JP2021113162A - Ubiquinol delivery agent having high photostability and low phototoxicity - Google Patents

Abstract

To provide a ubiquinol delivery agent having high photostability and low phototoxicity.SOLUTION: The present disclosure provides a ubiquinol delivery agent having high photostability and low phototoxicity, the ubiquinol delivery agent containing one of a ubiquinol carboxylate ester derivative represented by formula (1) (where R1 and R2 each represent a hydrogen atom or glycine, N-acyl glycine or the like, at least one of R1 and R2 denotes glycine, N-acylglycine, N-alkylglycine, or the like. n denotes an integer of 1-10) or a salt thereof or a counterion mixture of ubiquinol carboxylate ester.SELECTED DRAWING: Figure 1

Description

The present invention relates to a ubiquinol delivery agent which has high photostability and does not show phototoxicity even in a state where light shielding is not required and light shielding is difficult.

Coenzyme Q (CoQ, ubiquinone, UQ) is a compound having a polyisoprenyl group at the 6-position of 2,3-dimethoxy-5-methyl-1,4-benzoquinone, and is distinguished by the number of isoprene units (n). Will be done. N = 10 ubiquinone-10 in mammals and n = 9 ubiquinone-9 in rodents are the major ubiquinones (Non-Patent Document Crane, 2001). Ubiquinone constitutes a redox system between flavoproteins and cytochromes in the mitochondrial respiratory chain, functions as an electron transport chain by controlling oxidative phosphorylation, and plays an important role in ATP synthesis.

Ubiquinone is endogenous and two biosynthetic routes have been shown. One is ubiquinol, which is prenylated from 4-hydroxybenzoic acid and oligoprenylpyrrophosphate by the catalyst of the mitochondrial enzyme Coq2, and is mainly used for the mitochondrial respiratory chain (Non-Patent Document 1). The other is ubiquinol produced from 4-hydroxybenzoic acid and oligoprenylpyrrophosphate catalyzed by the Golgi apparatus membrane enzyme UBIAD1 (non-mitochondrial prenylation enzyme), which mainly oxidizes Golgi apparatus-derived membranes and lipoproteins. It functions as an antioxidant that protects against lipid peroxidation (Non-Patent Document 2). Since only reduced ubiquinol can exert its antioxidant activity, it is shown that it is biosynthesized as ubiquinol.

Ubiquinone has beneficial effects on the following conditions and illnesses: Periodontal disease, blood circulation disease, memory disorder, fatigue, abnormal heartbeat, hypertension, immune dysfunction, hepatitis C and other liver diseases, aging, prevention of migraine, improvement of human ability in sports, etc. It is known that even if ubiquinone (oxidized form) is administered, most of it exists as reduced ubiquinol in the body, and most of its excellent actions are considered to be due to the excellent antioxidant action of reduced ubiquinol. There is.

JP 2005-2005 Patent No. 4255716 JP 2002-104922 Patent No. 5096653

Forsgren et al., Biochem J, 2004; 519-526. Mugoni et al., Cell, 2013; 152: 504-518. Imada I et al., Chem Pharm Bull, 1964; 12 (9): 1042-1046. Onoue S et al., Eur J Pharm, 2012; 46: 492-499. Kwon SS et al., Colloids Surf A Physicochem Eng Asp, 2002; 210: 95-104. Wang J et al., J Nanosci Nanotechnol, 2012; 12 (3): 2136-48. Onoue S et al., E Eur J Pharm, 2014; 53: 118-125. Qin B et al., J Agric Food Chem, 2017; 65: 3360-3367.

Currently, oxidized ubiquinone and reduced ubiquinol are used to ensure body levels of ubiquinol. However, both ubiquinone and ubiquinol are known to be extremely unstable to light (Non-Patent Document 3). Further, as shown in Examples 3 and 4 of the present application and Non-Patent Document 4, in the light irradiation of ubiquinone, direct phototoxicity caused by active oxygen species generated by type I photochemical reaction and type II photochemical reaction Two types of phototoxicity, indirect phototoxicity, are induced through the reaction between the singlet oxygen produced in the above and biological components. That is, when these ubiquinone or ubiquinol are used as a ubiquinol delivery agent, they are chemically stabilized by light until the administration to the patient is completed through the raw material drug, the formulation process, the distribution process, and the storage period at a medical institution. When sexual and toxicological stability (onset of side effects due to decomposition products and foreign substances, irritation) is affected, not only the decrease in effectiveness due to the decrease in stability but also the occurrence of undesired side effects may occur. Therefore, the ubiquinol delivery agent needs to be able to ensure light stability and avoid phototoxicity throughout these processes.

To improve the stability of oxidized ubiquinone against photodegradation, self-emulsifying drug delivery system (SEDDS) (Non-Patent Document 4), nanoparticulation by poly (methylcry) (PMMA) (Non-Patent Document 5), nanolipid Carrier formation (Non-Patent Document 6), nanocrystal dispersion SEDDS (s-SEDDS) (Non-Patent Document 7), PEGylated solanenol micelle (Non-Patent Document 8), inclusion with cyclodextrin (Patent Documents 1 and 3) ), Shading with a caramel dye or the like (Patent Document 2), and other formulations have been attempted. In addition, inclusion with cyclodextrin is disclosed for improving the photostability of reduced ubiquinol (Patent Document 3). Self-emulsifying drug delivery system (SEDDS) (Non-Patent Document 4) has shown photostabilization and suppression of phototoxicity (generation of singlet oxygen and formation of ROS), but the half-life of photolysis is 15 min. Is about 40 minutes, which is not enough to ensure stability.

In particular, when ubiquinone or ubiquinol is applied as an external preparation for skin or eye drops, it is difficult to shield the application site from light after application, and light stability cannot be ensured by shading. Therefore, ubiquinone or ubiquinol is used as an external preparation for skin. Is greatly impaired. In addition, since oxidized ubiquinone generates ROS and singlet oxygen by sunlight or UVA irradiation, there are concerns about direct phototoxicity due to ROS and indirect phototoxicity due to peroxidation of skin surface lipids such as squalane due to singlet oxygen. Will be done. In addition, porphyrin, tetracycline, ketoprofen, furalene 60 and the like are known as photosensitizers that generate singlet oxygen by ultraviolet rays. Against this background, an active ubiquinol delivery agent that does not require light-shielding and exhibits phototoxicity while ensuring photostability even in a state where light-shielding is difficult is desired.

We have found and disclosed that a specific ubiquinol carboxylic acid ester derivative exhibits excellent water solubility and reduced ubiquinol release property in vivo, and functions as a ubiquinol delivery agent in various dosage forms (Patent Document 4). .. However, it has not been clarified whether or not the ubiquinol carboxylic acid ester derivative is effective as a ubiquinol delivery agent which has high photostability and does not show phototoxicity even in a state where light shielding is not required and light shielding is difficult.

As described above, the present inventors have found and disclosed that a ubiquinol carboxylic acid ester derivative having a specific structure can exhibit excellent water solubility and reduced ubiquinol release property in vivo (Patent Document 4). ). As a result of continued examination of its usefulness, the ubiquinol carboxylic acid ester derivative is effective as an active ubiquinol delivery agent without requiring light shielding or showing high photostability and phototoxicity even in a state where light shielding is difficult. We found that there was something, and came to complete the present invention.

