JP2022140917A - Ionic liquid, solvent, preparation, and percutaneous absorption agent - Google Patents

Abstract

To provide a percutaneous absorption agent that can facilitate the penetration of a nucleic acid into skin.SOLUTION: An ionic liquid comprises a cation comprising an ionized amino group of β-alanine ethyl ester, and an anion represented by the general formula R-COO-. In the general formula, R is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkenyl group, where at least one ethylene group constituting the alkenyl group is optionally substituted with a vinylene group.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to ionic liquids, solvents, formulations and transdermal absorbers.

Nucleic acid drugs and mRNA drugs are attracting attention as new drug discovery modalities for diseases that have been difficult to eradicate with low-molecular-weight drugs and antibody drugs. Nucleic acid medicines bind to target RNA or disease-causing proteins and reduce the protein or inhibit its function. mRNA drugs treat diseases by expressing target proteins in cells. Since nucleic acid drugs and mRNA drugs are both composed of nucleic acids, screening for highly effective base sequences is easier than with low-molecular-weight drugs. It has the advantage that it can be manufactured at low cost.

mRNA medicines include mRNA vaccines. Since mRNA vaccines can express various antigenic proteins in vivo by changing the nucleotide sequence of nucleic acids, they are expected to have high therapeutic efficacy and application to various diseases. Furthermore, since mRNA vaccines do not use antigens derived from pathogens and have no risk of genome insertion or mutation, high safety can be expected.

For example, Non-Patent Document 1 discusses anti-tumor immunity by mRNA vaccines. The mRNA vaccine is envisioned for administration by injection. Injectable vaccines are hindered by their widespread use in developing countries where infectious diseases are a serious problem, such as hygiene and the need for medical personnel. Moreover, even in developed countries, there is a risk of needlestick accidents, which are accompanied by pain, which poses a problem that the quality of life (QOL) of patients is greatly reduced. In contrast, transdermal vaccines that are simply applied to the skin are a very attractive alternative due to their superior safety, convenience and minimal invasiveness.

It is not easy to efficiently permeate the hydrophilic nucleic acid from the hydrophobic skin surface into the body. In order to put transdermal vaccines into practical use, it is necessary to develop a transdermal drug delivery system (DDS) that allows nucleic acids to penetrate through the skin surface with high efficiency. Non-Patent Documents 2, 3 and 4 discuss liposomes, electroporation and creams, respectively, as transdermal DDS for nucleic acids. Liposomes as a transdermal DDS are easily taken up by the reticuloendothelial system and accumulate in the liver, which is a barrier to practical use. Electroporation is very expensive and complicated to operate. A drawback of creams is their low drug delivery efficiency.

Non-Patent Document 5 discusses transdermal DDS using a fatty acid-based amino acid ionic liquid.

Stefano Persano, 6 others, "Lipopolyplex potentiates anti-tumor immunity of mRNA-based vaccination", Biomaterials, 2017, 125, 81-89 Barbara Geusens, 5 others, "Ultradeformable cationic liposomes for delivery of small interfering RNA (siRNA) into human primary melanocytes", Journal of Controlled Release, 2013, 2013, 2302 Ｔｏｍｏｙｕｋｉ Ｉｎｏｕｅ，外８名，「Ｍｏｄｕｌａｔｉｏｎ ｏｆ ｓｃｒａｔｃｈｉｎｇ ｂｅｈａｖｉｏｒ ｂｙ ｓｉｌｅｎｃｉｎｇ ａｎ ｅｎｄｏｇｅｎｏｕｓ ｃｙｃｌｏｏｘｙｇｅｎａｓｅ－１ ｇｅｎｅ ｉｎ ｔｈｅ ｓｋｉｎ ｔｈｒｏｕｇｈ ｔｈｅ ａｄｍｉｎｉｓｔｒａｔｉｏｎ ｏｆ ｓｉＲＮＡ」，Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｇｅｎｅ Ｍｅｄｉｃｉｎｅ，２００７年，９（１１），９９４－１００１ M Takanashi, 10 others, "Therapeutic silencing of an endogenous gene by siRNA cream in an arthritis model mouse", Gene Therapy, 2009, 16, 982-989 Ｒａｈｍａｎ Ｍｄ Ｍｏｓｈｉｋｕｒ，外５名，「Ｄｅｓｉｇｎ ａｎｄ Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ Ｆａｔｔｙ Ａｃｉｄ－Ｂａｓｅｄ Ａｍｉｎｏ Ａｃｉｄ Ｅｓｔｅｒ ａｓ ａ Ｎｅｗ “Ｇｒｅｅｎ” Ｈｙｄｒｏｐｈｏｂｉｃ Ｉｏｎｉｃ Ｌｉｑｕｉｄ ｆｏｒ Ｄｒｕｇ Ｄｅｌｉｖｅｒｙ」，ＡＣＳ Ｓｕｓｔａｉｎａｂｌｅ Ｃｈｅｍｉｓｔｒｙ ＆ Ｅｎｇｉｎｅｅｒｉｎｇ，２０２０年，８，３６，１３６６０－１３６７１

