Method for purifying a composition

Abstract

A method for purifying a composition that comprises a step for dissolving the composition containing a compound represented by formula(1) [in formula (1): R1 represents -N(R2)-R2 (wherein R2 represents a C1-C4 alkyl group); R3 and R4 represent a C3-C8 alkanediyl group; R5 represents a hydroxyl group; R6 represents -R7-OH (wherein R7 represents a C4-C12 alkanediyl group) or a hydrogen atom; and n is an integer of 0 or 1] in an aqueous layer and performing liquid-liquid extraction, wherein an oil layer used in the liquid-liquid extraction contains one or more liquids selected from the group consisting of a ketone liquid, an ester liquid and an ether liquid each having a solubility parameter (SP value) of 14.8-20.5 (MPa1 / 2).

Description

Technical Field

[0001] The present invention relates to a method for purifying a composition. More specifically, the present invention relates to a method for purifying a composition, a composition, a compound, and lipid nanoparticles. This application claims priority based on Japanese Patent Application No. 2021-204594 filed in Japan on December 16, 2021, and incorporates its content herein by reference.

Background Art

[0002] Most of the drugs administered to a living body are metabolized in the liver and excreted from the kidneys before reaching the target sites such as receptors and genes (on-target). In addition, they may act on off-target sites and cause side effects.

[0003] A drug delivery system (DDS) is a drug discovery technology for controlling the pharmacokinetics of a drug in order to maximize the effect of the drug. By DDS, a drug can be delivered to a target site (on-target) at an appropriate concentration and for an appropriate period.

[0004] As DDS, lipid nanoparticles encapsulating a drug are known. For example, Patent Document 1 and Non-Patent Document 1 describe lipid nanoparticles encapsulating siRNA. In addition, Patent Document 1 and Non-Patent Document 1 describe that lipid nanoparticles encapsulating siRNA are useful for cancer immunotherapy by knockdown using siRNA targeting immunosuppressive factors of dendritic cells as a target site.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, the inventors of the present invention have found that the lipids constituting the lipid nanoparticles described in Patent Document 1 and Non-Patent Document 1 contain impurities, and these impurities have an adverse effect on the in vivo dynamics of the lipid nanoparticles encapsulating siRNA to the target site, and also reduce the knockdown efficiency of the target gene. Therefore, an object of the present invention is to provide a technique for removing impurities contained in a composition containing lipids.

Means for Solving the Problems

[0008] The present invention includes the following embodiments. [1] A method for purifying a composition, comprising a step of dissolving a composition containing a compound represented by the following formula (1) in an aqueous layer and performing liquid-liquid extraction to purify the compound represented by the following formula (1), wherein the oil layer used in the liquid-liquid extraction has a solubility parameter (SP value) of 14.8 to 20.5 (MPa 1 / 2 ), and contains one or more liquids selected from the group consisting of ketone-based liquids, ester-based liquids, and ether-based liquids.

Chemical Formula

Advantages of the Invention

[0009] According to the present invention, it is possible to provide a technique for removing impurities contained in a composition containing a lipid.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0011] Hereinafter, embodiments will be shown to explain the present invention in more detail, but the present invention is not limited to the following embodiments at all.

[0012] [Purification Method of Composition] In one embodiment, the present invention provides a method for purifying a composition, which includes dissolving a composition containing a compound represented by the following formula (1) in an aqueous layer and performing liquid-liquid extraction to purify the compound represented by the following formula (1), wherein the oil layer used in the liquid-liquid extraction contains one or more liquids selected from the group consisting of ketone-based liquids, ester-based liquids, and ether-based liquids having a solubility parameter (SP value) of 14.8 to 20.5 (MPa 1 / 2 ).

[0013]

Chemical Formula

[0014] In formula (1), R 1 represents -N(R 2 )-R 2 (wherein R 2 each independently represents a C1-C4 alkyl group), R 3 represents a C3-C8 alkanediyl group, R 4 represents a C3-C8 alkanediyl group, R 5 represents a hydroxyl group, R 6 represents -R 7 -OH (wherein R 7 represents a C4-C12 alkanediyl group) or a hydrogen atom, and n represents an integer of 0 or 1.

[0015] As described above, the inventors have found that the lipids constituting the lipid nanoparticles described in Patent Document 1 and Non-Patent Document 1 contain impurities, and these impurities have an adverse effect on the in vivo dynamics of lipid nanoparticles encapsulating siRNA to the target site and also reduce the knockdown efficiency of the target gene. The chemical structure of this impurity is difficult to identify and is still unknown.