That is, the ubiquinol delivery agent according to the present invention is
General formula (1)

（式中Ｒ_１及びＲ_２はそれぞれ水素原子またはグリシン、N −アシルグリシン、N -アルキルグリシン、N,N -ジアルキルグリシン、アシル、ジカルボン酸ヘミエステル及びその塩から選ばれる置換基を意味し、R₁, R₂の少なくとも一方はグリシン、N -アシルグリシン、N -アルキルグリシン、N,N -ジアルキルグリシン、アシル、ジカルボン酸ヘミエステル、及びその塩である。ｎは１〜１０の整数を意味する。）で表されるユビキノールのカルボン酸エステル誘導体またはその塩の1種類を含有する、光曝露下に適用されるものである。
また、前記送達剤において、上記一般式（１）中Ｒ_１及びＲ_２はそれぞれ水素原子またはジカルボン酸ダブルエステルを意味し、Ｒ_１, Ｒ_２の少なくとも一方はジカルボン酸ダブルエステルで表されるユビキノールカルボン酸エステル誘導体であることが好適である。
また、前記送達剤において、上記一般式（１）中Ｒ_１及びＲ_２はそれぞれ水素原子またはグリシン、N -アシルグリシン、N -アルキルグリシン、N,N -ジアルキルグリシン、ジカルボン酸ヘミエステル、及びその塩から選ばれる置換基を意味し、Ｒ_１, Ｒ_２の少なくとも一方はグリシン、N -アシルグリシン、N -アルキルグリシン、N,N -ジアルキルグリシン、ジカルボン酸ヘミエステル、及びその塩で表されるユビキノールのカルボン酸エステル誘導体にその対イオンあるいは対イオンの塩をモル比１：２〜１：１０で含有し、水溶液とした時、粒子サイズ10 nm以下のミセル溶液を与える固形剤及び液剤であることが好適である。

(In the formula, R ₁ and R ₂ mean hydrogen atoms or substituents selected from N-acyl glycine, N-alkyl glycine, N, N-dialkyl glycine, acyl, dicarboxylic acid hemiesters and salts thereof, respectively, and R. _At least one of 1, R ₂ is glycine, N-acyl glycine, N-alkyl glycine, N, N-dialkyl glycine, acyl, dicarboxylic acid hemiester, and salts thereof. N means an integer of 1 to 10. ) Contains one of the carboxylic acid ester derivatives of ubiquinol or a salt thereof, and is applied under light exposure.
Further, in the delivery agent, R ₁ and R ₂ in the general formula (1) mean a hydrogen atom or a dicarboxylic acid double ester, respectively _{, and at least one of R 1} and R ₂ is a ubiquinol represented by a dicarboxylic acid double ester. It is preferably a carboxylic acid ester derivative.
Further, in the delivery agent, R ₁ and R ₂ in the above general formula (1) are hydrogen atoms or glycine, N-acyl glycine, N-alkyl glycine, N, N-dialkyl glycine, dicarboxylic acid hemiester, and salts thereof, respectively. Means a substituent selected from _{, and at least one of R 1} and R ₂ is a ubiquinol represented by glycine, N-acyl glycine, N-alkyl glycine, N, N-dialkyl glycine, dicarboxylic acid hemiester, and a salt thereof. It is a solid agent or a liquid agent that contains a counterion or a salt of the counterion in a carboxylic acid ester derivative at a molar ratio of 1: 2 to 1:10 and gives a micelle solution having a particle size of 10 nm or less when prepared as an aqueous solution. Suitable.

The external preparation for skin according to the present invention is characterized by containing a ubiquinol carboxylic acid ester derivative represented by the general formula (1) or a salt thereof.
Further, the eye drop according to the present invention is characterized by containing a ubiquinol carboxylic acid ester derivative represented by the general formula (1) or a salt thereof.
Further, the eye ointment according to the present invention is characterized by containing a ubiquinol carboxylic acid ester derivative represented by the general formula (1) or a salt thereof.
Further, in the ubiquinol carboxylic acid ester derivative according to the present invention, in the above general formula (1), R ₁ and R ₂ are HOH ₂ C- (CH (OH)) ₄ CH ₂ NHCH ₃ · HOOCCH ₂ CH ₂ CO-. , CH ₃ CH ₂ OOCCH ₂ CH ₂ CO- and (CH ₃ ) ₂ CH00CCH ₂ CH ₂ CO-, which are carboxylic acid residues selected from the group.

As described above, according to the active ubiquinol delivery agent, which does not require light shielding and does not require light shielding, high photostability is ensured even in a state where light shielding is difficult, and does not show phototoxicity, the ubiquinolcarboxylic acid ester or By using the salt or a counterion mixture of a ubiquinol carboxylic acid ester, it is possible to deliver active ubiquinol with high photostability and no phototoxicity even in a state where light shielding such as skin administration and instillation is difficult. Can be done. In addition, the photostability does not change significantly through the formulation process, distribution process, storage in medical institutions, and administration procedure from handling in the ward to patient administration, not limited to administration forms that are difficult to block light. Moreover, it is possible to avoid the possibility of phototoxicity and enable active ubiquinol delivery. Since the present invention does not require an additive for shading or a specific formulation form, it can be applied to a wide range of formulations.

Explanatory drawing of light stability of ubiquinol carboxylic acid ester derivative with respect to artificial sunlight. ■: Ubiquinone, □: Ubiquinol, ▲: Compound 1, △: Compound 2, ▼: Compound 3, ●: Compound 9 Explanatory drawing of time change of composition in compound 9 ethanol solution under shading. ●: Compound 9 (bisester), ○: Monoester, ■: Ubiquinol, □: Ubiquinone Explanatory drawing of time change of composition in compound 9 ethanol solution in artificial sunlight irradiation. ●: Compound 9 (bisester), ○: Monoester, ■: Ubiquinol, □: Ubiquinone Explanatory drawing of wavelength characteristic of photolysis of ubiquinone. ●: 279 nm, ○: 310 nm, ■: 341 nm, □: 373 nm Explanatory drawing of wavelength characteristic of photolysis of ubiquinol. ●: 279 nm, ○: 310 nm, ■: 341 nm, □: 373 nm Explanatory drawing of wavelength characteristic (297nm) of photodecomposition of ubiquinol carboxylic acid ester derivative. ■: Ubiquinone, □: Ubiquinol, ▲: Compound 1, △: Compound 2, ▼: Compound 3 Explanatory drawing of wavelength characteristic (310 nm) of photodecomposition of ubiquinol carboxylic acid ester derivative. ■: Ubiquinone, □: Ubiquinol, ▲: Compound 1, △: Compound 2, ▼: Compound 3 Explanatory drawing of active oxygen species (ROS) generation state by type I photochemical reaction in artificial sunlight irradiation. ■: Ubiquinone, □: Ubiquinol, ▲: Compound 1, △: Compound 2, ▼: Compound 3, ●: Compound 9, ▽: Sulisobenzone, ○: Quinine Explanatory drawing of singlet oxygen generation state by type II photochemical reaction in artificial sunlight irradiation. ■: Ubiquinone, □: Ubiquinol, ▲: Compound 1, △: Compound 2, ▼: Compound 3, ●: Compound 9, ▽: Sulisobenzone, ○: Quinine Explanatory drawing of active oxygen species (ROS) generation state by type I photochemical reaction in UVA irradiation and UVB irradiation. ■: Ubiquinone, □: Ubiquinol, ▲: Compound 1, △: Compound 2, ▼: Compound 3, ●: Compound 9, ▽: Sulisobenzone, ○: Quinine Explanatory drawing of singlet oxygen generation state by type II photochemical reaction in UVA irradiation and UVB irradiation. ■: Ubiquinone, □: Ubiquinol, ▲: Compound 1, △: Compound 2, ▼: Compound 3, ●: Compound 9, ▽: Sulisobenzone, ○: Quinine Explanatory drawing of particle size of aqueous solution of cationic ubiquinol carboxylic acid ester and anionic counterion mixture. Explanatory drawing of particle size of an aqueous solution of an anionic ubiquinol carboxylic acid ester and a cationic counterion mixture. Explanatory drawing of evaluation of delivery property of intracellular ubiquinol to human epidermal keratinized cells by ubiquinone and ubiquinol carboxylic acid ester. ○: Control, ■: Ubiquinone, ●: Compound 9 Explanatory drawing of evaluation of delivery property of intracellular ubiquinone to human epidermal keratinized cells by ubiquinone and ubiquinol carboxylic acid ester. ○: Control, ■: Ubiquinone, ●: Compound 9 Explanatory drawing of plasma ubiquinol after intravenous administration of an aqueous solution of an anionic ubiquinol carboxylic acid ester and a cationic counterion mixture in rats. ●: Compound 10 (ubiquinone equivalent 5 mg / kg), Δ: Compound 10 (ubiquinone equivalent 1 mg / kg) Explanatory drawing of evaluation of suppression effect of ubiquinol carboxylic acid ester on cytotoxicity to human epidermal keratinized cells by H ₂ O _{2 stimulation.} □: H ₂ O ₂ not added, ■: H ₂ O ₂ added (300 μM) Control (H ₂ O ₂ added) vs * p <0.05, * * p <0.05 by Dunnett's test Explanatory drawing of intracellular ubiquinone delivery by ubiquinol carboxylic acid ester to H ₂ O _{2 stimulated human epidermal keratinized cells.} □: H ₂ O ₂ not added, ■: H ₂ O ₂ added Illustration of intracellular ubiquinol delivery by ubiquinol carboxylic acid ester to H ₂ O _{2 stimulated human epidermal keratinized cells.} □: H ₂ O ₂ not added, ■: H ₂ O ₂ added Illustration of the inhibitory effect of ubiquinol carboxylic acid ester on MMP-1 mRNA expression in human epidermal keratinized cells by H ₂ O _{2 stimulation.} □: H ₂ O ₂ not added, ■: H ₂ O ₂ added Control (H ₂ O ₂ added) vs * * p <0.05 by Dunnett's test