Non-Patent Document 5 shows that the above ionic liquid improves the skin permeability of compounds and peptides, but does not examine the skin permeability of nucleic acids. There is a demand for a technique that allows efficient permeation of nucleic acids into the skin.

The present invention has been made in view of the circumstances described above, and an object thereof is to provide an ionic liquid, a solvent, a formulation, and a transdermal absorber capable of improving the permeability of nucleic acids into the skin.

The ionic liquid according to the first aspect of the present invention is
A cation in which the amino group of β-alanine ethyl ester is ionized;
an anion represented by the general formula R ^- COO-;
including.
[In the general formula, R represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkenyl group, and at least one of the ethylene groups constituting the alkenyl group may be substituted with a vinylene group. ]

In this case, the carbon number of R in the general formula is 8 to 22,
You can do it.

Further, R in the general formula has an unsaturated bond,
You can do it.

Further, R in the general formula is a group consisting only of carbon atoms and hydrogen atoms,
You can do it.

The ionic liquid according to the first aspect of the present invention is
is hydrophobic,
You can do it.

The solvent according to the second aspect of the present invention is
The ionic liquid according to the first aspect of the present invention is included.

The formulation according to the third aspect of the present invention is
The ionic liquid according to the first aspect of the present invention is included.

The transdermal absorber according to the fourth aspect of the present invention is
The ionic liquid according to the first aspect of the present invention is included.

The transdermal absorbent according to the fourth aspect of the present invention is
further comprising an oil phase;
You can do it.

The transdermal absorbent according to the fourth aspect of the present invention is
further comprising a nucleic acid;
You can do it.

According to the present invention, it is possible to improve the permeability of nucleic acids into the skin.

FIG. 3 is a diagram showing the amount of single strand DNA (ssDNA) permeated into the skin, quantified in Examples. PBS indicates phosphate buffered saline. IPM stands for isopropyl myristate. IL indicates an ionic liquid. IL-IPM indicates a mixture of IL and IPM. FIG. 4 is a diagram showing the results of qualitative analysis in Examples. (A), (B), (C), (D) and (E) show results for untreated, PBS, IPM, IL and IL-IPM, respectively.

An embodiment according to the present invention will be described with reference to the drawings. The present invention is not limited by the following embodiments and drawings. In the following embodiments, expressions such as "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of". Further, the ^isotopic species of the ^hydrogen atoms present in the molecule of the compound used in the present invention is not particularly limited. (deuterium D).

(Embodiment)
The ionic liquid according to the present embodiment contains a cation obtained by ionizing the amino group of β-alanine ethyl ester shown in Formula 1 below, and an anion represented by the general formula R—COO 2 ^— .

In the general formula, R represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkenyl group, and at least one ethylene group constituting the alkenyl group may be substituted with a vinylene group.