[0016] However, by the purification method of the present embodiment, the compound represented by the above formula (1) can be purified to remove impurities, and the composition (lipid) described later can be produced. When lipid nanoparticles encapsulating siRNA are prepared with this lipid, compared with the case where the purification method of the present embodiment is not carried out, the in vivo dynamics of the lipid nanoparticles to the target site can be improved, and a decrease in the knockdown efficiency of the target gene can be suppressed. In this specification, improving the in vivo dynamics means that the ratio of the lipid nanoparticles delivered to the target site among the administered lipid nanoparticles increases.

[0017] The composition containing the compound represented by the above formula (1) can be obtained, for example, by the reaction of Scheme (I) shown in FIG. 1. In FIG. 1, R 2 , R 7 are the same as those in the above formula (1), R 2 each independently represents a C1-C4 alkyl group, R 7 each independently represents a C4-C12 alkanediyl group, and R 8 represents a protecting group. In FIG. 1, Compounds 2', 2'', 3', 3'', 4', 4'', 5', 5'' are by-products. However, the inventors have confirmed that even if these compounds are present, when the composition (lipid) described later is produced and lipid nanoparticles encapsulating siRNA are prepared, there is no adverse effect on the in vivo dynamics of the lipid nanoparticles to the target site and the knockdown efficiency of the target gene.

[0018] R 8As the protecting group, those usually used as protecting groups for hydroxyl groups can be appropriately used. Specifically, for example, tert-butyldimethylsilyl group, trimethylsilyl group, triethylsilyl group, benzyl group, tert-butyl group, methoxymethyl group, 2-tetrahydropyranyl group, acetyl group, benzoyl group, etc. can be mentioned.

[0019] Liquid-liquid extraction is a separation and concentration method that utilizes the distribution of solutes between two immiscible liquids. In the purification method of the present embodiment, a composition containing the compound represented by the above formula (1) is dissolved in an aqueous layer, and extraction is performed between this and an oil layer that does not mix with it. As the oil layer used in liquid-liquid extraction, a solubility parameter (SP value) of 14.8 to 20.5 (MPa 1 / 2 ) One or more liquids selected from the group consisting of ketone-based liquids, ester-based liquids, and ether-based liquids are used. Liquid-liquid extraction can be performed under normal conditions. Specifically, for example, after mixing the above aqueous layer and oil layer at room temperature, they are allowed to stand to separate the aqueous layer and the oil layer, and the aqueous layer is recovered. As a result, it can be obtained in the aqueous layer from which impurities have been removed and the compound represented by the above formula (1) has been recovered.

[0020] Here, examples of the ketone-based liquid include cyclohexanone, methyl isobutyl ketone, and diisopropyl ketone. Examples of the ester-based liquid include ethyl acetate and butyl acetate. Examples of the ether-based liquid include diethyl ether, dipropyl ether, cyclopentyl methyl ether, and propylene glycol monomethyl ether acetate. In this specification, a liquid having both a -C(O)- group and an ether bond, such as propylene glycol monomethyl ether acetate, is classified as an ether-based liquid as described above. These liquids may be used alone or in combination of two or more.

[0021] [Composition] In one embodiment, the present invention provides a compound represented by the following formula (2), wherein n is 0, and R 9 are both -R 7 -O-C(O)-R 10and R 5 is a hydroxyl group, the compound (2-1) where n is 0 and R 9 one of which is -R 7 -O-C(O)-R 10 and the other of R 9 is -O-C(O)-R 10 and R 5 is a hydrogen atom, the compound (2-2), and n is 1 and R 9 both are -R 7 -O-C(O)-R 10 and R 5 is a hydroxyl group, the compound (2-3), and provides a composition containing one or more compounds selected from the group consisting of the compound (2-1), the compound (2-2), and the compound (2-3), wherein the total content ratio of the compound (2-1), the compound (2-2), and the compound (2-3) contained in the composition is 90% by mass or more.

[0022]

Chemical formula

[0023] In formula (2), R 1 is -N(R 2 )-R 2 (wherein R 2 each independently represents a C1-C4 alkyl group.), R 3 represents a C3-C8 alkanediyl group, R 4 represents a C3-C8 alkanediyl group, R 5 represents a hydroxyl group or a hydrogen atom, R 9 each independently is -R 7 -O-C(O)-R 10 or -O-C(O)-R 10 (wherein R 7 represents a C4-C12 alkanediyl group, R 10 represents a C4-C25 alkenyl group.), and here, at least one R 9 is -R 7 -O-C(O)-R 10 and n represents an integer of 0 or 1.

[0024] The general formulas of compound (2-1), compound (2-2) and compound (2-3) are shown below.