Hereinafter, preferred embodiments of the present invention will be described in detail, but it goes without saying that the present invention is not limited thereto.
The present invention does not require light-shielding using the compound represented by the above general formula (1) or a salt thereof or a counterion mixture of a ubiquinolcarboxylic acid ester, and has high photostability and phototoxicity even in a state where light-shielding is difficult. With respect to active ubiquinol delivery agents not shown. The compound represented by the general formula (1) can be contained alone in the preparation, or can be blended in the preparation as a salt thereof or a counterion mixture of a ubiquinolcarboxylic acid ester.

In the present invention, the following are exemplified as _{carboxylic acid residues R 1} and R _{2 having a nitrogen substituent.} A hydrogen atom or one or two alkyl group or acyl group bonded to a nitrogen atom. Examples of the alkyl group are linear or branched alkyl groups having 1 to 6 carbon atoms, and the following are exemplified. Methyl group, ethyl group, n-propyl, n-butyl group, n-pentyl group, n-hexyl group, isobutyl group, 1-methylpropyl group, tert-butyl group, 1-ethylpropyl group, isoamyl group. As the alkyl group, a methyl group and an ethyl group are preferable. Further, the hydrocarbon chain having an acyl group can be similarly defined.

The amino group and the carbonyl group are preferably bonded by a linear, branched or cyclic alkylene group having 1 to 7 carbon atoms. The branched alkylene group is an alkylene group derived from an alkyl group such as isopropyl, isobutyl, tert-butyl, or 1-ethylpropyl. The cyclic alkylene group means an alkylene group containing a cyclopentane ring, a cyclohexane ring, a methylcyclohexane ring, or the like in the structure. Particularly preferred as the alkylene group is a methylene group or an ethylene group.

As the hydrohalide, hydrochloride, hydrobromide and the like are preferable. In the present invention, the hydrohalide has an advantage that the melting point is often higher than that of the drug substance quinone compound, and it is easy to handle in the formulation. Examples of other salts include the following. Alkyl sulfonates include methane sulfonates, and sugar salts include gluconates, glucoheptanes, and lactobionates.

In the present invention, the dicarboxylic acid hemiester residues R ₁ and R ₂ are selected from the dicarboxylic acid hemiester and its alkali metal salt or meglumine salt. The carbonyl groups of the dicarboxylic acid residue are bonded by a linear alkylene group having 2 to 4 carbon atoms. As the alkylene group, an ethylene group or a propylene group is particularly preferable. Sodium salt and potassium salt are preferable as the alkali metal salt.

In the present invention, the dicarboxylic acid double ester residue is bonded between the carbonyl groups of the dicarboxylic acid by a linear alkylene group having 2 to 4 carbon atoms. As the alkylene group, an ethylene group or a propylene group is particularly preferable. The ester is preferably ethyl ester, isopropyl ester or the like.

In the present invention, the counter ion of the counter ion mixture of the ubiquinol carboxylic acid ester having a carboxylic acid residue having a nitrogen substituent is preferably an endogenous bile acid or a salt thereof, particularly preferably sodium taurocholate, and is paired with the ubiquinol carboxylic acid ester. The mixing molar ratio of the ion mixture is preferably 1: 2 to 1:10. The counterion of the counterion mixture of the ubiquinolcarboxylic acid ester having a dicarboxylic acid hemiester residue is preferably meglumine, and the mixed molar ratio of the ubiquinolcarboxylic acid ester and the counterion mixture is preferably 1: 2 to 1:10.

Various methods for producing the compound of the present invention represented by the general formula (1) can be considered, and typical methods are as follows. Ubiquinone represented by the general formula (2) is reduced with a reducing agent to obtain ubiquinol represented by the general formula (3), and this ubiquinol and a carboxylic acid having a nitrogen substituent, a reactive acid derivative thereof, or a halogen thereof. The target substance (1) of the present invention can be obtained by carrying out an esterification reaction with a hydrohalide by a conventional method. A carboxylic acid hemiester is obtained by a conventional method of ubiquinone represented by the general formula (2), zinc powder, acetic acid and acid anhydride. A ubiquinolcarboxylic acid hemiester and an alcohol are esterified by a conventional method to obtain a ubiquinolcarboxylic acid alcohol ester.

Examples of the reducing agent used here include sodium borohydride, sodium hydrosulfite, trin-butylphosphine, zinc chloride, stannous chloride and the like.

The esterification reaction of ubiquinol follows a conventional method, but each functional group of an amino acid having a primary or secondary amino group or a hydroxyl group or a thiol group in the side chain is a tert-butoxycarbonyl group (hereinafter abbreviated as t-BOC group) or benzyl. Protect and use with an appropriate protective group such as an oxycarbonyl group (hereinafter abbreviated as Z group) and 9-fluorenylmethoxycarbonyl group (hereinafter abbreviated as FMOC group), and N, N-dialkylamino acids or pyridinecarboxylic acids are halogens. Dicyclohexylcarbodiimide (hereinafter abbreviated as DCC), 1-methyl-3- (3-dimethylaminopropyl) -carbodiimide (hereinafter abbreviated as EDC), N, N-disuccinimid oxalate (hereinafter abbreviated as DCC) using hydride. It is preferable to carry out the reaction in the presence of an active esterification reagent such as DSO) to give preferable results. At this time, pyridine is preferable as the solvent.

Moreover, in the method using a reactive acid derivative, the method using an acid halide, particularly an acid chloride, gives preferable results. At this time, the solvent is preferably an anhydrous benzene-anhydrous pyridine mixture. Hydrogen halides and alkylsulfonates are produced by reacting a free amino acid ester with hydrohalic acid or alkylsulphonic acid by a conventional method. A hydrohalide can be produced by producing an N-acylamino acid ester and then deprotecting with a hydrohalic acid by a conventional method.

Ubiquinolcarboxylic acid hemiester is produced by heating and reacting ubiquinone, zinc, and dicarboxylic acid anhydride. The reaction in acetic acid-sodium acetate gives favorable results.

The ubiquinolcarboxylic acid double ester is produced by esterifying the ubiquinolcarboxylic acid hemiester by a conventional method.
[Skin external preparation]
The ubiquinol carboxylic acid ester derivative according to the present invention can be used as an external preparation for skin because of its excellent photostability and low phototoxicity.
The concentration of the ubiquinol carboxylic acid ester derivative when used as an external preparation for skin is preferably 0.01 to 1% by mass (hereinafter, simply abbreviated as "%"), particularly preferably 0.05 to 0.5%. Within this range, the ubiquinol carboxylic acid ester derivative can be stably blended, and excellent medicinal effects can be exhibited.

