Method for producing lipid nanoparticles

Abstract

The present invention addresses the problem of providing a method for producing lipid nanoparticles, which enables the production of lipid nanoparticles having a narrow particle size distribution. According to the present invention, there is provided a method for producing lipid nanoparticles, the method comprising a step 1 for preparing a first solution that is an acidic aqueous solution containing an active ingredient, a step 2 for preparing a second solution that is a solution containing a specific lipid and an alcohol; a step 3 for preparing a lipid nanoparticle dispersion by mixing the first solution with the second solution in a flow channel, a pH adjustment step for adjusting the pH of the lipid nanoparticle dispersion, and an alcohol removal step, wherein the alcohol content in the lipid nanoparticle dispersion obtained in step 3 is 12.5-33 vol%, and the lipid nanoparticle dispersion is held for 1 minute or more after the completion of the step 3.

Description

Method for producing lipid nanoparticles

[0001] The present invention relates to a method for producing lipid nanoparticles, which comprises preparing a lipid nanoparticle dispersion by mixing an acidic aqueous solution containing an active ingredient and an alcoholic solution containing lipids in a flow channel.

[0002] Lipid nanoparticles (LNPs) are used to deliver nucleic acids to living organisms or cells, and have been put to practical use in disease treatments, vaccines, etc. LNPs are also known to be usable as gene transfer reagents.

[0003] A known method for producing nucleic acid-encapsulated LNPs involves mixing a lipid solution and a nucleic acid solution in a flow channel.

[0004] Patent Document 1 describes a lipid composition that can achieve excellent nucleic acid delivery for a wide variety of nucleic acids, and a method for producing the same. Patent Document 2 describes a lipid composition that can achieve high nucleic acid encapsulation rate and excellent nucleic acid delivery, and a method for producing the same. However, the process conditions and post-treatment methods, which are important especially when the production volume is increased, are not fully described. Patent Document 3 describes that performing tangential flow filtration (TFF) after neutralization in the LNP production process improves physical property changes and TFF processability. Patent Document 4 describes a method for producing LNPs with specified channel inner diameters and flow rates.

[0005] Patent Document 1: WO2021 / 095876 Patent Document 2: WO2022 / 230964 Patent Document 3: U.S. Patent No. 11,564,893 Patent Document 4: JP2023-542643A

[0006] The pH-adjusted LNP particles produced using conventional technology have a wide particle size distribution, with many larger particles. This can lead to further particle size increases during post-processing steps such as concentration and solvent removal, or changes in the component composition due to filtration.

[0007] An object of the present invention is to provide a method for producing lipid nanoparticles that can produce lipid nanoparticles with a narrow particle size distribution.

[0008] As a result of intensive research to solve the above problems, the inventors have confirmed that the above problems can be solved by adjusting the alcohol content of the lipid nanoparticle dispersion obtained by mixing an acidic aqueous solution and an alcohol solution in a flow channel to 12.5 to 35% by volume and holding the mixture for 1 minute or more after completion of the mixing. The present invention was completed based on the above findings. According to the present invention, the following inventions are provided.

[0009] <1> A method for producing lipid nanoparticles, comprising: step 1 of preparing a first solution, which is an acidic aqueous solution containing an active ingredient; step 2 of preparing a second solution, which is a solution containing an alcohol and at least one lipid selected from the group consisting of a lipid represented by formula (1), a lipid represented by formula (2), and a lipid represented by formula (3); step 3 of preparing a lipid nanoparticle dispersion by mixing the first solution prepared in step 1 with the second solution prepared in step 2 in a flow path; a pH adjustment step of adjusting the pH of the lipid nanoparticle dispersion to a pH higher than the pKa of the lipid nanoparticles after step 3; and an alcohol removal step of removing alcohol from the lipid nanoparticle dispersion after step 3, either after the pH adjustment step, during the pH adjustment step, or before the pH adjustment step, wherein the lipid nanoparticle dispersion obtained in step 3 has an alcohol content of 12.5 to 35% by volume, and the lipid nanoparticle dispersion is held for 1 minute or more after completion of step 3. In formula (1), X is -NR １ - or -O-, R １ is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R ２１ -L １ -R ２２ represents a group represented by -, and R ２１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L １ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or indicates R ２２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms; R ２ and R ３ are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R ３１ -L２ -R ３２ represents a group represented by -, and R ３１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L ２ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or indicates R ３２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms; R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , and R １２ each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms; R ４ and R ５ , R １０ and R ５ , R ５ and R １２ , R ４ and R ６ , R ５ and R ６ , R ６ and R ７ , R ６ and R １０ , R １２ and R ７ , and R ７ and R ８ may be bonded to each other to form a 4- to 7-membered ring which may contain an O atom, and the substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR ４５ R ４６ an amino group represented by the formula: ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and the substituent on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, —NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; a, b, c, and d each independently represent an integer of 0 to 3, with the proviso that a+b is 1 or more and c+d is 1 or more. In formula (2), R １ and R ２ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ represents a hydrocarbon group having 2 to 8 carbon atoms, and R １ , R ２ and R ３ The hydrocarbon group represented by is —OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , and -O-R ５６ and R ４ represents a hydrocarbon group having 1 to 8 carbon atoms, and R ５ and R ６ are each independently a hydrocarbon group having 1 to 8 carbon atoms, or -R ８ -L １ -R ９ where R ５ and R ６ and R are both hydrocarbon groups having 1 to 8 carbon atoms, ７ is -R １０ -L ２ -R １１ -L ３-R １２ indicates, R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ５３ , R ５４ , R ５５ , and R ５６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms; R ５３ , R ５４ , R ５５ , and R ５６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ５８ The aryl group having 6 to 20 carbon atoms may be substituted with —OH, —COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ or -(C1-C12 hydrocarbon group)-R ５７ and R ５８ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ５７ is -OH, COOH, -NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , -O-R ６６ Indicates. ６１ and R ６２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ６３ , R ６４ , R ６５ , and R ６６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms; R ６３ , R ６４ , R ６５ , and R ６６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ６８ The aryl group having 6 to 20 carbon atoms may be substituted with —OH, —COOH, —NR ６１ R ６２ , -OC(O)OR ６３, -C(O)O-R ６４ , —OC(O)—R ６５ , -O-R ６６ or -(C1-C12 hydrocarbon group)-R ６７ and R ６８ represents a hydrocarbon group having 1 to 12 carbon atoms; L １ , L ２ , and L ３ R each independently represents —OC(O)O—, —C(O)O—, —OC(O)—, or —O—. ８ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ９ represents a hydrocarbon group having 1 to 24 carbon atoms, R １０ represents a hydrocarbon group having 1 to 8 carbon atoms, and R １１ represents a hydrocarbon group having 1 to 24 carbon atoms, R １２ represents a hydrocarbon group having 1 to 24 carbon atoms, R ９ , and R １２ The hydrocarbon group represented by is an aryl group, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or -S-R ５８ and R ５３ , R ５４ , R ５５ , and R ５８ is defined as above, and R １１ The hydrocarbon group represented by is —OC(O)O—R ５３ , -C(O)O-R ５４ or —OC(O)—R ５５ and R ５３ , R ５４ , and R ５５ The definition of is as above. In formula (3), R １ , R ２ , R ３ and R ４ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R １ , R ２ , R ３ and R ４The substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by the formula (I) are each independently —C(O)O—R １１ , —OC(O)—R １２ , -O-R １３ , -CO-R １４ , -OC(O)OR １５ , or -S-S-R １６ indicates, R １１ , R １２ , R １３ , R １４ , R １５ and R １６ are each independently -S-R １７ represents a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with １７ represents a hydrocarbon group having 1 to 12 carbon atoms, R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by the formula (I) are each independently —OH, —COOH, —NR ２１ R ２２ , -OC(O)OR ２３ , -C(O)O-R ２４ , —OC(O)—R ２５ , -O-R ２６ , —C(O)NR ２７ R ２８ , -NR ２９ C(O)R ３０ , -N(R ３１ ) S (O) ２ R ３２ , -N(R ３３ )C(O)N(R ３４ ) R ３５ , -N(R ３６ ) C(S)N(R ３７ ) R ３８ , -OC(O)N(R ３９ ) R ４０ , or -N(R ４１ )C(O)OR ４２ indicates, R ２１ and R ２２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; R２３ , R ２４ , R ２５ , R ２６ , R ２７ , R ２８ , R ２９ , R ３０ , R ３１ , R ３２ , R ３３ , R ３４ , R ３５ , R ３６ , R ３７ , R ３８ , R ３９ , R ４０ , R ４１ , and R ４２ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R ２３ , R ２４ , R ２５ , R ２６ , R ２７ , R ２８ , R ２９ , R ３０ , R ３１ , R ３２ , R ３３ , R ３４ , R ３５ , R ３６ , R ３７ , R ３８ , R ３９ , R ４０ , R ４１ , and R ４２ The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by is an aryl group having 6 to 20 carbon atoms, a heterocyclic group, —OH, —COOH, or NR ５１ R ５２ indicates R ５１ and R ５２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; R ７ , R ８ , and R ９ each independently represents a hydrocarbon group having 2 to 8 carbon atoms; R ５ and R ６ , or R ５ and R ７may combine to form a 4- to 7-membered ring. <2> The method for producing lipid nanoparticles according to <1>, further comprising step 4 of mixing the mixture obtained in step 3 with a third solution to prepare a lipid nanoparticle dispersion, wherein the time period from step 3 to step 4 is 1 minute or more. <3> The method for producing lipid nanoparticles according to <1>, wherein the time period from step 3 to step 4, whichever is earlier, is 1 minute or more. <4> The method for producing lipid nanoparticles according to any one of <1> to <3>, wherein the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is 20 to less than 30% by volume. <5> The method for producing lipid nanoparticles according to <2>, wherein the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is 20 to less than 27.5% by volume, and the time period from step 3 to step 4 is 1 minute or more but 24 hours or less. <6> The method for producing lipid nanoparticles according to any one of <1> to <5>, wherein the alcohol is ethanol. <7> The method for producing lipid nanoparticles according to any one of <1> to <6>, wherein the second solution in step 2 further contains at least one lipid selected from the group consisting of neutral lipids, lipids having nonionic hydrophilic polymers, and sterols. <8> The method for producing lipid nanoparticles according to any one of <1> to <7>, wherein the produced lipid nanoparticles have an average particle size of less than 200 nm. <9> The method for producing lipid nanoparticles according to any one of <1> to <8>, wherein the produced lipid nanoparticles have an average particle size with a polydispersity index PDI of less than 0.20.