The alkyl group for R may be linear, branched or cyclic. The alkyl group for R preferably has 8 to 22 carbon atoms, more preferably 12 to 22 carbon atoms. For example, as the alkyl group for R, linear alkyl groups such as dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, henicosyl group and docosyl group, and their Examples include branched alkyl groups and cyclic alkyl groups thereof. An amino group, a benzyl group, a halogen atom, etc. can be mentioned as a substituent with which an alkyl group can be substituted. These substituents may be further substituted with a substituent. When the alkyl group is substituted with a substituent, the total number of carbon atoms in the alkyl group and the substituent is preferably 8-22, more preferably 12-22.

Alkenyl groups in R may be either linear or branched. The alkenyl group for R preferably has 8 to 22 carbon atoms, more preferably 12 to 22 carbon atoms. For example, as the alkenyl group for R, linear alkenyl groups such as dodecenyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icosenyl group, henicosenyl group and docosenyl group, and these can be exemplified by a branched alkenyl group of Substituents that can be substituted on the alkenyl group include an amino group, a benzyl group, a halogen atom (eg, a fluorine atom), and the like. These substituents may be further substituted with a substituent. When the alkenyl group is substituted with a substituent, the total number of carbon atoms in the alkenyl group and the number of carbon atoms in the substituent is preferably 8-22, more preferably 12-22. At least one of the alkenyl groups in R may be substituted with a vinylene group to form a polyene structure. The number of double bonds in the polyene structure is preferably 2-6, more preferably 2-4. The positions of the double bonds are not particularly limited, but it is preferable that the double bonds are arranged with at least two single bonds interposed therebetween.

R is preferably a group having an unsaturated bond, more preferably a substituted or unsubstituted alkenyl group or a group having a substituted or unsubstituted polyene structure. In addition, the alkyl group, alkenyl group, and group having a polyene structure in R may be substituted with a substituent, but even in that case, R preferably consists only of carbon atoms and hydrogen atoms. That is, when the alkyl group, alkenyl group and polyene structure are substituted with substituents, the substituents are preferably composed only of carbon atoms and hydrogen atoms.

Examples of R—COO 2 ^— include carboxylate ions in which a hydrogen ion is dissociated from the carboxy group of a fatty acid, or derivatives thereof. A fatty acid that produces a carboxylate ion may be a saturated fatty acid or an unsaturated fatty acid. Examples of saturated fatty acids include myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0) and lauric acid (C12:0). , oleic acid (C18:1), linoleic acid (C18:2), α-linolenic acid (C18:3), γ-linolenic acid (C18:3), arachidonic acid (C20:4), icosapentaenoic acid (C20: 5), docosahexaenoic acid (C22:6) and erucic acid (C22:1). Here, the numbers in parentheses are the number of carbon atoms and the number of double bonds in each fatty acid. For example, (C18:2) of linoleic acid indicates that the number of carbon atoms is 18 and the number of double bonds is two.

Specific examples of anions in the ionic liquid according to the present embodiment include carboxylate ions of linoleic acid, oleic acid, acetic acid, and stearic acid.

Next, preferable characteristics of the ionic liquid according to this embodiment will be described. The ionic liquid according to the present embodiment is preferably hydrophobic. Here, "hydrophobic" means that it dissolves in isopropyl myristate (IPM) in an amount of 0.1% by mass or more.

The ionic liquid according to the present embodiment preferably dissolves in the IPM in an amount of 0.1% by mass or more (it is hydrophobic), more preferably in an amount of 5% by mass or more, and in an amount of 20% by mass or more. is more preferred. Here, dissolving the ionic liquid in the IPM includes forming a homogeneous system and dissolving the ionic liquid in the IPM, as well as forming reverse micelles and dissolving. The fact that the ionic liquid forms reverse micelles in the IPM can be confirmed by observing a peak in the particle size distribution measured by DLS.