[0025]

Chemical formula

[0026]

Chemical formula

[0027]

Chemical formula

[0028] The composition of this embodiment is a lipid, and lipid nanoparticles encapsulating a drug can be produced. The composition of this embodiment is the R in the composition containing the compound represented by the above formula (1) purified by the above-described purification method 6 to R 9 (wherein R 9 represents -R 7 -O-C(O)-R 10 or -O-C(O)-R 10 ). Here, R 9 represents -R 7 -O-C(O)-R 10 or -O-C(O)-R 10 ).

[0029] The composition of this embodiment can be obtained, for example, by the reaction of Scheme (II) shown in FIG. 2. Scheme (II) is a reaction consecutive to Scheme (I) shown in FIG. 1. In FIG. 2, R 2 , R 7 are the same as those in the above formula (1), R 2 each independently represents a C1-C4 alkyl group, and R 7 each independently represents a C4-C12 alkanediyl group. R 10 each independently represents a C4-C25 alkenyl group.

[0030] In FIG. 2, compound 6 is an example of the above compound (2-1), compound 6' is an example of the above compound (2-2), and compound 6'' is an example of the above compound (2-3). Here, although compounds 6' and 6'' are by-products, the inventors have confirmed that even if these compounds are present, when lipid nanoparticles encapsulating siRNA are prepared, there is no adverse effect on the in vivo dynamics of the lipid nanoparticles to the target site and the knockdown efficiency of the target gene.

[0031] In FIG. 2, by reacting the composition containing compounds 5, 5', and 5'' with X-C(O)-R 10 (where X is a halogen atom), R 6 is converted to R9 (Here, R 9 represents -R 7 -O-C(O)-R 10 or -O-C(O)-R 10 .) is being converted.

[0032] The composition of this embodiment contains one or more compounds selected from the group consisting of the above compounds (2-1), (2-2), and (2-3). The total content ratio of compound (2-1), compound (2-2), and compound (2-3) contained in the composition is 90% by mass or more, preferably 95% by mass or more, more preferably 98% by mass or more, and still more preferably 99% by mass or more, and the remainder contains impurities. The composition of this embodiment can be obtained by implementing the purification method of the above-described composition to remove impurities.

[0033] In the composition of this embodiment, the content ratio of the above compound (2-1) contained in the composition is preferably 85 to 99% by mass. Also, the total content ratio of the above compound (2-2) and the above compound (2-3) contained in the composition is preferably 0.1 to 15% by mass.

[0034] [Compound] In one embodiment, the present invention provides a compound (2-2) in the above formula (2) where n is 0, one of R 9 is -R 7 -O-C(O)-R 10 and the other of R 9 is -O-C(O)-R 10 and R 5 is a hydrogen atom. The general formula of compound (2-2) is as described above.

[0035] In another embodiment, the present invention provides a compound (2-3) in the above formula (2) where n is 1, both of R 9 are -R 7 -O-C(O)-R 10 and R 5 is a hydroxyl group. The general formula of compound (2-3) is as described above.

[0036] [Lipid Nanoparticle] In one embodiment, the present invention provides lipid nanoparticles encapsulating a drug, which are formed from a composition containing one or more compounds selected from the group consisting of the above compounds (2-1), (2-2) and (2-3), and the total content ratio of compound (2-1), compound (2-2) and compound (2-3) contained in the composition is 90% by mass or more, preferably 95% by mass or more, more preferably 98% by mass or more, and still more preferably 99% by mass or more.

[0037] The lipid nanoparticles herein refer to particles with a particle size of 10 nm to 1,000 nm mainly composed of lipids. Lipid nanoparticles are also referred to as "Lipid Nanoparticle" (LNP).

[0038] The lipid nanoparticles of this embodiment have good in vivo dynamics at the target site because the incorporation of adverse impurities is reduced. Also, when the drug is siRNA, the knockdown efficiency of the target gene is high. The lipid nanoparticles of this embodiment can be formed from a composition obtained by implementing the above-described purification method of the composition to remove impurities.

[0039] As the lipid component of the lipid nanoparticles, only one or more compounds selected from the group consisting of the above compounds (2-1), (2-2) and (2-3) may be used. Generally, for example, one or more lipids selected from the group consisting of phospholipids, glycolipids, sterols, saturated or unsaturated fatty acids, and saturated or unsaturated fatty acid esters are combined to form lipid nanoparticles. The combination of multiple lipids and their blending ratios can be appropriately adjusted according to the purpose.

[0040] Examples of phospholipids and phospholipid derivatives include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramide phosphorylglycerol phosphate, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholine, distearoyl phosphatidylcholine, plasmalogen, phosphatidic acid, etc. These can be used alone or in combination of two or more. The fatty acid residues in these phospholipids are not particularly limited, and examples include saturated or unsaturated fatty acid residues having 12 to 20 carbon atoms. Specifically, acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid can be mentioned. In addition, phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used.