[0010] According to the method for producing lipid nanoparticles of the present invention, particles with a narrow particle size distribution can be produced.

[0011] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively.

[0012] <Method for producing lipid nanoparticles> The method for producing lipid nanoparticles of the present invention comprises: Step 1 of preparing a first solution, which is an acidic aqueous solution containing an active ingredient; Step 2 of preparing a second solution, which is a solution containing an alcohol and at least one lipid selected from the group consisting of a lipid represented by formula (1), a lipid represented by formula (2), and a lipid represented by formula (3); Step 3 of preparing a lipid nanoparticle dispersion by mixing the first solution prepared in step 1 with the second solution prepared in step 2 in a flow path; After step 3, a pH adjustment step of adjusting the pH of the lipid nanoparticle dispersion to a pH higher than the pKa of the lipid nanoparticles; and After step 3, an alcohol removal step of removing alcohol from the lipid nanoparticle dispersion after, during, or before the pH adjustment step. In this method, the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is 12.5 to 35% by volume, and the lipid nanoparticle dispersion is held for 1 minute or more after completion of step 3. According to the present invention, it is possible to produce particles with a narrow particle size distribution in the LNP intermediate after pH adjustment, and it is possible to reduce changes in particle size before and after the pH adjustment process and changes in lipid composition before and after the filtration process. Furthermore, the method for producing lipid nanoparticles of the present invention can improve the delivery efficiency of the encapsulated active ingredient.

[0013] In step 1, the first solution can be obtained by dissolving an active ingredient (e.g., a nucleic acid) in water or a buffer solution. The concentration of the active ingredient, such as a nucleic acid, is not particularly limited, but is preferably 1 to 1,000 μg / mL, and more preferably 10 to 500 μg / mL. If necessary, components such as a buffer component or an antioxidant can be added to adjust the pH. The pH of the aqueous phase is preferably 3.0 to 6.5, more preferably 3.5 to 6.0, and even more preferably 4.0 to 5.5. To adjust the pH to the above range, buffer components such as acetate buffer, citrate buffer, malate buffer, phosphate buffer, MES, Bis-Tris, and PIPES can be preferably used. The concentration of these buffer components is preferably 2 to 200 mmol / L, and more preferably 5 to 100 mmol / L. If necessary, salts such as sodium chloride and potassium chloride can be added to adjust the salt strength, and sugars or sugar alcohols such as sucrose, trehalose, and mannitol can be added to adjust the osmotic pressure.

[0014] In step 2, the second solution can be obtained by dissolving at least one lipid selected from the group consisting of lipids represented by formula (1), lipids represented by formula (2), and lipids represented by formula (3) in alcohol. Among alcohols, alcohols that are miscible with water in any ratio are more preferred, ethanol or 2-propanol are even more preferred, and ethanol is most preferred. The second solution may also contain components other than alcohol, such as water.

[0015] The total lipid concentration in the second solution is not particularly limited, but is preferably 1 mmol / L to 100 mmol / L, preferably 5 mmol / L to 80 mmol / L, and more preferably 10 mmol / L to 60 mmol / L.

[0016] In step 3, particles are formed by self-assembly when the first solution and the second solution are mixed, triggering the complexation of the components contained therein and the decrease in the solubility of each component due to the decrease in ethanol content. Since the mixing ratio of the first solution and the second solution affects particle formation, it is preferable to use a flow path that can control the mixing ratio so that it is always constant and highly reproducible. Specifically, microflow paths such as branch mixers, T-shaped mixers, herringbone mixers, and baffle mixers described in U.S. Patent No. 10,688,456, U.S. Patent No. 5,192,515, U.S. Patent No. 10,342,761, U.S. Patent No. 6,450,742, and Shepherd S. J. et al., Biomaterials, 274, 120826 (2021) can be preferably used. In each case, the flow path shape is devised to achieve precise mixing suitable for the production of lipid nanoparticles.

[0017] Furthermore, when high productivity is required, an impingement jet mixer (IJM) capable of rapid macro-mixing, a turbulent jet mixer which mixes using concentric jet flows, or even a membrane emulsification method can be used, and the optimal mixer can be selected depending on the required production scale and particle properties.

[0018] The mixing ratio (volume ratio) of the first solution to the second solution is preferably 7:1 to 2:1, and more preferably 4:1 to 2.3:1. The flow rate and flow rate can be adjusted by appropriately configuring the shape of the microchannel and are not particularly limited. Generally, when the flow rate and flow rate of the mixture of the first solution and the second solution are mixed, if the flow rate during mixing is low and the mixing efficiency is poor, the resulting particle size tends to be large. The flow rate of the mixture of the first solution and the second solution is preferably 5.0 m / s or more, more preferably 7.5 m / s or more, and most preferably 10.0 m / s or more. The flow rate is preferably 4 to 4,000 mL / min, and more preferably 8 to 2,000 mL / min.

[0019] In the present invention, step 4 may be further included in which the mixture obtained in step 3 is mixed with a third solution to prepare a lipid nanoparticle dispersion. When step 4 is performed, it is preferable that the time from step 3 to step 4 is 1 minute or more. When step 4 is not performed, it is preferable that the time from step 3 to step 4 is 1 minute or more before moving to the step that is performed first, either the pH adjustment step or the alcohol removal step.

[0020] When step 4 is performed, the method for producing lipid nanoparticles of the present invention includes: step 1 of preparing a first solution, which is an acidic aqueous solution containing an active ingredient; step 2 of preparing a second solution, which is a solution containing at least one lipid selected from the group consisting of lipids represented by formula (1), lipids represented by formula (2), and lipids represented by formula (3), and an alcohol; step 3 of preparing a lipid nanoparticle dispersion by mixing the acidic aqueous solution prepared in step 1 with the alcohol solution prepared in step 2 in a flow path; step 4 of preparing lipid nanoparticle dispersion B by mixing the mixed solution obtained in step 3 with a third solution; a pH adjustment step of adjusting the pH of lipid nanoparticle dispersion B obtained in step 4 to a pH higher than the pKa of the lipid nanoparticles; and an alcohol removal step of removing alcohol from the lipid nanoparticle dispersion after, during, or before the pH adjustment step. This is a method for producing lipid nanoparticles, wherein the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is 12.5 to 35% by volume, and the time from step 3 to step 4 is 1 minute or more.

[0021] When step 4 is not performed, the method for producing lipid nanoparticles of the present invention includes: step 1 of preparing a first solution, which is an acidic aqueous solution containing an active ingredient; step 2 of preparing a second solution, which is a solution containing at least one lipid selected from the group consisting of lipids represented by formula (1), lipids represented by formula (2), and lipids represented by formula (3), and an alcohol; step 3 of preparing a lipid nanoparticle dispersion by mixing the acidic aqueous solution prepared in step 1 with the alcohol solution prepared in step 2 in a flow path; a pH adjustment step after step 3 of adjusting the pH of the lipid nanoparticle dispersion to a pH higher than the pKa of the lipid nanoparticles; and an alcohol removal step after step 3, which is after the pH adjustment step, during the pH adjustment step, or before the pH adjustment step, of removing alcohol from the lipid nanoparticle dispersion. In this method, the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is 12.5 to 35% by volume, and the time between step 3 and the step which is performed first, either the alcohol removal step or the pH adjustment step, is 1 minute or more.

[0022] Step 4 may be carried out continuously after step 3, or the lipid nanoparticle dispersion obtained in step 3 may be stored in a container and then carried out again. When step 4 is carried out continuously after step 3, the piping volume from the place where the first solution and the second solution are mixed to the place where the third solution is mixed may be designed to be equal to or greater than the flow rate per minute of the lipid nanoparticle dispersion obtained in step 3. When the lipid nanoparticle dispersion obtained in step 3 is stored in a container and then carried out again, step 4 may be started one minute or more after the lipid nanoparticle dispersion is prepared in step 3.

[0023] In step 4, the third solution is added to reduce the alcohol content to an appropriate range, and therefore preferably contains water as its main component, and may contain other additives such as a buffer component for adjusting the pH, and / or a salt such as sodium chloride or potassium chloride for adjusting the salt strength, and / or a sugar or sugar alcohol (sucrose, trehalose, mannitol, etc.) for adjusting the osmotic pressure, as needed.

[0024] Furthermore, in step 4, the mixing ratio of the lipid nanoparticle dispersion A obtained in step 3 to the third solution affects particle formation, so it is preferable to mix them so that the mixing ratio can be controlled to be constant with good reproducibility. The method for this is not limited, but it is preferable to mix them using a flow path such as a T-tube or Y-tube, and each liquid may be added to a receiving container at a constant rate.

[0025] The pH of the third solution is preferably 4.0 to 8.0, more preferably 5.0 to 7.8, and even more preferably 6.0 to 7.6. The pH and buffer component concentration of the third solution are preferably selected so that the pH of lipid nanoparticle dispersion B obtained in step 4 by mixing the third solution with the lipid nanoparticle dispersion A obtained in step 3 is lower than its pKa.

