration of 5 μg / mL, and PI-negative cells were calculated as living cells using MAQSQuant Analyzer (Miltenyi Biotec).

The same experiment was performed three times to determine the mean and standard deviation. The results are shown in FIG. In addition, "*" in FIG. 3 indicates that it is statistically significant as compared with the example in which the control solution was added (indicated as 0 μM in each compound).

As shown in FIG. 3, when menthol was added, no significant cytotoxicity was shown in the range up to 1000 μM. In addition, when menthol valine ester or menthol isoleucine ester was added, no significant cytotoxicity was shown in the range up to 500 μM.

From this result, the menthol valine esters and _{IC 50} values of menthol isoleucine esters shown in FIG. 2 (7.54μM and 4.89μM), cytotoxicity was found to be caused.

Claims (4)

An anti-inflammatory agent containing an ester of monoterpene alcohol and a branched chain amino acid or a salt thereof as an active ingredient. The anti-inflammatory agent according to claim 1, wherein the monoterpene alcohol is menthol. The anti-inflammatory agent according to claim 1 or 2, wherein the branched chain amino acid is a compound represented by the following formula (1).

[In the formula, R represents a branched alkyl group having 3 to 6 carbon atoms. ] The anti-inflammatory agent according to any one of claims 1 to 3, wherein the ester is a compound represented by the following formula (2).

[In the formula, R represents a branched alkyl group having 3 to 6 carbon atoms. ]

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