[0041] Examples of glycolipids include glyceroglycolipids (e.g., sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride), sphingoglycolipids (e.g., galactosyl cerebroside, lactosyl cerebroside, ganglioside), etc.

[0042] Examples of sterols include sterols derived from animals (e.g., cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), sterols derived from plants (phytosterols) (e.g., stigmasterol, sitosterol, campesterol, brassicasterol), sterols derived from microorganisms (e.g., thymosterol, ergosterol), etc.

[0043] Examples of saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

[0044] Examples of saturated or unsaturated fatty acid esters include glycerin fatty acid esters in which one or two hydroxyl groups of glycerol are ester-bonded to fatty acids. The fatty acid residues in the glycerin fatty acid esters include, for example, acyl groups derived from saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid. Specifically, dimyristoyl glycerol (DMG), distearoyl glycerol (DSG), etc. can be mentioned.

[0045] The method for producing lipid nanoparticles is not particularly limited. For example, all lipid components are dissolved in an organic solvent such as chloroform, and a lipid film is formed by performing vacuum drying using an evaporator or spray drying using a spray dryer. Then, an aqueous solvent is added to the dried mixture, and further emulsification is performed using an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure jet emulsifier, etc., so that it can be produced. In addition, lipid nanoparticles can also be produced, for example, by the reverse phase evaporation method or the like. When it is desired to control the size of lipid nanoparticles, extrusion (extrusion filtration) can be performed under high pressure using a membrane filter with uniform pore size, etc.

[0046] The size of the dispersed lipid nanoparticles can be appropriately selected according to the purpose. For example, it can be about 60 to 140 nm in average particle diameter, for example, about 80 to 120 nm in average particle diameter, for example, about 20 to 50 nm in average particle diameter. The particle diameter can be measured, for example, by the DLS (dynamic light scattering) method. In the present specification, the average particle diameter of lipid nanoparticles means the number average particle diameter measured by DLS. The measurement by DLS can be performed by a conventional method using a commercially available DLS device or the like. The polydispersity index (PDI) is about 0.05 to 0.1, preferably about 0.06 to 0.08, and more preferably about 0.07.

[0047] The composition of the aqueous solvent (dispersion medium) is not particularly limited, and examples thereof include buffers such as phosphate buffer, citrate buffer, phosphate buffered saline, physiological saline, and media for cell culture. These aqueous solvents (dispersion media) can stably disperse lipid nanoparticles. Furthermore, sugars (aqueous solutions) such as monosaccharides of glucose, galactose, mannose, fructose, inositol, ribose, xylose, disaccharides of lactose, sucrose, cellobiose, trehalose, maltose, trisaccharides of raffinose, melibiose, etc., polysaccharides such as cyclodextrin, sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, maltitol, etc., and polyhydric alcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol, etc. may be added. In order to stably store lipid nanoparticles dispersed in an aqueous solvent for a long period of time, it is preferable to exclude electrolytes in the aqueous solvent as much as possible from the aspect of physical stability such as aggregation inhibition. Also, from the aspect of the chemical stability of the lipid, it is preferable to set the pH of the aqueous solvent from weakly acidic to near neutral (about pH 3.0 to 8.0), and it is preferable to remove dissolved oxygen by nitrogen bubbling or the like.

[0048] When the aqueous dispersion of the obtained lipid nanoparticles is freeze-dried or spray-dried, for example, monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, maltose; trisaccharides such as raffinose, melibiose, and polysaccharides such as cyclodextrin; sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, maltitol; etc. (aqueous solutions of sugars) may be used to improve stability in some cases. Also, when the above aqueous dispersion is frozen, for example, the above-mentioned saccharides, glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, polyhydric alcohols such as 1,3-butylene glycol (aqueous solutions) may be used to improve stability in some cases.

[0049] In the lipid nanoparticles of this embodiment, the encapsulated drug is not particularly limited. For example, in addition to nucleic acids such as siRNA, microRNA, mRNA, plasmid, the active ingredient of any medicine such as an antitumor agent, an anti-inflammatory agent, an antibacterial agent, an antiviral agent, any substance such as saccharides, peptides, low molecular compounds, metal compounds, etc. can be encapsulated in the lipid nanoparticles. These may be encapsulated alone or in combination of two or more.