[0026] The buffer component used to adjust the pH to that contained in the third solution is not particularly limited, but known buffer components such as ACES, BES, Bicine, Bis-Tris, CAPS, CHES, DIPSO, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, PIPES, TAPS, TAPSO, TES, Tricine, Tris buffer, phosphate buffer, acetate buffer, citrate buffer, and malate buffer can be preferably used.

[0027] In one example of the present invention, the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is 20 to less than 30% by volume. In one example of the present invention, the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is 20 to less than 27.5% by volume, and the time from step 3 to step 4 is 1 minute or more to 24 hours or less.

[0028] In another example of the present invention, the alcohol content of the lipid nanoparticle dispersion A obtained in step 3 is 12.5 to less than 27.5% by volume, and can be held for 1 minute to 24 hours after completion of step 3. In yet another example of the present invention, the alcohol content of the lipid nanoparticle dispersion A obtained in step 3 is 27.5 to less than 30.0% by volume, and can be held for 1 minute to 150 minutes after completion of step 3. In yet another example of the present invention, the alcohol content of the lipid nanoparticle dispersion A obtained in step 3 is 30.0 to less than 35.0% by volume, and can be held for 1 minute to 10 minutes after completion of step 3.

[0029] The method for producing lipid nanoparticles of the present invention includes a pH adjustment step of adjusting the pH of a lipid nanoparticle dispersion to a pH higher than the pKa of the lipid nanoparticles, and a step of removing alcohol contained in the lipid nanoparticle dispersion.

[0030] The method for adjusting the pH is not particularly limited, and the lipid nanoparticle dispersion may be mixed with a liquid having a high pH. The pH adjustment by the above-mentioned mixing may be carried out in multiple steps. When the pH adjustment is carried out in multiple steps, the lipid nanoparticle dispersion may be stored between the pH adjustment steps. "Multiple times" means two or more times, preferably two to five times, more preferably two or three times, and even more preferably two times. Alternatively, the pH may be adjusted during dialysis using a dialysis solution having a higher pH than that of the lipid nanoparticle dispersion.

[0031] There are no particular limitations on the method for removing alcohol, but at the laboratory level, it is common to use a cassette-type dialysis kit or a centrifugal ultrafiltration kit, etc. For large-scale production, tangential flow filtration (TFF) can be used.

[0032] The timing of removing alcohol may be any of before the pH adjustment step, during the pH adjustment step, and after the pH adjustment step. When removing alcohol before or after the pH adjustment step, dialysis may be performed using a dialysis solution having a pH lower or higher than the pKa of the lipid nanoparticle dispersion to be treated, respectively. In addition, when removing alcohol during the pH adjustment step, dialysis may be performed using a dialysis solution having a pH higher than the pKa of the lipid nanoparticle dispersion, with a pH lower than the pKa of the lipid nanoparticle dispersion. In addition, alcohol removal and pH adjustment may be performed continuously by the TFF method.

[0033] <pKa of Lipid Nanoparticles> The lipids represented by formula (1), formula (2), and formula (3) of the present invention have amino groups, and when the pH of the dispersion becomes acidic, they are protonated, causing the lipid nanoparticles to become cationic. This is a phenomenon known for lipid nanoparticles containing amino lipids similar to the above lipids. As described in Fig. 2 of Angew. Chem. Int. Ed. Engl. 2012, Vol. 51, pp. 8259-8533, the degree of protonation of the amino lipid in the lipid nanoparticles can be evaluated using a probe called 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). When the pH of the dispersion is low, the surface of the lipid nanoparticles becomes cationic, and TNS interacts with the lipid nanoparticles through electrostatic interactions, causing TNS to emit fluorescence. When the fluorescence intensity of TNS is plotted on the vertical axis, a plot similar to a titration curve is obtained. Here, the pH at which the fluorescence intensity of TNS is intermediate between that at a sufficiently low pH and that at a sufficiently high pH is referred to as the pKa of the lipid particle nanoparticle. It is generally known that lipid nanoparticles become cationic at physiological pH, reducing safety, and that if the pH is too low, the efficiency of intracellular payload delivery decreases. Therefore, the components and their ratios of lipid nanoparticles are designed to achieve a preferred pKa depending on the target organ / cell and application. While not particularly limited, the pKa is preferably 5.5 or higher and 7.0 or lower. During the production of lipid nanoparticles, it is preferable to promote particle formation by protonating lipids containing amino groups at a pH lower than the pKa and allowing them to interact with anionic nucleic acids, etc. Furthermore, since the final formulation is generally preferably near physiological pH, the pH of the lipid nanoparticle dispersion is adjusted across the pKa range during the production process. Near the pKa, the charge of the lipid nanoparticles becomes near neutral, reducing dispersion stability. This makes the lipid nanoparticles prone to coarsening and aggregation, which can affect post-treatment processes. Therefore, pH control in the production process is important.

[0034] The concentration can be adjusted in the step of post-treating the lipid nanoparticle dispersion. When diluting, a solution containing phosphate buffered saline, physiological saline, or other additives (e.g., those described above in pH adjustment, salt strength adjustment, and osmotic pressure adjustment) can be used as a diluent to dilute to an appropriate concentration. When concentrating, it can be concentrated by ultrafiltration using an ultrafiltration membrane. It is preferable to use the concentrated dispersion as it is, or it is also preferable to adjust the concentration to the desired level using the diluent after concentration.

[0035] In some embodiments, the TFF method can be used to continuously remove alcohol, adjust pH, and concentrate the solution. In this process, the organic solvent removal step and the concentration adjustment step may be performed in any order. If necessary, the organic solvent removal step and the concentration adjustment step may each be performed multiple times.

[0036] Solutions that can be used for alcohol removal, pH adjustment, and concentration adjustment in the post-treatment process of lipid nanoparticle dispersions may contain excipients, cryoprotectants, buffers, and antioxidants. Examples of excipients and cryoprotectants include, but are not limited to, sugars and sugar alcohols. Examples of sugars include sucrose, trehalose, maltose, glucose, lactose, and fructose, and examples of sugar alcohols include mannitol, sorbitol, inositol, and xylitol. Examples of buffers include, but are not limited to, ACES, BES, Bicine, CAPS, CHES, DIPSO, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, TAPS, TAPSO, TES, Tricine, Tris buffer, phosphate buffer, acetate buffer, and citrate buffer. Examples of antioxidants include EDTA, ascorbic acid, and tocopherol.

[0037] In order to prepare a lipid nanoparticle dispersion as a pharmaceutical composition, it is preferable to perform sterile filtration. As a filtration method, insoluble materials can be removed from the lipid particle dispersion using a hollow fiber membrane, a reverse osmosis membrane, a membrane filter, or the like. In the present invention, although not particularly limited, filtration is preferably performed using a filter having a sterilizable pore size (preferably a 0.2 μm filtration sterilization filter). In addition, it is preferable to perform sterile filtration after the pH adjustment step and the alcohol removal step. Furthermore, if necessary, the lipid nanoparticle dispersion can be frozen or lyophilized. The lipid nanoparticle dispersion can be frozen or lyophilized by a general method, and the method is not particularly limited.

[0038] The steps described above can be performed in any order to post-treat the lipid nanoparticle dispersion. Examples of post-treatment combinations are listed below, but are not limited thereto.

[0039] - When adjusting the pH before removing the alcohol: (1) Mix a liquid with a high pH and adjust the pH → Concentrate → Remove the alcohol by dialysis → Filter (2) Mix a liquid with a high pH and adjust the pH → Remove the alcohol by dialysis → Concentrate → Filter

[0040] - When alcohol removal and pH adjustment are performed simultaneously (3) Concentration → Alcohol removal and pH adjustment are performed simultaneously by dialysis → Filtration (4) Alcohol removal and pH adjustment are performed simultaneously by dialysis → Concentration → Filtration

[0041] - When adjusting the pH after removing the alcohol: (5) Concentrate → Remove the alcohol without adjusting the pH → Adjust the pH with the dialysate → Filter (6) Remove the alcohol without adjusting the pH → Concentrate → Adjust the pH with the dialysate → Filter (7) Remove the alcohol without adjusting the pH → Adjust the pH with the dialysate → Concentrate → Filter

[0042] <At least one lipid selected from the group consisting of lipids represented by formula (1), lipids represented by formula (2), and lipids represented by formula (3)> In step 2, a second solution is prepared, which is a solution containing at least one lipid selected from the group consisting of lipids represented by formula (1), lipids represented by formula (2), and lipids represented by formula (3) and alcohol. The lipids represented by formula (1), lipids represented by formula (2), and lipids represented by formula (3) used in the present invention are described below.

[0043] <<Lipid represented by formula (1)>> In formula (1), X is -NR １ - or -O-, R １ is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R ２１ -L １ -R ２２ represents a group represented by -, and R ２１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L １ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or indicates R ２２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms; R ２ and R ３ are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R ３１ -L ２ -R ３２ represents a group represented by -, and R ３１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L ２ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or indicates R ３２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms; R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , and R １２ each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms; R ４and R ５ , R １０ and R ５ , R ５ and R １２ , R ４ and R ６ , R ５ and R ６ , R ６ and R ７ , R ６ and R １０ , R １２ and R ７ , and R ７ and R ８ may be bonded to each other to form a 4- to 7-membered ring which may contain an O atom, and the substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR ４５ R ４６ an amino group represented by the formula: ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and the substituent on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, —NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６each independently represents a hydrocarbon group having 1 to 18 carbon atoms; a, b, c, and d each independently represent an integer of 0 to 3, with the proviso that a+b is 1 or more and c+d is 1 or more.