The ubiquinol carboxylic acid ester derivative of the present invention can be used alone as an external preparation for skin, but an external preparation for skin can also be prepared by mixing with one or more kinds of additives. Additives that are added as needed include water (purified water, hot spring water, deep water, etc.), alcohol, oils, surfactants, and metals that are commonly used in the formulation of skin cosmetics and external medicines. Drugs, gelling agents, powders, alcohols, water-soluble polymers, film-forming agents, resins, UV protective agents, inclusion compounds, antibacterial agents, fragrances, deodorants, salts, pH adjusters, refreshing agents, animals -Microbial extract, plant extract, blood circulation promoter, astringent, anti-fat leak agent, whitening agent, anti-inflammatory agent, active oxygen scavenger other than the single-term oxygen scavenger of the present invention, cell activator, moisturizer , Chelating agents, keratolytic agents, enzymes, hormones, vitamins and the like. The preparation of the external preparation for skin can be carried out according to a conventional method, and the blending amount of the additive can also be determined according to a conventional method as long as the effect of the present invention is not impaired.

The form of the external preparation for skin is not limited, and forms belonging to skin cosmetics such as milky lotion, cream, lotion, beauty liquid, pack, washing pigment, makeup cosmetic, etc .; shampoo, hair treatment, hair styling agent, It may be any of a form relating to a hair cosmetic such as a hair restorer and a hair restorer; and a form of an external preparation such as a dispersion, an ointment, an aerosol, a patch and a poultice.

[Eye drops]
The ubiquinol carboxylic acid ester derivative of the present invention can also be used as an eye drop as a dosage form because of its excellent photostability and low phototoxicity. By appropriately blending an isotonic agent, a buffer, a pH adjuster, a solubilizer, a thickener (dispersant), a stabilizer (antioxidant), a preservative (preservative), etc. as additives. , Can be formulated by a well-known method. In addition, a stable eye drop can be obtained by suspending the drug by adding a pH adjuster, a thickener, a dispersant, or the like.

The tonicity agent is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. For example, the ionic tonicity agent includes sodium chloride and potassium chloride. , Calcium chloride, magnesium chloride and the like, and examples of the nonionic tonicity agent include glycerin, propylene glycol, sorbitol, mannitol and the like.

The buffer is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and is, for example, phosphoric acid, phosphate, citric acid, acetic acid or ε-aminocaproic acid. And so on.

The pH regulator is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and is, for example, hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and water. Examples thereof include potassium oxide, boric acid, boar sand, sodium carbonate, sodium hydrogen carbonate and the like. The pH of the eye drop may be within the range acceptable for the ophthalmic preparation, but is in the range of 4.0 to 9.0, more preferably 5.5 to 8.5.

The solubilizer is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and is, for example, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, or polyoxy. Examples thereof include ethylene sorbitan fatty acid ester, vitamin E TPGS, polyoxyethylene fatty acid ester, polyoxyethylene polyoxypropylene glycol, sucrose fatty acid ester and the like.

The thickener and dispersant are not particularly limited as long as they are pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and are cellulosic, for example, hydroxypropylmethylcellulose or hydroxypropylcellulose. Molecular; polyvinyl alcohol; or polyvinylpyrrolidone and the like can be mentioned.

The stabilizer is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and is, for example, edetic acid, monosodium edetate, disodium edetate, edetic acid. Examples thereof include tetrasodium and sodium citrate, and sodium edetate may be a hydrate.

The antioxidant is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and is, for example, ascorbic acid, vitamin E, dibutylhydroxytoluene, butylhydroxyanisole, and erythorbine. Examples thereof include sodium acid, propyl gallate, and sodium sulfite.

The preservative (preservative) is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and is, for example, benzalkonium chloride, benzalkonium bromide, or benzethonium. Examples thereof include chloride, sorbic acid, potassium sorbate, methyl paraoxybenzoate, propyl paraoxybenzoate, chlorobutanol and the like, and these preservatives can also be used in combination.

[Eye ointment]
Examples of the dosage form of the ubiquinol carboxylic acid ester derivative of the present invention include eye ointments, which can be prepared using a general-purpose base such as white petrolatum, plastic base or liquid paraffin.

In order to show the usefulness of the compound of the present invention as a ubiquinol delivery agent having high photostability and low phototoxicity, first, an experiment in which the photostability in artificial sunlight irradiation was evaluated by comparison with oxidized ubiquinone and reduced ubiquinol. Let me give you an example. In addition, an example in which the wavelength characteristics of light stability by light irradiation with a spectroscope are evaluated by comparison with oxidized ubiquinone will be given. Next, the generation of reactive oxygen species by the type I photochemical reaction that causes direct phototoxicity by UVA irradiation and UVB irradiation and the production of singlet oxygen by the type II photochemical reaction that causes indirect phototoxicity are oxidized ubiquinone. Here is an example evaluated by comparison with. Further, an example of a ubiquinol carboxylic acid ester and a counterion mixture in which the aqueous solution can form mixed micelles having a particle size of nm order will be given. Furthermore, an example in which the delivery property of ubiquinol to human epidermal keratinized cells of the compound of the present invention was evaluated will be given. Finally, water was added to an anionic ubiquinolcarboxylic acid ester and a counterion mixture to make an immediate solution, which was intravenously administered to rats, and the plasma dynamics after administration was examined to evaluate the feasibility of injection administration. Let me give you an example.

Example 1
The ubiquinol derivatives shown in Tables 1 and 2 were produced by the methods shown in the following production methods A to H. The mass spectra of the obtained substances (ionization method; FD method and FAB method) and ¹ H-NMR spectra are shown in Tables 3 to 4.

Manufacturing method A
Dissolve 0.1 mol of amino acid in 100 ml of distilled water-dioxane (1: 1, v / v), add 30 ml of triethylamine, gradually add di-tert-butyl dicarbonate, and stir at room temperature for 30 minutes. Dioxane is distilled off under reduced pressure, 50 ml of aqueous sodium hydrogen carbonate solution (0.5 M) is added, and the mixture is washed with 100 ml of ethyl acetate. Wash the ethyl acetate layer with 50 ml of sodium hydrogen carbonate aqueous solution, combine the aqueous layers, add citric acid aqueous solution (0.5 M) under ice cooling to make it acidic (pH 3), saturate sodium chloride, and then use ethyl acetate. Extract (100 ml x 3 times). After dehydrating the extract with anhydrous sodium sulfate, the solvent is distilled off under reduced pressure, and isopropyl ether is added to the oily residue or cooled to crystallize to obtain Nt-BOC-amino acid. 1.16 mmol of ubiquinone-10 (ubiquinone) is dissolved in 100 ml of isopropyl ether, 2.8 mmol of sodium borohydride is suspended in 15 ml of methanol, and the mixture is stirred at room temperature until the yellow color of the solution becomes colorless. The isopropyl ether layer is washed by adding 100 ml of distilled water saturated with argon gas to the reaction solution. After separation, the isopropyl ether layer is dehydrated with anhydrous sodium sulfate and the solvent is distilled off under reduced pressure to obtain ubiquinol-10 (ubiquinol). Nt-BOC amino acid 2.8 mmol, DCC 2.8 mmol, and anhydrous pyridine 30 ml are added to ubiquinol to replace the atmosphere with argon gas, and then the mixture is stirred at room temperature for 24 hours. The solvent is evaporated under reduced pressure, ethyl acetate is added to the residue, and the soluble fraction is extracted (100 ml x 2 times). The extract is concentrated under reduced pressure, and the residue is separated and purified by silica gel flash chromatography (elution solvent; n-hexane: ethyl acetate, 85:15) to obtain a ubiquinol-1,4-bis-Nt-BOC-amino acid ester. .. Ubiquinol-1,4-bis-Nt-BOC-amino acid ester is dissolved in a small amount of acetone, and hydrochloric acid-dioxane (2.5 to 4.0 N) is deprotected by adding hydrochloric acid equivalent amount of about 20 times the amount of ester bond. I do. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was recrystallized from acetone to obtain a hydrochloride of ubiquinol-1,4-bis-amino acid ester.