[0050] siRNA (small interfering RNA) is a short double-stranded RNA consisting of 21 to 23 base pairs, which is involved in RNA interference (RNAi) and suppresses gene expression sequence-specifically by destroying mRNA. Since genes can be knocked down by RNA interference using siRNA, it is expected to be applied in the fields of pharmaceutical use and treatment of cancer and the like. The types of siRNA that can be used are not particularly limited, and any siRNA can be used as long as it can cause RNA interference. Generally, however, a double-stranded RNA of 21 to 23 base pairs having a structure in which the 3' part of the RNA strand protrudes by 2 bases, and each strand has a phosphate group at the 5' end and a hydroxyl group at the 3' end can be used as siRNA. In addition, siRNA in which the hydroxyl group at the 2' position of the ribose backbone is a methoxy group, a fluoro group or a methoxyethyl group and the phosphodiester bond is partially substituted with a phosphorothioate bond is also included.

[0051] The form of the lipid nanoparticles of the present embodiment is not particularly limited, and examples thereof include a form dispersed in an aqueous solvent (for example, water, physiological saline, phosphate-buffered saline, etc.), a form obtained by freeze-drying an aqueous dispersion, and the like.

Examples

[0052] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples. All animal experiments were carried out in accordance with a protocol reviewed by the Institutional Animal Care and Use Committee of Hokkaido University and approved by the President of Hokkaido University. In addition, the structure of the compounds contained in the composition was identified by 1 1H NMR (manufactured by Bruker, AVANCE III 600).

[0053] [Experimental Example 1] (Preparation of the composition before purification) According to Scheme (III) shown in Figure 3, a composition before purification (Composition (1-1)) containing Compound (4-1-1), Compound (4-2-1), and Compound (4-3-1) was prepared.

[0054] [Experimental Example 1-1] (Preparation of a composition containing compound (2-1-1), compound (2-2-1), and compound (2-3-1)) 26.0 g (88.0 mmol) of (6-bromohexyloxy)tert-butyldimethylsilane was dissolved in 26 mL of dry diethyl ether to prepare a solution. Under an argon atmosphere, 1 mL of the above solution, 4 mL of dry diethyl ether, and 2.35 g (96.8 mmol) of magnesium turnings were added to a flask, followed by the addition of a small piece of iodine. After allowing the solution to stand at room temperature until the whole solution turned brown, it was stirred while heating to 40 °C in an oil bath. After confirming decolorization, the above (6-bromohexyloxy)tert-butyldimethylsilane solution was added dropwise to the flask. After reacting at 40 °C for 2 hours, it was ice-cooled. Subsequently, 3.67 mL (39.6 mmol) of δ-valerolactone dissolved in 3.67 mL of dry diethyl ether was added dropwise to the flask and reacted overnight at room temperature. It was ice-cooled, diluted by adding diethyl ether, and the remaining magnesium was dissolved by adding saturated aqueous citric acid dropwise to the flask. The organic layer was separated and washed with water and saturated brine. Subsequently, anhydrous sodium sulfate was added to the organic layer for dehydration. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a composition containing compound (2-1-1), compound (2-2-1), and compound (2-3-1) (the composition of Experimental Example 1-1). The content ratios of compound (2-1-1), compound (2-2-1), and compound (2-3-1) contained in the composition (1-1) were compound (2-1-1):compound (2-2-1):compound (2-3-1) = 99.0:0.2:0.8.

[0055] [Experimental Example 1-2] (Preparation of a composition (composition (1-2)) containing compound (3-1-1), compound (3-2-1), and compound (3-3-1)) In a flask, 14.0 g of the composition of Experimental Example 1-1 was dissolved in 50 mL of dichloromethane, 321 mg (2.63 mmol) of N, N-dimethyl-4-aminopyridine (DMAP) and 5.50 mL (39.5 mmol) of diisopropylethylamine were added, and the mixture was cooled in ice. After gradually adding 6.02 g (31.6 mmol) of p-toluenesulfonyl chloride to the flask, the reaction was carried out overnight at room temperature. After distilling off the solvent using a rotary evaporator, the residue was suspended in ethyl acetate and washed by liquid separation with water and saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration, and after filtration, the solvent was distilled off using a rotary evaporator to obtain a composition containing compound (3-1-1), compound (3-2-1), and compound (3-3-1) (the composition of Experimental Example 1-2).

[0056] [Experimental Example 1-3] (Preparation of a composition containing compound (4-1-1), compound (4-2-1), and compound (4-3-1) (composition (1-3))) In a flask, 30 mL of tetrahydrofuran was added to 12.4 g of the composition of Experimental Example 1-2, and the mixture was cooled to 4°C. Subsequently, 7.38 mL (54.0 mmol) of dipropylamine was added to the flask, and the reaction was carried out for 11 days at room temperature. After distilling off the solvent using a rotary evaporator, the residue was suspended in ethyl acetate and washed by liquid separation with 0.5 N aqueous sodium hydroxide solution and saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtration, the solvent was distilled off using a rotary evaporator to obtain a composition containing compound (4-1-1), compound (4-2-1), and compound (4-3-1) (the composition of Experimental Example 1-3).