[0044] R １ a hydrocarbon group having 6 to 24 carbon atoms in R ２ and R ３The hydrocarbon group having 3 to 24 carbon atoms in the formula (I) is preferably an alkyl group, an alkenyl group, or an alkynyl group, and more preferably an alkyl group or an alkenyl group. The alkyl group having 6 to 24 carbon atoms and the alkyl group having 3 to 24 carbon atoms may be linear or branched, and may be chain-like or cyclic. The alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 6 to 20 carbon atoms, and more preferably the alkyl group having 3 to 24 carbon atoms is an alkyl group having 6 to 20 carbon atoms. Specific examples include hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, nonadecyl, and icosyl. The alkenyl group having 6 to 24 carbon atoms and the alkenyl group having 3 to 24 carbon atoms may be linear or branched, and may be linear or cyclic. The alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 6 to 20 carbon atoms, and the alkenyl group having 3 to 24 carbon atoms is more preferably an alkenyl group having 6 to 20 carbon atoms. Specifically, hexenyl group, heptenyl group, octenyl group, nonenyl group, decenyl group, undecenyl group, dodecenyl group, dodecadienyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group (preferably, (Z)-hexadec-9-enyl group), hexadecadienyl group, heptadecenyl group (preferably, (Z)-heptadeca-8-enyl group), heptadecadienyl group (preferably, (8Z, (11Z)-heptadeca-8,11-dienyl group), octadecenyl group (preferably, (Z)-octadec-9-enyl group), octadecadienyl group (preferably, (9Z,12Z)-octadeca-9,12-dienyl group), nonadecenyl group, icosenyl group (preferably, (Z)-icos-11-enyl group), icosadienyl group (preferably, (11,14)-icosa-11,14-dienyl group), and the like.The alkynyl group having 6 to 24 carbon atoms is preferably an alkynyl group having 6 to 20 carbon atoms, and the alkynyl group having 3 to 24 carbon atoms is more preferably an alkynyl group having 6 to 20 carbon atoms. Specific examples include a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group. Each of the above alkenyl groups preferably has one or two double bonds, and each of the alkynyl groups preferably has one or two triple bonds.

[0045] R ２１ and R ３１The hydrocarbon group having 1 to 24 carbon atoms in the above formula (I) is preferably an alkyl group having 10 to 24 carbon atoms, an alkenyl group having 10 to 24 carbon atoms, or an alkynyl group having 10 to 24 carbon atoms. The alkyl group having 10 to 24 carbon atoms may be linear or branched, and may be linear or cyclic. The alkyl group having 10 to 24 carbon atoms is preferably an alkyl group having 12 to 24 carbon atoms. Specific examples thereof include a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, a 2-butyloctyl group, a 1-pentylhexyl group, a 2-pentylheptyl group, a 3-pentyloctyl group, a 1-hexylheptyl group, and a 1-hexylnonyl group. Examples include a 2-hexyloctyl group, a 2-hexyldecyl group, a 3-hexylnonyl group, a 1-heptyloctyl group, a 2-heptylnonyl group, a 2-heptylundecyl group, a 3-heptyldecyl group, a 1-octylnonyl group, a 2-octyldecyl group, a 2-octyldodecyl group, a 3-octylundecyl group, a 2-nonylundecyl group, a 3-nonyldodecyl group, a 2-decyldodecyl group, a 2-decyltetradecyl group, a 3-decyltridecyl group, and a 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group. The alkenyl group having 10 to 24 carbon atoms may be linear or branched, open-chain or cyclic.Specific examples include a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably, a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), a pentadecenyl group (preferably, a (Z)-pentadecen-8-enyl group), a hexadecenyl group (preferably, a (Z)-hexadecan-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably, a (Z)-heptadecan-8-enyl group), a heptadecadienyl group (preferably, a (8Z,11Z)-heptadecan-8,11-dienyl group), an octadecenyl group (preferably, a (Z)-octadec-9-enyl group), and an octadecadienyl group (preferably, a (9Z,12Z)-octadecan-9,12-dienyl group). The alkynyl group having 10 to 24 carbon atoms may be linear or branched, and may be linear or cyclic. Specific examples include decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, and octadecynyl. Each of the above alkenyl groups preferably has one or two double bonds, and each of the alkynyl groups preferably has one or two triple bonds.

[0046] R ２２ and R ３２ Regarding the above, the divalent linking group and hydrocarbon linking group having 1 to 18 carbon atoms is preferably an alkylene group having 1 to 18 carbon atoms or an alkenylene group having 2 to 18 carbon atoms. The alkylene group having 1 to 18 carbon atoms may be linear or branched, and may be chain-like or cyclic. The number of carbon atoms is preferably 1 to 12, more preferably 1 to 10, and even more preferably 2 to 10. Specific examples include a methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, nonamethylene group, decamethylene group, undecamethylene group, and dodecamethylene group. The alkenylene group having 2 to 18 carbon atoms may be linear or branched, and may be chain-like or cyclic. The number of carbon atoms is preferably 1 to 12, and more preferably 2 to 10.

[0047] L １The preferred range of is —O(CO)O—, —O(CO)—, or —(CO)O—, and —O(CO)— or —(CO)O— is more preferred. ２ The preferred range is —O(CO)O—, —O(CO)—, or —(CO)O—, and —O(CO)— or —(CO)O— is more preferred.

[0048] R ４ , R ６ , R ９ , R １０ , R １１ , and R １２ The alkyl group having 1 to 18 carbon atoms in the optionally substituted alkyl group having 1 to 18 carbon atoms may be linear or branched, and may be linear or cyclic. The number of carbon atoms is preferably 1 to 12. Specific examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, and a dodecyl group. When the alkyl group has a substituent, the substituent may be a hydroxyl group, a carboxyl group, or a -O(CO)O-R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ A group represented by —O(CO)—R ４２ or -(CO)O-R ４３ A group represented by the following formula is more preferred.

[0049] R ５ , R ７ , and R ８The alkyl group having 1 to 18 carbon atoms in the optionally substituted alkyl group having 1 to 18 carbon atoms may be linear or branched, chain-like or cyclic. The number of carbon atoms is preferably 1 to 12, and more preferably 1 to 8. Specific examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, and a dodecyl group. When the alkyl group has a substituent, the substituent may be a hydroxyl group, a carboxyl group, or a -O(CO)O-R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ A group represented by —O(CO)—R ４２ , -(CO)O-R ４３ A group represented by the following formula is more preferred.

[0050] Examples of the 4- to 7-membered ring which may contain an O atom include an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring, and an azepane ring, and a 6-membered ring is preferred, with a piperidine ring and a morpholine ring being more preferred.

[0051] R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , and R １２ In the case where the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a substituted or unsubstituted aryl group, the aryl group preferably has 6 to 22 carbon atoms, more preferably 6 to 18 carbon atoms, and even more preferably 6 to 10 carbon atoms. Specific examples include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group. Examples of the substituent on the aryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, and -NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２, -(CO)O-R ４３ , or -O-R ４４ A group represented by the formula: is preferred, and a hydroxyl group or a carboxyl group is more preferred. Specific examples of the substituted aryl group include a hydroxyphenyl group and a carboxyphenyl group.

[0052] R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , and R １２ In the case where the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a substituted or unsubstituted heteroaryl group, the heteroaryl group preferably has 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Specific examples include a pyridyl group, a pyrazolyl group, an imidazolyl group, a benzimidazolyl group, a thiazolyl group, and an oxazolyl group. Examples of the substituent on the heteroaryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, and -NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ A group represented by the formula: is preferred, and a hydroxyl group or a carboxyl group is more preferred. Specific examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxypyridyl group, and a pyridonyl group.

[0053] R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６The hydrocarbon group having 1 to 18 carbon atoms in the above formula is preferably an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 2 to 18 carbon atoms, or an alkynyl group having 2 to 18 carbon atoms, and more preferably an alkyl group having 1 to 18 carbon atoms or an alkenyl group having 2 to 18 carbon atoms. The alkyl group having 1 to 18 carbon atoms may be linear or branched, and may be linear or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Specific examples include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl groups. The alkenyl group having 2 to 18 carbon atoms may be linear or branched, open-chain, or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18. Specifically, an allyl group, a prenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group (preferably, a (Z)-2-nonenyl group or an (E)-2-nonenyl group), a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably, a (Z)-tridec-8-enyl group), a tetradecenyl group (preferably, a tetradec-9-enyl group), a pentadecenyl group (preferably, a (Z)-pentadecen-8-enyl group), , a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadeca-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadec-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group), and the like. The alkynyl group having 2 to 18 carbon atoms may be linear or branched, and may be linear or cyclic. The number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18.Specific examples include a propargyl group, butynyl group, pentynyl group, hexynyl group, heptynyl group, octynyl group, nonynyl group, decynyl group, undecynyl group, dodecynyl group, tetradecynyl group, pentadecynyl group, hexadecynyl group, heptadecynyl group, and octadecynyl group.

[0054] X is -NR １ When indicating -, R １ is a hydrocarbon group having 6 to 24 carbon atoms, or R ２１ -L １ -R ２２ In this case, R ２ and R ３ is a hydrogen atom; ２ and R ３ the other is a hydrocarbon group having 6 to 24 carbon atoms, or R ３１ -L ２ -R ３２ It is preferred that the group is represented by -.

[0055] When X represents —O—, R ２ and R ３ are each independently a hydrocarbon group having 6 to 24 carbon atoms, or R ３１ -L ２ -R ３２ It is preferred that the group is represented by -.

[0056] R ４ , R ６ , R ９ , R １０ , R １１ , and R １２ is preferably a hydrogen atom.