The anion in the ionic liquid according to this embodiment has a structure represented by the above general formula, and since R contains an alkyl group or an alkenyl group, it can be easily made hydrophobic. Hydrophobic ionic liquids exhibit high compatibility with hydrophobic solvents, oil bases, hydrophobic drugs, and the like, and can be easily mixed with them. In addition, ionic liquids have high permeability to the permeation barrier of the stratum corneum and can be easily percutaneously absorbed. Hydrophobic ionic liquids are therefore particularly useful for transdermal DDS.

The ionic liquid according to the present embodiment preferably has a melting point of 100° C. or lower, more preferably 60° C. or lower, and even more preferably 40° C. or lower. Here, the melting point is determined by differential scanning calorimetry. An ionic liquid having a melting point within the above range can be suitably used as a solvent because it exhibits a liquid state over a wide temperature range.

Another embodiment provides a solvent comprising the ionic liquid according to the present embodiments. The solvent according to this embodiment may be composed only of the ionic liquid according to this embodiment, or may contain other solvents. Other solvents are not particularly limited and can be appropriately selected from known solvents.

The anion in the ionic liquid according to the present embodiment has a structure represented by the above general formula, and contains an alkyl group or an alkenyl group in R, so is highly compatible with hydrophobic substances. In addition, since the ionic liquid has carboxylate ions and cations, it behaves like a surfactant with respect to hydrophilic substances. Therefore, the ionic liquid according to the present embodiment can be uniformly mixed in combination with a hydrophobic solvent or a hydrophilic solvent, and can dissolve both hydrophobic and hydrophilic solutes. For example, if the ionic liquid according to the present embodiment is used as a solvent, it becomes possible to dissolve a poorly soluble pharmacologically active substance. By using the ionic liquid, it is possible to realize a solvent exhibiting high solubility regardless of the solvent and solute to be combined. In addition, it is possible to realize formulations and percutaneous absorbable agents with excellent biocompatibility.

In another embodiment, there is provided a formulation comprising an ionic liquid according to this embodiment. In addition to the ionic liquid according to the present embodiment, the formulation according to the present embodiment may contain ingredients commonly used in formulations, such as active ingredients, additives, excipients, and bases. Moreover, the form of the formulation according to the present embodiment is not particularly limited, and may be, for example, any form such as an internal medicine, an external medicine, and an injection.

The ionic liquid according to the present embodiment is a combination of an anion having a fatty acid as a basic skeleton and a cation ionized from β-alanine ethyl ester, and both basic skeletons are bio-related substances. Therefore, it has low toxicity and high biocompatibility. In addition, the ionic liquid according to the present embodiment has high compatibility with hydrophobic substances because it contains an alkyl group or an alkenyl group in R, and has a carboxylate ion and a cation, so it is not compatible with hydrophilic substances. It exhibits surfactant-like behavior. Therefore, by using the ionic liquid according to the present embodiment, it is possible to easily prepare a safe preparation having the advantages of the ionic liquid.

In another embodiment, there is provided a percutaneous absorber containing the ionic liquid according to this embodiment. The ionic liquid according to the present embodiment has low toxicity and high biocompatibility, and exhibits hydrophobicity by containing an alkyl group or an alkenyl group in the molecule, so that it can permeate the stratum corneum of the skin. . Therefore, the ionic liquid according to the present embodiment has the advantages of the ionic liquid, is safe, and can realize a transdermal absorbent that exhibits excellent transdermal absorbability.

The transdermal absorber according to this embodiment preferably further contains an oil phase. The oil phase may be liquid or solid with fluidity. The base of the oil phase is not particularly limited as long as it is an oily base that is acceptable for use in pharmaceutical preparations, preferably for use in pharmaceutical preparations for transdermal delivery. Either an oil that is liquid at room temperature (25° C.) or a fat that is solid at room temperature can be used. There are no restrictions on the origin of the raw material for the oil phase, and for example, both natural products and synthetic products can be used. The oil phase includes soybean oil, cottonseed oil, rapeseed oil, sesame oil, corn oil, peanut oil, safflower oil, sunflower oil, olive oil, rapeseed oil, perilla oil, fennel oil, cacao oil, cinnamon oil, peppermint oil and bergamot. It may be vegetable, such as oil, or animal, such as beef tallow, pork oil and fish oil. The oil phase may also be neutral lipids such as glycerides, triolein, trilinolein, tripalmitin, tristearin, trimyristin, triarachidonin, or synthetic lipids.