[0057] [Experimental Example 1-4] (Preparation of a composition containing compound (5-1-1), compound (5-2-1), and compound (5-3-1)) In a flask, 2.23 mL (39 mmol) of acetic acid and 26 mL of a 1.0 M solution of tetrabutylammonium fluoride (TBAF) in tetrahydrofuran were added to 7.27 g of the compositions of Experimental Examples 1-3, and the mixture was reacted overnight at room temperature. The solvent was distilled off using a rotary evaporator to obtain a composition (Composition (1-1)) containing Compound (5-1-1), Compound (5-2-1), and Compound (5-3-1).

[0058] [Experimental Example 2] (Preparation of the composition before purification (Composition (1-2))) In Experimental Example 1-1, a composition containing Compound (2-1-1):Compound (2-2-1):Compound (2-3-1) in a content ratio of 92.5:7.0:0.5 was obtained in the same manner except that 3.34 mL (36.0 mmol) of δ-valerolactone was used. Subsequently, Composition (1-2) was obtained in the same manner as in Experimental Example 1-2, Experimental Example 1-3, and Experimental Example 1-4.

[0059] [Experimental Example 3] (Preparation of the composition before purification (Composition (1-3))) In Experimental Example 1-1, a composition containing Compound (2-1-1):Compound (2-2-1):Compound (2-3-1) in a content ratio of 85.0:0.0:15.0 was obtained in the same manner except that 4.04 mL (43.6 mmol) of δ-valerolactone was used. Subsequently, Composition (1-3) was obtained in the same manner as in Experimental Example 1-2, Experimental Example 1-3, and Experimental Example 1-4.

[0060] [Experimental Example 4] (Purification of the composition) Compositions (Composition (1-1), Composition (1-2), and Composition (1-3)) (9.5 g each) containing Compound (5-1-1), Compound (5-2-1), and Compound (5-3-1) were each dissolved in 0.5 N hydrochloric acid aqueous solution (150 mL). After adding the solvents (100 mL) described in Table 1 respectively, the mixtures were stirred for 10 minutes. Subsequently, liquid separation was performed using a separatory funnel, and the aqueous layer was recovered. At this time, liquid separation and washing were repeated until the impurities contained in the organic layer disappeared or there was no change as determined by TLC analysis. Thereafter, 5 M sodium hydroxide aqueous solution (30 mL) was added to the recovered aqueous layer, and the mixture was stirred for 10 minutes. Further, ethyl acetate (100 mL) was added, and the mixture was stirred for 10 minutes. Then, the solution was separated using a separatory funnel, and the organic layer was recovered. The recovered organic layer was washed once by liquid separation with saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtration, the solvent was distilled off using a rotary evaporator to obtain a purified composition.

[0061]

Table 1

[0062] [Experimental Example 5] (Production of Compositions Containing Compound (6-1-1), Compound (6-2-1), and Compound (6-3-1)) According to Scheme (III) shown in Figure 3, compositions (Composition (3-1) to Composition (3-11)) containing Compound (6-1-1), Compound (6-2-1), and Compound (6-3-1) were produced.

[0063] [Experimental Example 5-1] (Production of Composition (3-1) to Composition (3-11)) In a flask, each of the compositions purified in Experimental Example 4 (Composition (2-1) to Composition (2-11)) was dissolved in 5 mL of dichloromethane. Subsequently, 900 mg (3.0 mmol) of oleyl chloride was added, and then the mixture was cooled to 4 °C. 697 μL (5.0 mmol) of triethylamine (TEA) was added dropwise to the flask, and the reaction was carried out at room temperature for 3 hours. After evaporating the solvent using a rotary evaporator, the residue was suspended in ethyl acetate, and the insoluble matter was removed by filtration. The filtrate was washed by liquid separation with a 0.5 N aqueous sodium hydroxide solution and saturated brine. Anhydrous sodium sulfate was added to the organic layer for dehydration. After filtration, the solvent was evaporated using a rotary evaporator to obtain a crude product. The crude product was purified by silica gel chromatography (the elution solvent was a continuous gradient of dichloroethane and ethanol) to obtain compositions (Composition (3-1) to Composition (3-11)) containing Compound (6-1-1), Compound (6-2-1), and Compound (6-3-1).

[0064] [Experimental Example 5-2] (Evaluation of the content ratios of Compound (6-1-1), Compound (6-2-1), Compound (6-3-1), and impurities contained in Composition (3-1) to Composition (3-11)) Compositions (Composition (3-1) to Composition (3-11)) containing Compound (6-1-1), Compound (6-2-1), and Compound (6-3-1) were separated and analyzed by UHPLC (CAD) using a column (C18 column, 1.7 μm, inner diameter 2.1 mm × 150 mm) and an eluent (a continuous gradient of 20 mM aqueous ammonium acetate solution and 20 mM ethanol solution of ammonium acetate). The evaluation results are shown in Table 2 below.