Manufacturing method B
1.16 mmol of ubiquinone is dissolved in 100 ml of isopropyl ether, 2.8 mmol of sodium borohydride is suspended in 15 ml of methanol, and the mixture is stirred at room temperature until the yellow color of the solution becomes colorless. The isopropyl ether layer is washed by adding 100 ml of distilled water saturated with argon gas to the reaction solution. After separation, the isopropyl ether layer is dehydrated with anhydrous sodium sulfate and the solvent is distilled off under reduced pressure to obtain ubiquinol. After adding 1.4 mmol of Nt-BOC amino acid, 1.4 mmol of DCC, and 30 ml of anhydrous pyridine to ubiquinol and replacing the atmosphere with argon gas, the mixture is stirred at room temperature for 24 hours. The solvent is evaporated under reduced pressure, ethyl acetate is added to the residue, and the soluble fraction is extracted (100 ml x 2 times). The extract is concentrated under reduced pressure, and the residue is separated and purified by silica gel flash chromatography (elution solvent; n-hexane: ethyl acetate, 85:15) to ubiquinol-1-Nt-BOC-amino acid ester and ubiquinol-4-Nt. -BOC-Obtain an amino acid ester. Ubiquinol-1-Nt-BOC-amino acid ester or ubiquinol-4-Nt-BOC-amino acid ester is dissolved in a small amount of acetone, and hydrochloric acid-dioxane (2.5 to 4.0 N) is added to hydrochloric acid in an amount of about 20 times the amount of ester bond. Deprotection basement is performed by adding an amount corresponding to the amount. After completion of the reaction, the solvent was evaporated under reduced pressure, and the residue was recrystallized from acetone to obtain hydrochlorides of ubiquinol-1-amino acid ester and ubiquinol-4-amino acid ester.

Manufacturing method C
1.16 mmol of ubiquinone is dissolved in 100 ml of isopropyl ether, 2.8 mmol of sodium borohydride is suspended in 15 ml of methanol, and the mixture is stirred at room temperature until the yellow color of the solution becomes colorless. The isopropyl ether layer is washed by adding 100 ml of distilled water saturated with argon gas to the reaction solution. After separation, the isopropyl ether layer is dehydrated with anhydrous sodium sulfate and the solvent is distilled off under reduced pressure to obtain ubiquinol. N, N-dialkylamino acid hydrochloride 2.8 mmol, DCC 2.8 mmol, and anhydrous pyridine 30 ml are added to ubiquinol to replace the atmosphere with argon gas, and then the mixture is stirred at room temperature for 24 hours. The solvent is distilled off under reduced pressure, the residue is suspended in distilled water, sodium hydrogen carbonate is added to adjust the pH to 7 to 8, and then the soluble fraction is extracted with ethyl acetate (100 ml × 3 times). The extract was dehydrated with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was separated and purified by silica gel flash chromatography (elution solvent; n-hexane: ethyl acetate, 85:15), and ubiquinol-1,4-bis. Obtain a -N, N-dialkyl amino acid ester. Ubiquinol-1,4-bis-N, N-dialkylamino acid ester was dissolved in a small amount of n-hexane, a double molar amount of hydrochloric acid-dioxane (2.5 to 4.0 N) was added, and the solvent was evaporated under reduced pressure. The residue is recrystallized from acetone to give ubiquinol-1,4-bis-N, N-dialkylamino acid ester hydrochloride.

Manufacturing method D
1.16 mmol of ubiquinone is dissolved in 100 ml of isopropyl ether, 2.8 mmol of sodium borohydride is suspended in 15 ml of methanol, and the mixture is stirred at room temperature until the yellow color of the solution becomes colorless. The isopropyl ether layer is washed by adding 100 ml of distilled water saturated with argon gas to the reaction solution. After separation, the isopropyl ether layer is dehydrated with anhydrous sodium sulfate and the solvent is distilled off under reduced pressure to obtain ubiquinol. N, N-dialkylamino acid hydrochloride 2.8 mmol, DCC 2.8 mmol, and anhydrous pyridine 30 ml are added to ubiquinol to replace the atmosphere with argon gas, and then the mixture is stirred at room temperature for 24 hours. The solvent is distilled off under reduced pressure, the residue is suspended in distilled water, sodium hydrogen carbonate is added to adjust the pH to 7 to 8, and then the soluble fraction is extracted with ethyl acetate (100 ml × 3 times). The extract was dehydrated with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was separated and purified by silica gel flash chromatography (elution solvent; n-hexane: ethyl acetate, 85:15), and ubiquinol-1-N, N. -Dialkyl amino acid ester and ubiquinol-4-N, N-dialkyl amino acid ester are obtained. Ubiquinol-1-N, N-dialkylamino acid ester or ubiquinol-4-N, N-dialkylamino acid ester is dissolved in a small amount of n-hexane, and a double molar amount of hydrochloric acid-dioxane (2.5 to 4.0 N) is added. , The solvent was evaporated under reduced pressure, and the residue was recrystallized from acetone to obtain hydrochlorides of ubiquinol-1-N, N-dialkylamino acid ester and ubiquinol-4-N, N-dialkylamino acid ester.

Manufacturing method E
2 mmol of ubiquinol-1,4-bis-N, N-dialkylamino acid ester is dissolved in 20 ml of dichloromethane, 4.1 mmol of alkyl sulfonic acid is added, and the mixture is stirred. The precipitated crystals are collected by filtration to obtain an alkyl sulfonate of ubiquinol-1,4-bis-N, N-dialkyl amino acid ester.

Manufacturing method F
1.16 mmol of ubiquinone, 8.26 mmol of zinc, 20.0 mmol of dicarboxylic acid anhydride, 7.31 mmol of sodium acetate, and 66.6 mmol of acetic acid are refluxed at 85 ° C. for 3 hours. 200 ml of ethyl acetate and 100 ml of purified water are added to the white solid produced after cooling to room temperature, and the ethyl acetate-soluble fraction is extracted (100 ml x 3 times). The extract is dehydrated with anhydrous sodium sulfate, the solvent is distilled off under reduced pressure, and the residue is separated and purified by silica gel flash chromatography (elution solvent; n-hexane: ethyl acetate, 7: 3), and ubiquinol-1,4-bis. -Obtain a dicarboxylic acid hemiester.

Manufacturing method G
2 mmol of ubiquinol-1,4-bis-dicarboxylic acid hemiester is dissolved by adding 2 times mol of 1 N sodium hydroxide aqueous solution or 2 times mol of meglumine aqueous solution, and lyophilized. Recrystallize with methanol-acetonitrile to give ubiquinol-1,4-bis-dicarboxylic acid hemiester sodium salt or ubiquinol-1,4-bis-dicarboxylic acid hemiester meglumine salt.

Manufacturing method H
Ubiquinol-1,4-bis-dicarboxylic acid hemiester is dissolved in primary or secondary alcohol, hydrochloric acid is added, and the mixture is stirred at room temperature. The solvent is distilled off under reduced pressure to obtain a ubiquinol-1,4-bis-dicarboxylic acid alcohol ester.

Table 1 shows the chemical formulas of the compounds according to the present invention and the corresponding production methods. For Examples 1 to 6, mass spectrometry (m / z, FD-MS or FAB-MS) and nuclear magnetic resonance spectrum ( ¹ H-NMR, δ (ppm, internal standard TMS)) are shown in Table 2. ..