The oil phase may be a sterol derivative such as cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmidate and cholesteryl arachidate, IPM, octyldodecyl myristate, cetyl myristate, ethyl oleate, Long chain fatty acid esters such as ethyl linoleate, isopropyl linoleate, isopropyl palmitate and butyl stearate may also be used. In addition, the oil phase may be a carboxylic acid ester such as ethyl lactate, cetyl lactate, triethyl citrate, diisopropyl adipate, diethyl sebacate, diisopropyl sebacate and cetyl 2-ethylhexanoate, petrolatum, paraffin squalane. and hydrocarbons such as vegetable squalane, and silicones. The oil base may be used singly or in combination of two or more.

As the oil phase, triglyceride or a food oil containing triglyceride as a main component can be used. Practically, soybean oil is preferable, and highly purified soybean oil is more preferable. Neutral lipids or long-chain fatty acid esters can also be suitably used, with long-chain fatty acid esters being more preferred, and IPM being even more preferred.

The content of the oil phase in the transdermal absorbent according to the present embodiment varies depending on the type of oil component, other constituents, etc., but is preferably 50 to 99.5% by mass, more preferably 80 to 90% by mass. .

The transdermal absorber according to this embodiment may further contain a skin penetration enhancer. As the skin permeation enhancer, any compound that is recognized to promote absorption in the skin can be used. Examples include fatty acids having 6 to 20 carbon atoms, aliphatic alcohols, fatty acid amides, fatty acid ethers, aromatic Organic acids, aromatic alcohols, aromatic organic acid esters or ethers, lactic acid esters, acetic acid esters, monoterpene compounds, sesquiterpene compounds, glycerin fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters , polysorbate, polyethylene glycol fatty acid esters, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ethers, sucrose fatty acid esters and vegetable oils.

A preferred skin penetration enhancer is glycerin fatty acid ester, which is an ester of glycerin and fatty acid. The glycerin may be polyglycerin. Glycerin is preferably monoglycerin, diglycerin or triglycerin.

Fatty acids include caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, undecylenic acid, ricinoleic acid, oleic acid, linoleic acid, linolenic acid, erucic acid, beef tallow, lard , coconut oil, palm oil, palm kernel oil, olive oil, rapeseed oil, rice bran oil, soybean oil, and castor oil.

A glycerin fatty acid ester is a monoglycerin fatty acid ester, a diglycerin fatty acid ester, or a triglycerin fatty acid ester. Specifically, glycerin fatty acid esters include diglyceryl monostearate, glyceryl monostearate, glyceryl myristate, tri(caprylic-capric)glyceryl, glyceryl monooleate, monoolive fatty acid glyceryl (glyceryl oleate or MGOL -70), diglyceryl monooleate, glyceryl monocaprylate (Capmul 808G, manufactured by ABITEC), glyceryl monocaprate, glyceryl caprate, glyceryl caprylate, monodiglyceride caprate, diglyceride caprate, glyceryl monolaurate , glyceryl monoundecylenate, glyceryl monopalmitate, and glyceryl monomyristate. Preferably, the skin penetration enhancer is glyceryl oleate.

Skin penetration enhancers may be monohydric or polyhydric alcohols. Examples of monohydric alcohols include lower alcohols such as methanol, ethanol, 1-propanol and 2-propanol, higher alcohols, geraniol, menthol, borneol, isoborneol, nerol, citronellol, fenchyl alcohol, carveol and neomenthol. terpene having a hydroxyl group such as

Examples of polyhydric alcohols include ethylene glycol, propylene glycol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1, 5-pentanediol, 1,2-hexanediol, glycerin, dipropylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol 400 and the like.