[0065]

Table 2

[0066] [Experimental Example 6] (Production of lipid nanoparticles encapsulating siRNA) A lipid solution was prepared by dissolving 1.83 mg of composition (3-1), 0.158 mg of phospholipid (DSPC, catalog number "COATSOME MC-8080", Yuka Sangyo), 0.696 mg of cholesterol (catalog number "C8667", Sigma-Aldrich), and 0.0502 mg of PEG lipid (PEG-DMG, product name "SUNBRIGHT GM-020", Yuka Sangyo) in 0.5 mL of ethanol. A siRNA solution was prepared by dissolving 0.0594 mg of siRNA in 1.5 mL of acetate buffer (pH 4). The lipid solution and the siRNA solution were mixed by a flow reactor. The resulting solution was dialyzed against phosphate-buffered saline (PBS) to produce lipid nanoparticles (LNP1) encapsulating siRNA. As the siRNA, a hybrid of a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 and a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 2 was used. This siRNA targeted the mRNA of mouse blood coagulation factor VII.

[0067] Lipid nanoparticles (LNP2 - LNP11) encapsulating siRNA were produced in the same manner except that compositions (3-2) to (3-11) were used instead of composition (3-1).

[0068] [Experimental Example 7] (In Vivo Kinetics Evaluation of Lipid Nanoparticles Encapsulating siRNA) The in vivo kinetics of lipid nanoparticles encapsulating siRNA were evaluated. Specifically, (LNP1 - LNP11) produced in Experimental Example 6 were administered to mice (C57BL / 6NCrSlc, 6-week-old female, Sankyo Labo Service) via the tail vein such that the siRNA amount was 0.03 mg / kg body weight. Subsequently, the mice were euthanized 24 hours later, and the livers and spleens were excised and frozen in liquid nitrogen.

[0069] Subsequently, each of the excised organs was diluted with PBS containing 0.25% Triton X-100 and homogenized using a bead mill homogenizer. Subsequently, the samples were treated at 95°C for 10 minutes to heat-denature the proteins. Subsequently, each sample was allowed to stand on ice for 5 minutes and then centrifuged at 20,000×g at 4°C for 20 minutes. Subsequently, the supernatant of each sample was treated at 95°C for 10 minutes to perform reverse transcription of RNA. A commercially available kit (product name: "TaqMan MicroRNA Reverse Transcription Kit", Thermo Fisher Scientific) was used for the reverse transcription reaction. The reaction conditions were 16°C for 30 minutes, followed by 42°C for 30 minutes, and then 85°C for 5 minutes.

[0070] Subsequently, PCR reagents (product name: "TaqMan Universal Master Mix II, no UNG", Thermo Fisher Scientific) were added to each sample after the reverse transcription reaction, and the amount of siRNA in the liver and spleen was quantified by real-time PCR.

[0071] Subsequently, the in vivo pharmacokinetics of each lipid nanoparticle were evaluated according to the following evaluation criteria. The results are shown in Table 3 below. Among LNP1 to LNP9, the pharmacokinetics were evaluated by comparison with LNP9, which had the lowest delivery amount of siRNA into the liver and spleen. (Evaluation Criteria) ++: The total amount of siRNA in the liver and spleen is 65% or more of that of LNP9. -: The total amount of siRNA in the liver and spleen is less than 65% of that of LNP9.

[0072] [Experimental Example 8] (Evaluation of the knockdown activity in vivo of lipid nanoparticles encapsulating siRNA) The knockdown activity of lipid nanoparticles encapsulating siRNA in vivo was evaluated. Specifically, those produced in Experimental Example 6 (LNP1 to LNP11) were administered to mice (C57BL / 6NCrSlc, 6 weeks old, female, Sankyo Labo Service Co., Ltd.) via the tail vein so that the amount of siRNA was 0.03 mg / kg body weight. Subsequently, the mice were euthanized 24 hours later, and blood was collected from the inferior vena cava. Subsequently, the blood sample was centrifuged at 800×g at 4°C for 5 minutes to obtain a plasma sample. Subsequently, using the BIOPHEN FVII kit (Hyphen Biomed), the amount of blood coagulation factor FVII in each sample and the amount of blood coagulation factor FVII in the group not administered with lipid nanoparticles were measured, and the knockdown rate was calculated based on the following formula (1). Knockdown rate (%) = (1 - (amount of blood coagulation factor FVII in the sample / amount of blood coagulation factor FVII in the group not administered with lipid nanoparticles)) × 100 …(1)

[0073] Subsequently, the knockdown activity of each lipid nanoparticle was evaluated according to the following evaluation criteria. The results are shown in Table 3 below. (Evaluation criteria) ++: Knockdown rate is 70% or more +: Knockdown rate is 30% or more and less than 70% -: Knockdown rate is less than 30%

[0074]

Table 3

Industrial applicability

[0075] According to the present invention, a technique for removing impurities contained in a composition containing a lipid can be provided.