[0057] R ５ represents a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, or —O(CO)—R ４２ or -(CO)O-R ４３ Preferably, R is an alkyl group having 1 to 18 carbon atoms which may be substituted with an alkyl group having 1 to 18 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 18 carbon atoms which may be substituted with a hydroxyl group. When R is an alkyl group, ４ , R ６ , R １０ and R １２and may be linked together to form a ring which may contain an O atom. Among these, alkyl groups having 1 to 18 carbon atoms, —O(CO)—R ４２ or -(CO)O-R ４３ The alkyl group may be substituted with an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 12 carbon atoms, or an alkyl group having 1 to 8 carbon atoms, which may be substituted with a hydroxyl group. ４２ or -(CO)O-R ４３ It is more preferably an alkyl group having 1 to 18 carbon atoms which may be substituted with.

[0058] R ７ and R ８ each independently represents a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, or —O(CO)—R ４２ or -(CO)O-R ４３ an alkyl group having 1 to 18 carbon atoms which may be substituted with an alkyl group having 1 to 8 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group, or R ７ and R ８ are preferably linked to each other to form a 4- to 7-membered ring which may contain an O atom.

[0059] R ５ and R ７ or R ８ are not linked to each other and do not form a ring.

[0060] a+b is preferably 1 or 2, and more preferably 1. c+d is preferably 1 or 2, and more preferably 1.

[0061] The lipid represented by formula (1) is preferably a lipid represented by the following formula (1-1):

[0062] R ２４ is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R ２１ -L １ -R ２２ represents a group represented by -, and R ２１represents a hydrocarbon group having 1 to 24 carbon atoms, and L １ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or indicates R ２２ represents a divalent linking group and a hydrocarbon linking group having 1 to 18 carbon atoms. ２５ is a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R ３１ -L ２ -R ３２ represents a group represented by -, and R ３１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L ２ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or indicates R ３２ represents a divalent linking group and a hydrocarbon linking group having 1 to 18 carbon atoms. ４ , R ５ , R ６ , R ７ , R ８ , R １０ , and R １２ each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms; R ４ and R ５ , R １０ and R ５ , R ５ and R １２ , R ４ and R ６ , R ５ and R ６ , R ６ and R ７ , R ６ and R １０ , R １２ and R ７ , and R ７ and R ８ Any one or more pairs of may be linked to each other to form a 4- to 7-membered ring which may contain an O atom. ５ and R ７ or R ８ and do not bond to each other and do not form a ring. The substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a hydroxyl group, a carboxyl group, -NR ４５ R４６ an amino group represented by the formula: ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and the substituent on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, —NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms.

[0063] R in formula (1-1) ４ , R ５ , R ６ , R ７ , R ８ , R １０ , and R １２ The definition and preferred range of are the same as those of formula (1).

[0064] R in formula (1-1) ２４is preferably an alkyl or alkenyl group having 6 to 24 carbon atoms. The alkyl group having 6 to 24 carbon atoms may be linear or branched, and may be chain-like or cyclic. The alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 8 to 20 carbon atoms. Specific examples include an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably, a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably, a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a nonadecyl group, and an icosyl group. The alkenyl group having 6 to 24 carbon atoms may be linear or branched, and may be chain-like or cyclic. The alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 8 to 20 carbon atoms. Specifically, octenyl group, nonenyl group, decenyl group, undecenyl group, dodecenyl group, dodecadienyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group (preferably, (Z)-hexadec-9-enyl group), hexadecadienyl group, heptadecenyl group (preferably, (Z)-heptadeca-8-enyl group), heptadecadienyl group (preferably, (8Z,11Z)-heptadeca-8-enyl group), Examples of alkenyl groups include an octadecenyl group (preferably, (Z)-octadec-8,11-dienyl group), an octadecenyl group (preferably, (9Z,12Z)-octadeca-9,12-dienyl group), a nonadecenyl group, an icosenyl group (preferably, (Z)-icosa-11-enyl group), and an icosadienyl group (preferably, (11,14)-icosa-11,14-dienyl group). Each of the above alkenyl groups preferably has one or two double bonds.

[0065] R in formula (1-1) ２５is preferably an alkyl or alkenyl group having 6 to 24 carbon atoms. The alkyl group having 6 to 24 carbon atoms may be linear or branched, and may be chain-like or cyclic. The alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 7 to 20 carbon atoms. Specific examples include hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, trimethyldodecyl (preferably 3,7,11-trimethyldodecyl), tetradecyl, pentadecyl, hexadecyl, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, and octadecyl. The alkenyl group having 6 to 24 carbon atoms may be linear or branched, and may be chain-like or cyclic. The alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 8 to 20 carbon atoms. Specifically, octenyl group, nonenyl group, decenyl group, undecenyl group, dodecenyl group, dodecadienyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group (preferably, (Z)-hexadec-9-enyl group), hexadecadienyl group, heptadecenyl group (preferably, (Z)-heptadeca-8-enyl group), heptadecadienyl group (preferably, (8Z,11Z)-heptadeca-8-enyl group), Examples of alkenyl groups include an octadecenyl group (preferably, (Z)-octadec-8,11-dienyl group), an octadecenyl group (preferably, (9Z,12Z)-octadeca-9,12-dienyl group), a nonadecenyl group, an icosenyl group (preferably, (Z)-icosa-11-enyl group), and an icosadienyl group (preferably, (11,14)-icosa-11,14-dienyl group). Each of the above alkenyl groups preferably has one or two double bonds.

[0066] In a preferred embodiment, X represents —O—; ２ , R ３ , R ３１ , L ２ , and R ３２ is the same as that in formula (1), ４ , R ５ , R ６ , R ７ , R８ , R ９ , R １０ , R １１ , and R １２ each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, the substituents on the optionally substituted alkyl group having 1 to 18 carbon atoms, the substituents on the substituted or unsubstituted aryl group, and the substituents on the substituted or unsubstituted heteroaryl group are defined as in formula (1), a+b is 1, and c+d is 1 or 2.

[0067] In a more preferred embodiment, the lipid represented by formula (1) is a lipid represented by the following formula (1-2):

[0068] In the formula, R ２ and R ３ are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R ３１ -L ２ -R ３２ represents a group represented by -, R ３１ represents a hydrocarbon group having 1 to 24 carbon atoms; L ２ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or indicates, R ３２ represents a divalent linking group, a hydrocarbon linking group having 1 to 18 carbon atoms; R ５ represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms; R ７ and R ８ each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms, and the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a hydroxyl group, a carboxyl group, -NR ４５ R ４６ an amino group represented by the formula: ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３, R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and the substituent on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, —NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and e represents 2 or 3. ２ , R ３ , R ５ , R ７ and R ８ The definition of is the same as that of equation (1).

[0069] In formula (1-2), preferably, R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms; R ５ The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a substituted or unsubstituted aryl group, —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and the substituent on the substituted or unsubstituted aryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, -NR ４５ R ４６an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms.

[0070] In formula (1-2), more preferably, R ２ and R ３ are each independently a hydrocarbon group having 3 to 24 carbon atoms, or R ３１ -L ２ -R ３２ represents a group represented by -, and L ２ represents —O(CO)— or —(CO)O—, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms; R ５ The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted with respect to the above group is an unsubstituted aryl group, —O(CO)—R ４２ or -(CO)O-R ４３ and R ４２ and R ４３ each independently represents a hydrocarbon group having 1 to 18 carbon atoms.

[0071] In formula (1-2), more preferably, R ２ and R ３ each independently represents a hydrogen atom or a hydrocarbon group having 3 to 24 carbon atoms; R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms; R ５ The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted with respect to the above group is an unsubstituted aryl group, —O(CO)—R ４２ or -(CO)O-R ４３ and R ４２ , and R ４３each independently represents a hydrocarbon group having 1 to 18 carbon atoms.

[0072] In formula (1-2), preferably, R ２ and R ３ At least one of ３１ -L ２ -R ３２ represents a group represented by -, and L ２ represents —O(CO)— or —(CO)O—, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms; R ５ The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted with respect to the above group is an unsubstituted aryl group, —O(CO)—R ４２ or -(CO)O-R ４３ and R ４２ , and R ４３ each independently represents a hydrocarbon group having 1 to 18 carbon atoms.

[0073] In formula (1-2), more preferably, R ２ and R ３ are each independently R ３１ -L ２ -R ３２ represents a group represented by -, and L ２ represents —O(CO)— or —(CO)O—, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms; R ５ The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted with respect to the above group is an unsubstituted aryl group, —O(CO)—R ４２ or -(CO)O-R ４３ and R ４２ , and R ４３ each independently represents a hydrocarbon group having 1 to 18 carbon atoms.

[0074] In formula (1-2), preferably, R ２ and R ３ One of them is R ３１ -L ２ -R ３２ represents a group represented by -, and R２ and R ３ the other represents a hydrocarbon group having 3 to 24 carbon atoms, and L ２ represents —O(CO)— or —(CO)O—, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms; R ５ The substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted with respect to the above group is an unsubstituted aryl group, —O(CO)—R ４２ or -(CO)O-R ４３ and R ４２ , and R ４３ each independently represents a hydrocarbon group having 1 to 18 carbon atoms. In formula (1-2), R ２ and R ３ One of them is R ３１ -L ２ -R ３２ represents a group represented by -, and R ２ and R ３ the other represents a hydrocarbon group having 6 carbon atoms, and L ２ represents —O(CO)— or —(CO)O—, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms; R ５ The substituent on the alkyl group having 1 to 18 carbon atoms that may be substituted is —O(CO)—R ４２ or -(CO)O-R ４３ and R ４２ , and R ４３ each independently represents a hydrocarbon group having 1 to 18 carbon atoms.