Compound

Compound

Compound (MS, NMR)

Compound (MS, NMR)

Example 2
Evaluation of photostability of ubiquinol carboxylic acid ester derivative against artificial sunlight <br /> Ubiquinone (Uq-10) and ubiquinol (UqH)) were used as control examples, and the test compound was a ubiquinol carboxylic acid ester derivative (Compounds 1, 2). , 3, 5, 9, 11, 12), compound 1-taurocholate Na (1: 2.5) mixture, and compound 9-meglumin (1:10) mixture. An ethanol solution of each compound is placed in a quartz cell with a stopper, and artificial sunlight (SOLAX 100W XC-100B, seric) is irradiated from the vertical direction at a temperature of 25 ° C at an illuminance of 15000 lx, and the drug concentration is adjusted over time by LC / MS / MS. Measured in. The illuminance was measured using an illuminometer (digital illuminance meter LX-1108, mother tool). As typical examples of photodecomposition by artificial sunlight irradiation, ubiquinone (Uq-10), ubiquinol (UqH), and ubiquinol carboxylic acid ester derivatives, which are control examples, are shown in FIG. 1 with changes in the residual amount of the drug over time. All the measured compounds were degraded according to a pseudo-primary reaction and their half-lives are shown in Table 5. Ubiquinone (Uq-10) and ubiquinol (UqH) were decomposed by light in a short time, and their half-lives were 1.8 hours and 2.0 hours, respectively. It was clarified that the ubiquinol carboxylic acid ester greatly improved the photostability and the half-life was 15 to 50 times longer than that of ubiquinone, so that high photostability against artificial sunlight could be ensured. Figures 2 and 3 show the changes over time in the amounts of bisester (Compound 9), monoester, ubiquinol, and ubiquinone under the shade of a ubiquinol-1,4-bis-hemisuccinate (compound 9) solution and under artificial sunlight irradiation, respectively. rice field. In both cases, rapid disappearance of bisesters, rapid increase of monoesters, and gradual formation of ubiquinol were observed, and the half-life of bisesters under shading and artificial sunlight irradiation were 2.0 hours and 1 respectively. It was .2 hours and was accelerated by artificial sunlight irradiation. The hydrolysis of the monoester to ubiquinol is clearly slower than the hydrolysis of the bisester, and the half-lives are 43 hours and 31 hours, respectively, under shading and artificial sunlight irradiation, which is also a control example in light irradiation. It was stable in comparison. Although the bisester form of Compound 9 is easily hydrolyzed into a monoester form, it is clear that the monoester form maintains photostability, and as a result, phototoxicity is exhibited as shown in Examples 4 and 5. It is thought that it can be avoided.
LC / MS / MS Measurement conditions: Mass spectrometer: LCMS-8050 LIQUID CHROMATOGRAPH MASS SPECTROMETER (SHIMADZU), High performance liquid chromatography (HPLC) device: Shimadzu HPLC System [System controller (CBM-20A), Pump (LD-20AD) , Degasser (DGU-20As), UV detector (SPD-20A), Auto injector (SIL-20AC HT)] was used. The column used was Shim-pack XR-C8 3.0 x 75 mm, particle size 2.2 μm (SHIMADZU). As the mobile phase, a methanol solution of 10 mM ammonium acetate and 0.1% acetic acid and an ethanol solution of 10 mM ammonium acetate and 0.1% acetic acid were used in gradient mode. The flow rate was 0.2 mL / min, the sample cooler was 4 ° C, and the column oven was 40 ° C. Ionization was measured in MRM mode using the Electrospray ionization (ESI) method. MRM: Ubiquinone (863.5> 197.0), Ubiquinol (882.6> 196.5), Compound 1 (1035.6> 490.8), Compound 2 (950.6> 406.0), Compound 3 (950.6> 406.0), Compound 9 (1083.0> 297.0, mono-ester) (979.0> 197.0)), Compound 11 (1140.0> 325.0), Compound 12 (1168.0> 339.05), Compound 13 (1112.0> 311.10).

Example 3
Wavelength characteristics of photolysis of ubiquinol carboxylic acid ester derivative Ubiquinone (Uq-10) and ubiquinol (UqH)) were used as control examples, and the test compound was ubiquinol carboxylic acid ester derivative (Compound 1, 2, 3, 9, 11, 12). , 13), compound 1-taurocholate Na (1: 2.5) mixture, and compound 9-meglumin (1:10) mixture. An ethanol solution of each compound is placed in a quartz cell with a stopper, and irradiated with light having a wavelength of 279-435 nm using a spectrophotometer (MM-3 multi-wavelength irradiation spectroscope, spectroscope) at a temperature of 25 ° C. The drug concentration in the sample was measured by the above-mentioned LC / MS / MS. The logarithmic value of the drug residual rate was plotted against the irradiation energy to obtain a straight line. Control ubiquinone and ubiquinol are shown in FIGS. 3 (A) and 3 (B), respectively. Significant decomposition of ubiquinone was observed below 279 nm, significant decomposition of ubiquinol was observed at wavelengths below 310 nm, and it was clear that ubiquinol also decomposed at long wavelengths. As a typical example, the decompositions of ubiquinone, ubiquinol, and ubiquinol carboxylic acid ester derivatives (Compounds 1, 2, and 3) at 279 nm and 310 nm are shown in FIGS. 5A and 5B, respectively. Table 6 shows the irradiation energy at which the amount of drug at each wavelength is half of the initial concentration as half irradiation energy. At 279 nm, the monocarboxylic acid ester was decomposed faster than the ubiquinol bis ester, and the stability varied greatly depending on the number of ester derivatizations. At 310 nm, the stability of ubiquinol monoesters and bisesters was similar and was not affected by the number of ester derivatizations.

Example 4
Generation of reactive oxygen species (ROS) by type I photochemical reaction in artificial sunlight irradiation
ROS measurement: Test compound in phosphate buffer (20 mM, pH 7.4) containing 10% polyoxyethylene (40) cured castor oil (Nikko Chemicals), 2% ethanol, 1% glycerin (Fuji Film Wako) Dissolve (compound concentration 10 mM), add 12.5 μL of it to 125 μL of nitroblue tetrazolium phosphate buffer solution (400 μM, Fuji Film Wako), and add phosphate buffer to make the total amount 625 μL (test compound concentration). 200 μM). The test solution was dispensed into a 96-well plate with 200 μL / well and irradiated with 15000 lx of artificial sunlight (SOLAX 100W XC-100B, seric). The illuminance was measured using an illuminometer (digital illuminance meter LX-1108, mother tool). Quinine (Fujifilm Wako) was used as a positive control, and sulisobenzone (Combi-Blocks) was used as a negative control. The absorbance at 560 nm was measured over time to evaluate the formation of ROS (Fig. 6). Table 7 shows the ROS levels after 3 hours of irradiation as a percentage of the positive control quinine (100%). Although ubiquinone is lower than that of the positive control quinine, the ROS produced by the type I photochemical reaction is clearly increased over time, and there is concern about direct phototoxicity. It was revealed that the ROS formation of the ubiquinol carboxylic acid ester derivative (Compounds 1, 2, 3, 9) by artificial sunlight irradiation was clearly low, and the direct phototoxicity by the type I photochemical reaction was low. As shown in Example 2, the bisester form of Compound 9 is rapidly hydrolyzed to the monoester form by artificial sunlight irradiation, but the high chemical stability of the monoester makes it difficult to produce highly phototoxic ubiquinone. As a result, direct phototoxicity due to type I photochemical reactions is considered to be low.

Generation of reactive oxygen species by artificial sunlight irradiation (15000lx, 3h)

Example 5
Singlet oxygen production by type II photochemical reaction in artificial sunlight <br /> Measurement of singlet oxygen: Test compound 10% polyoxyethylene (40) hardened castor oil (Nikko Chemicals), 2% ethanol (Fujifilm Wako) ), Dissolve in a phosphate buffer (20 mM, pH 7.4) containing 1% glycerin (Fujifilm Wako) (compound concentration 10 mM) and add 20 μL to imidazole (200 μM, Fujifilm Wako) and p-nitrosodimethyl. In addition to 500 μL of a phosphate buffer containing aniline (200 μM, Tokyo Kasei), a phosphate buffer was further added to bring the total volume to 1000 μL (test compound concentration 200 μM). This test solution was dispensed into a 96-well plate with 200 μL / well and irradiated with 15000 lx of artificial sunlight (SOLAX 100W XC-100B, seric). The absorbance at 440 nm was measured over time to evaluate the production of singlet oxygen. Quinine was used as a positive control and sulisobenzone was used as a negative control. As shown in FIG. 7, ubiquinone, like the positive control quinine, had higher singlet oxygen over time, and ubiquinol carboxylic acid ester derivatives (compounds 1, 2, 3, and 9) had clearly lower singlet oxygen production. rice field. Table 8 shows the singlet oxygen level after irradiation for 3 hours as a percentage of the positive control quinine (100%). As a result, ubiquinone is concerned about indirect phototoxicity due to type II photochemical reaction, but ubiquinol carboxylic acid ester derivatives (compounds 1, 2, 3, 9) are indirect phototoxicity due to type II photochemical reaction by artificial sunlight irradiation. It is clear that can be avoided. Compound 9 is considered to be able to avoid indirect phototoxicity due to type II photochemical reactions for the same reasons as in Examples 2 and 4.