Examples of higher alcohols include saturated alcohols having 8 to 18 carbon atoms such as octanol, nonanol, decanol, undecyl alcohol, lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, heptadecyl alcohol, and stearyl alcohol; and unsaturated alcohols having 8 to 18 carbon atoms such as oleyl alcohol, linoleyl alcohol and linolenyl alcohol.

The content of the skin penetration enhancer in the transdermal absorber according to the present embodiment is appropriately determined from the viewpoint of improving the skin penetration of nucleic acids and the like. , 3 to 16% by weight, 4 to 15% by weight or 5 to 10% by weight,

As shown in the following examples, the transdermal absorber according to this embodiment can allow nucleic acids, which hardly penetrate the skin, to penetrate into the body through the skin. Therefore, the transdermal absorber is suitable for transdermal DDS of nucleic acids. The percutaneous absorption agent may further contain nucleic acids such as nucleic acid drugs and mRNA drugs. Nucleic acids may be DNA or RNA. Nucleic acids may be single-stranded or double-stranded. Nucleic acids include antisense and siRNA that target RNA, decoy nucleic acids that bind to transcription factors and suppress the transcription stage, aptamers that bind to proteins and inhibit their function, and Toll-like receptor 9 to act on immune system-activating CpG oligodeoxynucleotides and the like. Although the base length of the nucleic acid is not particularly limited, it is, for example, 10-80 bases, 15-50 bases, or 20-30 bases. Nucleic acids may include nucleic acids modified in their base sequence.

The dosage form of the percutaneous absorption agent is not particularly limited, and may be any dosage form such as a liquid (lotion, spray, etc.), ointment, cream, gel, milky lotion, patch, and the like. In addition, the transdermal absorber can contain a base in addition to the ionic liquid according to this embodiment. The base is appropriately selected from those commonly used in transdermal absorbers. In addition, the percutaneous absorber may include stabilizers, preservatives, solubilizers, emulsifiers, suspending agents, and pH adjusters used in pharmaceuticals such as external preparations, quasi-drugs, cosmetics, etc., if necessary. Additives such as agents and antioxidants may also be added.

The dosage of the percutaneous absorption agent according to this embodiment can be adjusted according to the patient's age, symptoms, type of applicable disease, and the like. For example, administration is usually carried out over several weeks to several months so that the amount of nucleic acid per administration per adult is 1 μg to 30 mg, preferably 10 μg to 10 mg, more preferably 30 μg to 3 mg. can be done.

Vertebrates are preferable, and mammals are more preferable as the application target of the transdermal absorbent according to the present embodiment. Mammals include, for example, humans, chimpanzees and other primates, domestic animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs, rats, mice and guinea pigs, pet animals and laboratory animals. mentioned. Humans are particularly preferred as mammals.

The transdermal absorbent according to this embodiment can be used for diseases that can be treated with nucleic acids or nucleic acid medicines. The disease is a disease that can be treated or prevented, or that the progression of the disease can be halted or inhibited, in particular by targeting RNA. Examples of diseases include skin diseases such as skin cancer, rheumatoid arthritis, and atopic dermatitis. In addition, the transdermal absorbable agent according to this embodiment is also suitable for transdermal DDS of mRNA vaccines.

In another embodiment, there is provided a method of treating the above disease, comprising using the above transdermal agent containing a therapeutically effective amount of nucleic acid in a subject in need thereof. The therapeutically effective amount of the nucleic acid is the amount indicated as the dose of the nucleic acid in the transdermal absorber, and is the dose set according to the disease. Also, in another embodiment, the above transdermal agent for treatment of the above diseases is provided.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited by the examples.