[0075] In formula (1-2), more preferably, R ２ and R ３ One of them is R ３１ -L ２ -R ３２ represents a group represented by -, and R ２ and R ３ the other represents a hydrocarbon group having 6 carbon atoms, and L ２ represents —O(CO)— or —(CO)O—, and R ５represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms.

[0076] In formula (1-2), more preferably, R ２ and R ３ One of them is R ３１ -L ２ -R ３２ represents a group represented by -, and R ２ and R ３ the other represents a hydrocarbon group having 6 carbon atoms, and L ２ represents —O(CO)— or —(CO)O—, and R ５ represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and e represents 2. In formula (1-2), R ２ and R ３ One of them is R ３１ -L ２ -R ３２ represents a group represented by -, and R ２ and R ３ the other represents a hydrocarbon group having 3 to 5 carbon atoms, and L ２ represents —O(CO)— or —(CO)O—, and R ５ represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms.

[0077] In formula (1-2), more preferably, R ２ and R ３ One of them is R ３１ -L ２ -R ３２ represents a group represented by -, and R ２ and R ３ the other represents a hydrocarbon group having 3 to 5 carbon atoms, and L ２ represents —O(CO)— or —(CO)O—, and R ５represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and e represents 2. In formula (1-2), R ２ and R ３ One of them is R ３１ -L ２ -R ３２ represents a group represented by -, and R ２ and R ３ the other represents a hydrocarbon group having 6 carbon atoms, and L ２ represents —O(CO)— or —(CO)O—, and R ５ represents a hydrogen atom or a substituted alkyl group having 1 to 18 carbon atoms, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and the substituent on the substituted alkyl group having 1 to 18 carbon atoms is —O(CO)—R ４２ or -(CO)O-R ４３ and R ４２ , and R ４３ each independently represents a hydrocarbon group having 1 to 18 carbon atoms.

[0078] In formula (1-2), more preferably, R ２ and R ３ One of them is R ３１ -L ２ -R ３２ represents a group represented by -, and R ２ and R ３ the other represents a hydrocarbon group having 6 carbon atoms, and L ２ represents —O(CO)— or —(CO)O—, and R ５ represents a hydrogen atom or a substituted alkyl group having 1 to 18 carbon atoms, and R ７ and R ８ each independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and the substituent on the substituted alkyl group having 1 to 18 carbon atoms is —O(CO)—R ４２ or -(CO)O-R ４３ and R ４２ , and R４３ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and e represents 2.

[0079] Specific examples of the lipid represented by formula (1) and a production method thereof are described in WO 2019 / 235635. The entire contents of WO 2019 / 235635 are incorporated herein by reference.

[0080] <<Lipid represented by formula (2)>> In formula (2), R １ and R ２ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ represents a hydrocarbon group having 2 to 8 carbon atoms, and R １ , R ２ and R ３ The hydrocarbon group represented by is —OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , and -O-R ５６ and R ４ represents a hydrocarbon group having 1 to 8 carbon atoms, and R ５ and R ６ are each independently a hydrocarbon group having 1 to 8 carbon atoms, or -R ８ -L １ -R ９ where R ５ and R ６ and R are both hydrocarbon groups having 1 to 8 carbon atoms, ７ is -R １０ -L ２ -R １１ -L ３ -R １２ indicates, R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ５３ , R ５４ , R ５５ , and R ５６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms; R ５３ , R ５４ , R５５ , and R ５６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ５８ The aryl group having 6 to 20 carbon atoms may be substituted with —OH, —COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ or -(C1-C12 hydrocarbon group)-R ５７ and R ５８ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ５７ is -OH, COOH, -NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , -O-R ６６ Indicates. ６１ and R ６２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ６３ , R ６４ , R ６５ , and R ６６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms; R ６３ , R ６４ , R ６５ , and R ６６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ６８ The aryl group having 6 to 20 carbon atoms may be substituted with —OH, —COOH, —NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , -O-R ６６ or -(C1-C12 hydrocarbon group)-R ６７ and R ６８ represents a hydrocarbon group having 1 to 12 carbon atoms; L １ , L ２ , and L ３R each independently represents —OC(O)O—, —C(O)O—, —OC(O)—, or —O—. ８ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ９ represents a hydrocarbon group having 1 to 24 carbon atoms, R １０ represents a hydrocarbon group having 1 to 8 carbon atoms, and R １１ represents a hydrocarbon group having 1 to 24 carbon atoms, R １２ represents a hydrocarbon group having 1 to 24 carbon atoms, R ９ , and R １２ The hydrocarbon group represented by is an aryl group, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or -S-R ５８ and R ５３ , R ５４ , R ５５ , and R ５８ is defined as above, and R １１ The hydrocarbon group represented by is —OC(O)O—R ５３ , -C(O)O-R ５４ or —OC(O)—R ５５ and R ５３ , R ５４ , and R ５５ The definition of is as above.

[0081] In formula (2), -(a hydrocarbon group having 1 to 12 carbon atoms)-R ６７ The hydrocarbon group having 1 to 12 carbon atoms in the formula (I) is preferably an alkylene group having 1 to 12 carbon atoms or an alkenylene group having 2 to 12 carbon atoms. The alkylene group having 1 to 12 carbon atoms and the alkenylene group having 2 to 12 carbon atoms may be linear or branched, and may be linear or cyclic. Specific examples include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a decamethylene group, and an undecamethylene group.

[0082] In formula (2), the aryl group preferably has 6 to 20 carbon atoms, more preferably 6 to 18 carbon atoms, and even more preferably 6 to 10 carbon atoms. Specific examples include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group.

[0083] In formula (2), R １ and R ２ are each independently preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. ３ represents a hydrocarbon group having preferably 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. １ , R ２ and R ３ The hydrocarbon group represented by may preferably be substituted with —OH.

[0084] In formula (2), L １ , and L ３ each independently preferably represents —C(O)O— or —OC(O)—. ２ preferably represents —OC(O)O—, —C(O)O—, or —OC(O)—.

[0085] In formula (2), R ８ represents a hydrocarbon group having preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms. ９ represents a hydrocarbon group having preferably 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms. １１ represents a hydrocarbon group having preferably 1 to 16 carbon atoms, and more preferably a hydrocarbon group having 1 to 9 carbon atoms. １２ represents a hydrocarbon group having preferably 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms. ９ , and R １２ The hydrocarbon group represented by is preferably an aryl group or an S—R ５８ where R ５８represents a hydrocarbon group preferably having 1 to 8 carbon atoms. １１ The hydrocarbon group represented by is preferably —C(O)O—R ５５ , or OC(O)-R ５６ where R ５５ , and R ５６ each independently represents a hydrocarbon group having 1 to 16 carbon atoms; ５５ , and R ５６ The hydrocarbon group represented by is preferably an aryl group having 6 to 20 carbon atoms or —S—R ５８ and R ５８ The definition of is as above.

[0086] The lipid represented by formula (2) is preferably, as a first example, a compound represented by the following formula (2-1): In formula (2-1), R １ and R ２ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ represents a hydrocarbon group having 2 to 8 carbon atoms, and R １ , R ２ and R ３ The hydrocarbon group represented by is —OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or O-R ５６ and R ４ represents a hydrocarbon group having 1 to 8 carbon atoms, and R ５ and R ６ are each independently a hydrocarbon group having 1 to 8 carbon atoms, or R ８ -L １ -R ９ where R ５ and R ６ and L are both hydrocarbon groups having 1 to 8 carbon atoms, １ represents —OC(O)O—, —C(O)O—, —OC(O)—, or —O—, and R ８ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ９represents a hydrocarbon group having 1 to 24 carbon atoms, and R ９ The hydrocarbon group represented by is an aryl group, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or S-R ５８ and R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ５３ , R ５４ , R ５５ , and R ５６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms; R ５３ , R ５４ , R ５５ , and R ５６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ５８ The aryl group having 6 to 20 carbon atoms may be substituted with —OH, —COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ or -(C1-C12 hydrocarbon group)-R ５７ and R ５８ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ５７ is -OH, COOH, -NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ Indicates. １３ represents a hydrocarbon group having 1 to 8 carbon atoms, and R １４ is -R １５ -L ５ -R １６ indicates R １５ represents a hydrocarbon group having 1 to 24 carbon atoms, and L ５ represents —OC(O)O—, —C(O)O—, —OC(O)—, or —O—, and R １６ represents a hydrocarbon group having 1 to 24 carbon atoms, R１５ The hydrocarbon group having 1 to 24 carbon atoms represented by is —OC(O)O—R ５３ , -C(O)O-R ５４ , or OC(O)-R ５５ and R ５３ , R ５４ , and R ５５ is defined as above, and R １６ The hydrocarbon group having 1 to 24 carbon atoms represented by is an aryl group having 6 to 20 carbon atoms, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or S-R ５８ and R ５３ , R ５４ , R ５５ , and R ５８ The definition of is as above.

[0087] In formula (2-1), R １ and R ２ are each independently preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. ３ represents a hydrocarbon group having preferably 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms. １ , R ２ and R ３ The hydrocarbon group represented by may preferably be substituted with —OH.

[0088] In formula (2-1), L １ is preferably —C(O)O— or —OC(O)—. ８ represents a hydrocarbon group having preferably 1 to 10 carbon atoms, and more preferably a hydrocarbon group having 1 to 8 carbon atoms. ９ preferably represents a hydrocarbon group having 1 to 18 carbon atoms, and R ９ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms, or S—R ５８ In formula (2-1), R １４ is preferably —R１５ -L ５ -R １６ indicates R １５ represents a hydrocarbon group having 1 to 18 carbon atoms; L ５ represents -OC(O)O-, and R １６ represents a hydrocarbon group having 1 to 18 carbon atoms. １５ The hydrocarbon group having 1 to 18 carbon atoms represented by is preferably —C(O)O—R ５５ , or OC(O)-R ５６ may be substituted with R ５５ , and R ５６ each independently represents a hydrocarbon group having 1 to 16 carbon atoms; R ５５ , and R ５６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ５８ and R ５８ The definitions of are as above. In formula (2-1), R １６ The hydrocarbon group having 1 to 18 carbon atoms represented by is preferably an aryl group or an S—R ５８ and R ５８ The definition of is as above.