Generation of singlet oxygen by artificial sunlight irradiation (15000lx, 3h)

Example 6
Generation of reactive oxygen species (ROS) by type I photochemical reaction in UVA irradiation and UVB irradiation Prepare a sample test solution in the same manner as in Example 4 above, and dispense 200 μL / well of the test solution into a 96-well plate. First, UVA (UV Crosslinker CL-1000L 365 nm, UVP) ^{was irradiated up to 10 J / cm 2} , and then UVB (UV Crosslinker CL-1000M 302 nm, UVP) was irradiated up ^{to 3 J / cm 2.} The absorbance at 560 nm was measured over time to evaluate the formation of ROS. As shown in FIG. 8, the positive control quinine produced ROS only on UVB irradiation. Since ubiquinone generated ROS depending on the irradiation dose at both UVA and UVB irradiation wavelengths, it is clear that ubiquinone may exhibit direct phototoxicity by type I photochemical reaction due to a wide range of UV light. became. It was clarified that the ubiquinol carboxylic acid ester derivative had low ROS generation at both UVA irradiation and UVB irradiation wavelengths (Fig. 8), and that direct phototoxicity due to UV light could be avoided.

Example 7
Singlet oxygen generation by type II photochemical reaction in UVA irradiation and UVB irradiation Prepare a sample test solution in the same manner as in Example 5 above, dispense 200 μL / well of the test solution into a 96-well plate, and first UVA. (UV Crosslinker CL-1000L 365 nm, UVP) ^{was irradiated up to 10 J / cm 2} , followed by UVB (UV Crosslinker CL-1000M 302 nm, UVP) up to 3 J / cm ² . The absorbance at 440 nm was measured over time to evaluate the production of singlet oxygen. As shown in FIG. 9, the positive control quinine produced singlet oxygen only in UVB irradiation. Since ubiquinone produced singlet oxygen depending on the irradiation dose at both UVA and UVB wavelengths, ubiquinone may exhibit indirect phototoxicity due to type II photochemical reactions due to a wide range of UV light. It became clear. It was clarified that the ubiquinol carboxylic acid ester derivative produced low singlet oxygen at both wavelengths of UVA irradiation and UVB irradiation (Fig. 9), and that indirect phototoxicity due to UV light could be avoided.

Example 8
Preparation of cationic ubiquinol carboxylic acid ester and anionic counterion mixture and particle size of the aqueous solution A mixture of compound 1 and sodium taurocholate having a molar ratio of 1: 1 to 1:10 was prepared with a methanol solution or an aqueous solution. A powder mixture was obtained by drying under reduced pressure or freeze-drying. The particle size of the aqueous solution of the mixture (compound 1 equivalent concentration 22.5 mmol / L) was measured at Zetasizer Nano ZS (MALVERN, Worcestershire, UK) at 25 ° C. An aqueous solution of a mixture having a mixing ratio of 1: 2.5 or more gave a solution having an average particle size of 5 nm (Fig. 10). The aqueous solution of the mixture was shown to have low phototoxicity even under artificial sunlight irradiation (Tables 7 and 8). It is clear that the mixture of Compound 1 and sodium taurocholate provides a clear mixed micelle with low phototoxicity and particle size on the order of nm.

Example 9
Preparation of anionic ubiquinol carboxylic acid ester and cationic counterion mixture and particle size of the aqueous solution A mixture of compound 9 and meglumine in a molar ratio of 1: 1 to 1:10 is prepared with a methanol solution or an aqueous solution, and the pressure is reduced. A powder mixture is obtained by bottom drying or freeze drying. A solution in which water is added to a mixture having a mixing ratio of 1: 2 or more (compound 9 equivalent concentration 23.5 mmol / L) gives a solution having an average particle size of 9 nm (FIG. 11). The aqueous solution of the mixture was shown to have low phototoxicity even under artificial sunlight irradiation (Tables 7 and 8). It is clear that the mixture of compound 9 and meglumine provides a clear mixed micelle with low phototoxicity and particle size on the order of nm.

Example 10
Evaluation of Deliverability of Ubiquinol to Human Epidermal Keratinocytes <br /> The purpose of clarifying that the compound of the present invention can function as a ubiquinol delivery agent having high photostability and low phototoxicity even in an administration form in which shading is difficult. Therefore, assuming skin administration, the ubiquinol delivery property of the compound of the present invention in human epidermal keratinocytes was evaluated. Human epidermal keratinized cell lines (HaCaT cells) were seeded on a 12-well plate (1.0 × 10 ⁵ cells / well), cultured for 24 hours, and then the test compound (10 μM) was dissolved in a medium of 10% FBS and added to the cells. The intracellular ubiquinol and ubiquinone concentrations were measured by LC / MS / MS over time. Extraction method: Add 1 ml of phosphate buffer to each cultured cell, scrape, add methanol (200 μl) and n-hexane (600 μl) to the cell homogenate solution (200 μl) obtained by sonicating, stir, and then stir at 4 ° C., 3000. Centrifugation was carried out at rpm for 10 minutes, 500 μl of the supernatant was concentrated with nitrogen gas, and the residue was redissolved in methanol (50 μl) to prepare an LC / MS / MS sample. The protein concentration was measured using the BCA protein assay kit (Thermo Fisher Scientific). The intracellular ubiquinol and ubiquinone when compound 9 and oxidized ubiquinone were added are shown in FIGS. 12A and 12B, respectively. Both ubiquinol and ubiquinone increased over time due to drug addition. It was clarified that the intracellular ubiquinol remained at a high concentration and the AUC increased 20 times by the administration of Compound 9 as compared with the administration of oxidized ubiquinone, and the intracellular ubiquinol functioned as an excellent ubiquinol delivery agent. It is clear that the compound of the present invention can function as a ubiquinol delivery agent having high photostability and low phototoxicity even in an administration form in which light shielding is difficult in skin administration.

Example 11
Evaluation of Deliverability of Hydroponic Counterion Mixture of Anionic Ubiquinol Carboxyl Ester after Intravenous Administration to Rats The mixture of compound 9 and meglumine shown in Example 9 (compound 10) is water soluble. It was highly potent and dissolved quickly when water and isotonic buffer were added. This solution was intravenously administered to male SD rats (8 weeks old), and the plasma ubiquinol concentration was measured over time by the method shown in Example 10. Plasma ubiquinol levels increased rapidly from 5 minutes after administration at both administration concentrations of ubiquinone equivalent 1 mg / kg rat body weight and 5 mg / kg rat body weight (Fig. 13). It is clear that a cationic counterion mixture of anionic ubiquinol carboxylic acid esters can also function as a ubiquinol delivery agent when administered intravenously.

Example 12
The effect of ubiquinol carboxylic acid ester on changes in human epidermal keratinocyte response with aging in hydrogen peroxide stimulation of human epidermal keratinocytes was evaluated.
Evaluation of inhibitory effect of ubiquinol carboxylic acid ester on cytotoxicity of human epidermal keratinized cells by H ₂ O ₂ stimulation <br /> Test compound: 0.1% polyoxyethylene (40) hardened castor oil (Nikko Chemicals), 1% It was dissolved in an FBS-free DMEM medium containing ethanol (Fujifilm Wako) and 0.05% glycerin (Fujifilm Wako) to prepare a test compound solution. Human epidermal keratinized cell lines (HaCaT cells) were ^{seeded on a 24-well plate (5.0 × 10 4} cells / well) and cultured for 24 hours, the medium was replaced with a test compound solution, and the cells were further cultured for 24 hours. The test solution _{was replaced with FBS-free medium to which H 2} O _{2 was} not added or added, and after culturing for 24 hours, the cell viability assay was measured using the cell titer blue viability assay (Promega). As shown in FIG. 14, the survival rate of the H ₂ O ₂ stimulation HaCaT cells were significantly reduced compared to the non-H ₂ O ₂ stimulation HaCaT cells viability by administration of ubiquinone or ubiquinol-carboxylic acid ester (Compound 9) Was significantly higher.