Claims

1. A method for purifying a composition, comprising the step of dissolving a composition containing a compound represented by the following formula (1) in an aqueous layer and performing liquid-liquid extraction to purify the compound represented by the following formula (1). The oil layer used in the liquid-liquid extraction has a solubility parameter (SP value) of 14.8 to 20.5 (MPa １／２ ), and a purification method comprising one or more liquids selected from the group consisting of ketone-based liquids, ester-based liquids, and ether-based liquids. 【Chemical 1】 [In formula (1), R １ represents -N(R ２ )-R ２ (wherein each R ２ independently represents a C1-C4 alkyl group.), R ３ represents a C4 alkanediyl group, R ４ represents a C3-C8 alkanediyl group, R ５ represents a hydroxyl group, R ６ each independently represents -R ７ -OH (wherein R ７ represents a C6 alkanediyl group.) or a hydrogen atom, and n represents an integer of 0 or 1.]

2. The step of dissolving a composition containing the compound represented by the formula (1) in an aqueous layer and performing liquid-liquid extraction to purify the compound represented by the formula (1) is a step of dissolving a composition containing the compounds represented by the following formulas (5-1-1), (5-2-1) and (5-3-1) in an aqueous layer and performing liquid-liquid extraction to purify the compounds represented by the following formulas (5-1-1), (5-2-1) and (5-3-1). The purification method according to Claim 1. [Chemical Formula 2]

3. The ketone-based liquid is cyclohexanone, methyl isobutyl ketone or diisopropyl ketone, the ester-based liquid is ethyl acetate or butyl acetate, and the ether-based liquid is diethyl ether, dipropyl ether, cyclopentyl methyl ether or propylene glycol monomethyl ether acetate. The purification method according to Claim 1 or 2.

4. A composition comprising one or more compounds selected from the group consisting of a compound (2-1) represented by the following formula (2-1), a compound (2-2) represented by the following formula (2-2) and a compound (2-3) represented by the following formula (2-3), and the total content ratio of the compound (2-1), the compound (2-2) and the compound (2-3) contained in the composition is 90% by mass or more. [Chemical Formula 3] [In formula (2-1), R1 represents -N(R2)-R2 (wherein R2 each independently represents an alkyl group having 1 to 4 carbon atoms), R3 represents an alkanediyl group having 4 carbon atoms, R7 each independently represents an alkanediyl group having 6 carbon atoms, and R10 each independently represents an alkenyl group having 17 carbon atoms.] 【Chemical Formula 4】 [In formula (2-2), R1 represents -N(R2)-R2 (wherein R2 each independently represents an alkyl group having 1 to 4 carbon atoms), R3 represents an alkanediyl group having 4 carbon atoms, R7 represents an alkanediyl group having 6 carbon atoms, and R10 each independently represents an alkenyl group having 17 carbon atoms.] 【Chemical Formula 5】 In formula (2-3), R1 represents -N(R2)-R2 (wherein each R2 independently represents a C1-C4 alkyl group), R3 represents a C4 alkanediyl group, R4 represents a C3-C8 alkanediyl group, each R7 independently represents a C6 alkanediyl group, and each R10 independently represents a C17 alkenyl group. Claim 5 The composition according to claim 4, wherein the content ratio of the compound (2-1) contained in the composition is 85 to 99% by mass. Claim 6 The composition according to claim 4 or claim 5, wherein the total content ratio of the compound (2-2) and the compound (2-3) contained in the composition is 0.1 to 15% by mass. Claim 7 The composition according to claim 4, wherein the compound (2-1) is the compound represented by the following formula (6-1-1), the compound (2-2) is the compound represented by the following formula (6-2-1), and the compound (2-3) is the compound represented by the following formula (6-3-1). 【Chemical Formula 6】 Claim 8 A lipid nanoparticle formed from the compound contained in the composition according to claim 4 or claim 5 and encapsulating a drug. Claim 9 A lipid nanoparticle formed from the compound contained in the composition according to claim 6 and encapsulating a drug. Claim 10 The lipid nanoparticle according to claim 8, wherein the drug is siRNA. Claim 11 The lipid nanoparticle according to claim 9, wherein the drug is siRNA.

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