[0089] A second example of the lipid represented by formula (2) is preferably a compound represented by the following formula (2-2): In formula (2-2), R １ and R ２ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ represents a hydrocarbon group having 2 to 8 carbon atoms, and R １ , R ２ and R ３ The hydrocarbon group represented by is —OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or O-R ５６ and R ４ and R ８ each independently represents a hydrocarbon having 1 to 8 carbon atoms; R ２１ and R ２２each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ２３ and R ２４ each independently represents a hydrocarbon group having 1 to 12 carbon atoms; R ２５ and R ２６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms; ２１ and L ２２ each independently represents —OC(O)O—, —C(O)O—, —OC(O)—, or —O—; R ２５ and R ２６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or -S-R ５８ and R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ５３ , R ５４ , R ５５ and R ５６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and the aryl group having 6 to 20 carbon atoms is OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ or -(C1-C12 hydrocarbon group)-R ５７ and R ５７ is -OH, COOH, -NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ Indicates. ５８ represents a hydrocarbon group having 1 to 12 carbon atoms.

[0090] In formula (2-2), R １ and R ２R each independently represents a hydrocarbon group having preferably 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. １ and R ２ The hydrocarbon group represented by may be substituted with —OH, but is more preferably a hydrocarbon group without any substituents.

[0091] In formula (2-2), R ３ represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms.

[0092] In formula (2-2), R ２１ and R ２２ are each independently preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably a hydrocarbon group having 1 to 6 carbon atoms. ２３ and R ２４ Each independently preferably represents a hydrocarbon group having 1 to 10 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms. ２５ and R ２６ each independently represents a hydrocarbon group having preferably 1 to 20 carbon atoms, more preferably a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably a hydrocarbon group having 1 to 12 carbon atoms. ２１ and L ２２ are each independently preferably —C(O)O— or —OC(O)—.

[0093] As a third example, the lipid represented by formula (2) is preferably a compound represented by the following formula (2-3): In formula (2-3), R １ and R ２ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ represents a hydrocarbon group having 2 to 8 carbon atoms, and R １ , R ２ and R ３ The hydrocarbon group represented by is —OH, COOH, —NR ５１ R ５２ , -OC(O)OR５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or O-R ５６ and R ４ and R ８ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ３１ , R ３２ , R ３３ , and R ３４ each independently represents a hydrocarbon group having 1 to 12 carbon atoms; R ３５ , R ３６ , R ３７ , and R ３８ each independently represents a hydrocarbon group having 1 to 24 carbon atoms; ３１ , L ３２ , L ３３ , and L ３４ each independently represents —OC(O)O—, —C(O)O—, —OC(O)—, or —O—; R ３５ , R ３６ , R ３７ , and R ３８ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or S-R ５８ and R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ５３ , R ５４ , R ５５ , and R ５６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and the aryl group having 6 to 20 carbon atoms is OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ or -(C1-C12 hydrocarbon group)-R ５７ and R ５７ is -OH, COOH, -NR ５１ R５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ Indicates. ５８ represents a hydrocarbon group having 1 to 12 carbon atoms.

[0094] In formula (2-3), R １ and R ２ R each independently represents a hydrocarbon group having preferably 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms. １ and R ２ The hydrocarbon group represented by may be substituted with —OH, but is more preferably a hydrocarbon group without any substituents.

[0095] In formula (2-3), R ３ represents a hydrocarbon group preferably having 2 to 6 carbon atoms, and more preferably a hydrocarbon group having 2 to 4 carbon atoms.

[0096] In formula (2-3), R ３１ , R ３２ , R ３３ , and R ３４ are each independently preferably a hydrocarbon group having 1 to 10 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms, and even more preferably a hydrocarbon group having 1 to 3 carbon atoms.

[0097] In formula (2-3), R ３５ , R ３６ , R ３７ , and R ３８ R each independently represents a hydrocarbon group having preferably 1 to 20 carbon atoms, more preferably a hydrocarbon group having 1 to 16 carbon atoms, and even more preferably a hydrocarbon group having 1 to 12 carbon atoms. ３５ , R ３６ , R ３７ , and R ３８ The hydrocarbon group represented by is preferably an aryl group having 6 to 20 carbon atoms, or S—R ５８ More preferably, it is substituted with -S-R ５８ In formula (2-3), R ３５ , R３６ , R ３７ , and R ３８ are each independently particularly preferably —S—R ５８ or a hydrocarbon group having 1 to 12 carbon atoms substituted with

[0098] In formula (2-3), L ３１ , L ３２ , L ３３ , and L ３４ are each independently preferably —C(O)O— or —OC(O)—.

[0099] In formula (2-3), R ５８ represents a hydrocarbon group preferably having 1 to 10 carbon atoms, and more preferably a hydrocarbon group having 1 to 8 carbon atoms.

[0100] Specific examples of the lipid represented by formula (2) and a method for producing the same are described in WO 2022 / 230964. The entire contents of WO 2022 / 230964 are incorporated herein by reference.

[0101] <<Lipid represented by formula (3)>> In formula (3), R １ , R ２ , R ３ and R ４ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R １ , R ２ , R ３ and R ４ The substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by the formula (I) are each independently —C(O)O—R １１ , —OC(O)—R １２ , -O-R １３ , -CO-R １４ , -OC(O)OR １５ , or -S-S-R １６ indicates, R １１ , R １２ , R １３ , R １４ , R １５ and R １６ are each independently -S-R １７represents a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with １７ represents a hydrocarbon group having 1 to 12 carbon atoms, R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by the formula (I) are each independently —OH, —COOH, —NR ２１ R ２２ , -OC(O)OR ２３ , -C(O)O-R ２４ , —OC(O)—R ２５ , -O-R ２６ , —C(O)NR ２７ R ２８ , -NR ２９ C(O)R ３０ , -N(R ３１ ) S (O) ２ R ３２ , -N(R ３３ )C(O)N(R ３４ ) R ３５ , -N(R ３６ ) C(S)N(R ３７ ) R ３８ , -OC(O)N(R ３９ ) R ４０ , or -N(R ４１ )C(O)OR ４２ indicates, R ２１ and R ２２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; R ２３ , R ２４ , R ２５ , R ２６ , R ２７ , R ２８ , R ２９ , R ３０ , R ３１ , R ３２ , R ３３ , R ３４ , R ３５ , R ３６ , R ３７ , R ３８ , R ３９ , R ４０ , R ４１ , and R４２ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R ２３ , R ２４ , R ２５ , R ２６ , R ２７ , R ２８ , R ２９ , R ３０ , R ３１ , R ３２ , R ３３ , R ３４ , R ３５ , R ３６ , R ３７ , R ３８ , R ３９ , R ４０ , R ４１ , and R ４２ The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by is an aryl group having 6 to 20 carbon atoms, a heterocyclic group, —OH, —COOH, or NR ５１ R ５２ indicates R ５１ and R ５２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; R ７ , R ８ , and R ９ each independently represents a hydrocarbon group having 2 to 8 carbon atoms; R ５ and R ６ , or R ５ and R ７ may be joined together to form a 4- to 7-membered ring.

[0102] In formula (3), preferably, R １ Ga-R １ａ -L １ -R １ｂ indicates R １ａ represents a hydrocarbon group having 1 to 18 carbon atoms; L １ represents —C(O)O—, —OC(O)—, —OC(O)O—, or —S—S—, and R １ｂ represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ Ga-R ３ａ -L ３ -R ３ｂ indicates R ３ａrepresents a hydrocarbon group having 1 to 18 carbon atoms; L ３ represents —C(O)O—, —OC(O)—, —OC(O)O—, or —S—S—, and R ３ｂ represents a hydrocarbon group having 1 to 18 carbon atoms; R ２ and R ４ each independently represents an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; R ２ and R ４ The substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by the formula (I) are each independently —C(O)O—R １１ , —OC(O)—R １２ , -O-R １３ , -CO-R １４ , -OC(O)OR １５ , or -S-S-R １６ indicates, R １１ , R １２ , R １３ , R １４ , R １５ and R １６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 12 carbon atoms; R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 12 carbon atoms represented by the formula (I) are each independently —OH, —O—R ２６ , —C(O)NR ２７ R ２８ , or -NR ２９ C(O)R ３０ indicates, R ２６ , R ２７ , R ２８ , R ２９ , and R ３０ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 12 carbon atoms; R ２６ , R ２７ , R ２８ , R ２９ , and R ３０ The substituent on the optionally substituted hydrocarbon group having 1 to 12 carbon atoms represented by R is an aryl group or heterocyclic group having 6 to 10 carbon atoms,７ , R ８ and R ９ are each independently -(CH ２ ) ｎ -, and n is an integer of 2 to 8.