Example 13
Evaluation of intracellular ubiquinone and ubiquinol delivery by ubiquinol carboxylic acid ester to H ₂ O ₂ stimulated human epidermal keratinized cells <br /> Test compound was 0.1% polyoxyethylene (40) hardened castor oil (Nikko Chemicals), 1 It was dissolved in an FBS-free DMEM medium containing% ethanol (Fujifilm Wako) and 0.05% glycerin (Fujifilm Wako) to prepare a test compound solution. Human epidermal keratinized cell lines (HaCaT cells) were ^{seeded on a 24-well plate (5.0 × 10 4} cells / well) and cultured for 24 hours, the medium was replaced with a test compound solution, and the cells were further cultured for 24 hours. The test solution _{was replaced with an FBS-free medium to which H 2} O _{2 was} not added or was added, and after culturing for 24 hours, an LC / MS / MS measurement sample was prepared by the same extraction operation as in Example 10, and LC was prepared by the same method as in Example 2. / MS / MS was measured. Ubiquinone was significantly higher than the control by the administration of Compound 9, the influence of H ₂ O ₂ significantly different not added or added seen not H ₂ O ₂ added was observed (Fig. 15A) .. Ubiquinol was significantly higher than the control with ubiquinone administration and compound 9 administration (Fig. 15B). Ubiquinol From what has been reduced either by H ₂ O ₂ added in comparison with the H ₂ O ₂ was not added, the causative oxidative stress under aging believed ubiquinol is lowered to function as an antioxidant.

Example 14
Suppressive effect of ubiquinol carboxylic acid ester on promotion of MMP-1 mRNA expression by _{H 2} O ₂ stimulation of human epidermal keratinocytes <br /> Real-time reverse transcription polymerase linkage of mRNA level of matrix metalloproteinase-1 (MMP-1) It was analyzed by reaction (RT-PCR). The primer sets for GAPDH and MMP-1 are shown in Table 2. Preparation of the test compound solution was described in Example 13. HaCaT cells were seeded on a 6-well plate (5.0 × 10 ⁵ cells / well) and cultured for 24 hours, the medium was replaced with a test compound solution, and the cells were further cultured for 24 hours. The test solution _{was replaced with FBS-free medium to which H 2} O _{2 was} not added or was added, and the cells were collected after culturing for 24 hours. RNase inhibitor-added RNA later solution (Thermo Fisher Scientific, Waltham, Massachusetts, USA) stored overnight at 4 ° C, and total RNA was extracted with an RNA extraction kit (High Pure RNA Isolation Kit, Roche, Basel, Switzerland). CDNA was synthesized using 500 ng of RNA and the ReverTra Ace qPCR RT kit (TOYOBO, Osaka, Japan). A Light Cycler 480 system (Roche, Basel, Switzerland) was used for mRNA quantification. Primer-Probe mix, Light Cycler 480 Probes Master (Roche), and 1 μL cDNA template were used. The standard PCR conditions are 95 ° C (10 minutes) for one cycle, 95 ° C (10 seconds), 60 ° C (30 seconds), 72 ° C (1 second) for 45 cycles, and 50 ° C (30 seconds) for one cycle. rice field. The mRNA expression level was determined by dividing by the amount of GAPDH in each sample. MMP-1 mRNA in H ₂ O _{2 treated HaCa T cells was significantly higher than that in H 2} O ₂ untreated cells, and administration of Compound 9 significantly reduced MMP-1 mRNA (Fig. 16). It was shown that H ₂ O ₂ treatment can suppress the development of wrinkles in the aging model.

Real-time RT-PCR primer set

The following is a formulation example in which the ubiquinol carboxylic acid ester derivative according to the present invention is blended with an external preparation for skin.
[Toner]
A solution in which the following components (3), (4) and (8) to (10) are mixed and dissolved, and a solution in which the following components (1), (2), (5) to (7) and (11) are mixed and dissolved. And were mixed to make it uniform, and a lotion was obtained.
(Ingredient) (%)
(1) Glycerin 5.0
(2) 1,3-butylene glycol 6.5
(3) Polyoxyethylene (20EO) sorbitan 1.2
Monolauric Acid Ester (4) Ethyl Alcohol 8.0
(5) Ubiquinol Carboxylate Derivative 0.05
(6) Lactic acid 0.05
(7) Sodium lactate 0.1
(8) Paramethoxycinnamate-2-ethylhexyl 3.0
(9) Preservatives Appropriate amount (10) Fragrances Appropriate amount (11) Purified water remaining amount

[Oil-type emulsion in water]
The following components (8) to (9) were added to the component (12) and swollen, and then the component (10) was added and mixed, and heated to 70 ° C. to prepare an aqueous phase. The following components (1) to (6) were heated to 70 ° C., and this was added to the aqueous phase to emulsify. The emulsion was cooled to room temperature, the following components (7), (11) and (13) were added and mixed uniformly to obtain an emulsion.
(Ingredient) (%)
(1) Polyoxyethylene (10EO) sorbitan 1.0
Monostearate (2) Polyoxyethylene (60EO.) Sorbitol 0.5
Tetraoleate (3) Glyceryl monostearate 1.0
(4) Stearic acid 0.5
(5) Behenyl alcohol 0.5
(6) Squalene 8.0
(7) Ubiquinol Carboxylate Derivative 0.1
(8) Preservative 0.1
(9) Carboxyvinyl polymer 0.1
(10) Sodium hydroxide 0.05
(11) Ethyl alcohol 5.0
(12) Remaining amount of purified water (13) Appropriate amount of fragrance

Claims (7)

General formula (1)

(In the formula, R ₁ and R ₂ mean hydrogen atoms or substituents selected from N-acyl glycine, N-alkyl glycine, N, N-dialkyl glycine, acyl, dicarboxylic acid hemiesters and salts thereof, respectively, and R. _At least one of 1, R ₂ is glycine, N-acyl glycine, N-alkyl glycine, N, N-dialkyl glycine, acyl, dicarboxylic acid hemiester, and salts thereof. N means an integer of 1 to 10. A ubiquinol delivery agent applied under light exposure, which comprises one of a carboxylic acid ester derivative of ubiquinol represented by) or a salt thereof. In the delivery agent according to claim 1, R ₁ and R ₂ in the above general formula (1) mean a hydrogen atom or a dicarboxylic acid double ester, respectively _{, and at least one of R 1} and R ₂ is represented by a dicarboxylic acid double ester. A ubiquinol delivery agent applied under light exposure, which is a ubiquinolcarboxylic acid ester derivative. In the delivery agent according to claim 1 or 2, R ₁ and R ₂ in the above general formula (1) are hydrogen atoms or glycine, N-acyl glycine, N-alkyl glycine, N, N-dialkyl glycine, dicarboxylic acid hemiester, respectively. , And a substituent selected from the salt thereof, and _{at least one of R 1} and R ₂ is represented by glycine, N-acyl glycine, N-alkyl glycine, N, N-dialkyl glycine, dicarboxylic acid hemiester, and a salt thereof. A solid agent and a liquid agent that give a micelle solution having a particle size of 10 nm or less when the ubiquinol carboxylic acid ester derivative is contained in a molar ratio of 1: 2 to 1:10 and a micelle solution having a particle size of 10 nm or less. A ubiquinol delivery agent applied under light exposure. An external preparation for skin containing a ubiquinol carboxylic acid ester derivative represented by the general formula (1) or a salt thereof. An eye drop containing a ubiquinol carboxylic acid ester derivative represented by the general formula (1) or a salt thereof. An eye ointment containing a ubiquinol carboxylic acid ester derivative represented by the general formula (1) or a salt thereof. A ubiquinol carboxylic acid ester derivative represented by the following general formula (4) or a salt thereof.

(In the general formula (4), R ₁ and R ₂ are HOH ₂ C- (CH (OH)) ₄ CH ₂ NHCH ₃ · HOOCCH ₂ CH ₂ CO-, CH ₃ CH ₂ OOCCH ₂ CH ₂ CO- and ( CH ₃ ) ₂ CH00CCH ₂ CH ₂ A carboxylic acid residue selected from the group consisting of CO-)

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