[Preparation of ionic liquid and sample]
An ionic liquid was prepared as follows. β-alanine was dissolved in ethanol, and thionyl chloride (1.5 molar equivalents) was added drop by drop while cooling with ice. After stirring for 10 minutes, the mixture was allowed to stand overnight at room temperature to allow the esterification reaction to proceed. By removing the solvent from this reaction solution with a rotary evaporator, white crystals containing Intermediate 1 (β-alanine ethyl ester chloride) were obtained. After dissolving the white crystals in Milli-Q water, diethyl ether was added to separate the phases, and the aqueous phase containing Intermediate 1 was recovered. This aqueous phase was neutralized by adding ammonia (2 molar equivalents) dropwise to Intermediate 1. Further, 50 mL of diethyl ether was added, and the mixture was stirred at room temperature for 2 hours to recover the diethyl ether phase containing intermediate 2 (β-alanine ethyl ester). After sufficient dehydration with sodium sulfate, intermediate 2 was obtained by solvent removal with a rotary evaporator and a vacuum pump. Linoleic acid (1 molar equivalent) was added to Intermediate 2, and the mixture was stirred overnight at 45°C in a dry nitrogen atmosphere shielded from light to react, thereby obtaining an ionic liquid.

A reagent was mixed with the composition shown in Table 1 to prepare a sample. SEQ ID NO: 1 shows the nucleotide sequence of FITC-labeled ssDNA (manufactured by Fasmac).

[Quantitative analysis]
Pig skin (Yucatan micropig skin, manufactured by Charles River Japan) was thawed, fat was removed, and cut into pieces of about 2 cm x 2 cm. 4 mL of PBS was added to the receiver phase of a vertical Franz cell (effective area: 0.785 cm ² ), and after setting the porcine skin, 1 mL of PBS was added so as to remove air from the Franz cell. The receiver phase was kept at 32.5° C. and stirred with a stirrer, and 400 μL of sample was added to each donor phase. After 48 hours, the pig skin was taken out and washed with PBS and 70% ethanol. The skin tissue was cut into small pieces, immersed in 500 μL of PBS, and shaken for 24 hours. Using a fluorometer, the fluorescence intensity (λex=490 nm, λem=520 nm) of each extract was measured, and the amount of ssDNA permeated into the skin was calculated.

[Qualitative analysis]
The porcine skin was exposed to 400 μL of the sample in the same manner as in the above quantitative analysis, and after 48 hours, the skin was removed and immersed in 4% paraformaldehyde/phosphate buffer for 4 hours or longer to fix the cells. Cut out to fit the effective area, and after cutting in half, O.D. C. T. Cryoembedded with Compound. After cutting into pieces of 20 μm with a cryostat microtome to prepare skin slices, observation was performed with a fluorescence microscope.

[result]
The results of a quantitative analysis of ssDNA skin penetration are shown in FIG. All samples except PBS showed high skin permeation. According to the qualitative fluorescence microscopy image shown in FIG. 2, deep skin fluorescence was observed in the samples containing the ionic liquid compared to the IPM-only samples. These results suggested that the ssDNA remained in the stratum corneum in the IPM-only sample, and that the use of IL promoted penetration into the deep skin.

The embodiments described above are for the purpose of explaining the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of equivalent inventions are considered to be within the scope of the present invention.

The present invention is suitable for transdermal formulations containing nucleic acids as active ingredients.

Claims (10)

A cation in which the amino group of β-alanine ethyl ester is ionized;
an anion represented by the general formula R ^- COO-;
ionic liquids, including
[In the general formula, R represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkenyl group, and at least one of the ethylene groups constituting the alkenyl group may be substituted with a vinylene group. ] The carbon number of R in the general formula is 8 to 22,
The ionic liquid according to claim 1. R in the general formula has an unsaturated bond,
The ionic liquid according to claim 1 or 2. R in the general formula is a group consisting only of carbon atoms and hydrogen atoms,
The ionic liquid according to any one of claims 1 to 3. is hydrophobic,
The ionic liquid according to any one of claims 1 to 4. A solvent containing the ionic liquid according to any one of claims 1 to 5. A formulation comprising the ionic liquid according to any one of claims 1 to 5. A transdermal absorbent comprising the ionic liquid according to any one of claims 1 to 5. further comprising an oil phase;
The percutaneous absorber according to claim 8. further comprising a nucleic acid;
The percutaneous absorber according to claim 8 or 9.

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