[0103] In formula (3), R is more preferably １ Ga-R １ａ -L １ -R １ｂ indicates R １ａ represents a hydrocarbon group having 1 to 18 carbon atoms; L １ represents —C(O)O— or —OC(O)—, and R １ｂ represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ Ga-R ３ａ -L ３ -R ３ｂ indicates R ３ａ represents a hydrocarbon group having 1 to 18 carbon atoms; L ３ represents —C(O)O— or —OC(O)—, and R ３ｂ represents a hydrocarbon group having 1 to 18 carbon atoms; R ２ and R ４ each independently represents a hydrocarbon group having 1 to 10 carbon atoms; R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 6 carbon atoms; R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 6 carbon atoms represented by the formula (I) are each independently —OH, —O—R ２６ , —C(O)NR ２７ R ２８ , or -NR ２９ C(O)R ３０ indicates, R ２６ , R ２７ , R ２８ , R ２９ , and R ３０ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 12 carbon atoms; R ２６ , R ２７ , R ２８ , R ２９ , and R ３０The substituent on the optionally substituted hydrocarbon group having 1 to 12 carbon atoms represented by R represents an aryl group having 6 to 10 carbon atoms, ７ , R ８ and R ９ are each independently -(CH ２ ) ｎ -, and n is an integer of 2 to 8.

[0104] In formula (3), R is most preferably １ Ga-R １ａ -L １ -R １ｂ indicates R １ａ represents a hydrocarbon group having 1 to 5 carbon atoms; L １ represents —C(O)O—, and R １ｂ represents a hydrocarbon group having 7 to 14 carbon atoms; R ３ Ga-R ３ａ -L ３ -R ３ｂ indicates R ３ａ represents a hydrocarbon group having 1 to 5 carbon atoms; L ３ represents —C(O)O—, and R ３ｂ represents a hydrocarbon group having 7 to 14 carbon atoms; R ２ and R ４ each independently represents a hydrocarbon group having 3 to 8 carbon atoms; R ５ and R ６ each independently represents a hydrocarbon group having 2 carbon atoms; R ７ , R ８ and R ９ are each independently -(CH ２ ) ｎ -, and n is an integer of 2 to 4.

[0105] A method for producing the lipid represented by formula (3) will be described. The lipid represented by formula (3) can be produced by combining known methods, and can be produced, for example, according to the production method shown below.

[0106] [Production Method 1] A method for producing a compound of formula [1] from a compound of formula [2].

[0107] In the formula, R １ , R ２ , R ３ , R４ , R ５ , R ６ , R ７ , R ８ and R ９ has the same meaning as above; R ８ａ , R ９ａ and R Ａ means a hydrocarbon group having 1 to 7 carbon atoms.

[0108] (1-1) The compound of formula [3A] can be produced by reacting the compound of formula [2] in the presence of water and an acid, with or without a solvent. The acid used in this reaction can be an inorganic or organic acid. Organic acids are preferred, and specific examples include formic acid, acetic acid, trifluoroacetic acid, 4-toluenesulfonic acid, and methanesulfonic acid, with formic acid being more preferred. The amount of acid used can be 1 to 100 times (v / w), preferably 1 to 10 times (v / w), relative to the compound of formula [2]. The amount of water used can be 0.1 to 100 times (v / w), preferably 0.1 to 10 times (v / w), relative to the compound of formula [2]. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. The amount of the solvent used is not particularly limited, but may be 0.1 to 50 times (v / w) the amount of the compound of formula [2]. This reaction may be carried out at −30 to 150° C., preferably 0 to 100° C., for 5 minutes to 48 hours.

[0109] (1-2) The compound of formula [1] can be produced by reacting a compound of formula [3A] with a compound of formula [4] in the presence of a reducing agent. Known examples of compounds of formula [4] include N,N-diethylethylenediamine and N,N-diethyl-1,3-diaminopropane. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples include halogenated hydrocarbons, alcohols, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include esters, with ethyl acetate being more preferred. The amount of solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of formula [3A]. Examples of reducing agents used in this reaction include sodium borohydride, sodium cyanoborohydride, pyridine borane, 2-picoline borane, and sodium triacetoxyborohydride, with sodium triacetoxyborohydride being more preferred. The amount of reducing agent used may be 1 to 100 times, preferably 1 to 10 times, the molar amount of the compound of formula [3A]. The amount of compound of formula [4] used may be 0.1 to 1 times the molar amount of the compound of formula [3A]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0110] (1-3) The compound of formula [5] can be produced by reacting the compound of formula [3A] with the compound of formula [4] in the presence of a reducing agent. This reaction can be carried out in accordance with the production method (1-2), and the compound of formula [4] can be used in an amount of 1 to 10 times the molar amount of the compound of formula [3A].

[0111] (1-4) The compound of formula [1] can be produced by reacting the compound of formula [3B] with the compound of formula [5] in the presence of a reducing agent. This reaction can be carried out in accordance with the production method (1-2), and the compound of formula [3B] can be used in an amount of 1 to 10 times the molar amount of the compound of formula [5].

[0112] In the compounds used in the above-mentioned production methods, when isomers (e.g., optical isomers, geometric isomers, tautomers, etc.) exist, these isomers can also be used. In addition, when solvates, hydrates, and various forms of crystals exist, these solvates, hydrates, and various forms of crystals can also be used.

[0113]

[0033] In the compounds used in the above-mentioned production methods, for example, compounds having an amino group, a hydroxyl group, or a carboxyl group can have these groups protected in advance with a conventional protecting group, and after the reaction, these protecting groups can be removed by a method known per se. The compounds obtained by the above-mentioned production methods can be derived into other compounds by subjecting them to a reaction known per se, such as condensation, addition, oxidation, reduction, rearrangement, substitution, halogenation, dehydration, or hydrolysis, or by an appropriate combination of these reactions.

[0114] In formulas (1) to (3), the hydrocarbon group is preferably an alkyl group, an alkenyl group, or an alkynyl group.

[0115] The alkyl group may be linear or branched, and may be linear or cyclic. Specific examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, and a 2-butyloctyl group. , 1-pentylhexyl group, 2-pentylheptyl group, 3-pentyloctyl group, 1-hexylheptyl group, 1-hexylnonyl group, 2-hexyloctyl group, 2-hexyldecyl group, 3-hexylnonyl group, 1-heptyloctyl group, 2-heptylnonyl group, 2-heptylundecyl group, 3-heptyldecyl group, 1-octylnonyl group, 2-octyldecyl group, 2-octyldodecyl group, 3-octylundecyl group, 2-nonylundecyl group, 3-nonyldodecyl group, 2-decyldodecyl group, 2-decyltetradecyl group, 3-decyltridecyl group, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like.

[0116] The alkenyl group may be linear or branched, linear or cyclic. Specific examples include allyl, prenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably, (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably, (Z)-tridec-8-enyl), tetradecenyl (preferably, tetradec-9-enyl), and pentadecenyl (preferably, (Z)-pentadecen-8-enyl). , a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadeca-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadec-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group), and the like.

[0117] The alkynyl group may be linear or branched, open-chain or cyclic, and specific examples thereof include a propargyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group.

[0118] Preferably, all of the above alkenyl groups have one or two double bonds, and preferably, all of the alkynyl groups have one or two triple bonds.

[0119] R in formula (3) ７ , R ８ , and R ９The hydrocarbon group having 2 to 8 carbon atoms represented by is preferably an alkylene group, an alkenylene group, or an alkynylene group. The alkylene group, alkenylene group, or alkynylene group having 2 to 8 carbon atoms may be linear or branched, and may be linear or cyclic. Specific examples include an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group.

[0120] The aryl group having 6 to 20 carbon atoms is preferably an aryl group having 6 to 18 carbon atoms, more preferably an aryl group having 6 to 10 carbon atoms. Specific examples include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group.

[0121] The heterocyclic group means a heteroaryl group or a heteroaliphatic ring group.

[0122] The heteroaryl group refers to an aromatic heterocyclic group, and may be an aromatic heterocyclic group fused with an aromatic hydrocarbon ring, a heteroaliphatic ring, or an aliphatic hydrocarbon ring, and is preferably a monocyclic nitrogen-containing heteroaryl group, a monocyclic oxygen-containing heteroaryl group, a monocyclic sulfur-containing heteroaryl group, a monocyclic nitrogen-containing oxygen-containing heteroaryl group, a monocyclic nitrogen-containing sulfur-containing heteroaryl group, a bicyclic nitrogen-containing heteroaryl group, a bicyclic oxygen-containing heteroaryl group, a bicyclic sulfur-containing heteroaryl group, a bicyclic nitrogen-containing oxygen-containing heteroaryl group, or a bicyclic nitrogen-containing sulfur-containing heteroaryl group. The five-membered heteroaryl group is a monocyclic heteroaryl group having five atoms constituting the ring.

[0123] Furthermore, the aromatic heterocycle means an aromatic ring having a heteroatom as a ring member, and may be a condensed aromatic heterocycle, an aromatic hydrocarbon ring, a heteroaliphatic ring, or an aliphatic hydrocarbon ring, and is preferably a monocyclic nitrogen-containing aromatic heterocycle, a monocyclic oxygen-containing aromatic heterocycle, a monocyclic sulfur-containing aromatic heterocycle, a monocyclic nitrogen-containing oxygen-containing aromatic heterocycle, a monocyclic nitrogen-containing sulfur-containing aromatic heterocycle, a bicyclic nitrogen-containing aromatic heterocycle, a bicyclic oxygen-containing aromatic heterocycle, a bicyclic sulfur-containing aromatic heterocycle, a bicyclic nitrogen-containing oxygen-containing aromatic heterocycle, or a bicyclic nitrogen-containing sulfur-containing aromatic heterocycle.

[0124] The term "monocyclic nitrogen-containing heteroaryl group" refers to a heteroaryl group having an aromatic ring containing at least one nitrogen atom, such as pyrrolinyl, pyrrolyl, tetrahydropyridyl, pyridyl, imidazolinyl, imidazolyl, pyrazolinyl, pyrazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazolyl, or tetrazolyl (which may be partially saturated). The heteroaryl group may be further fused with another aromatic ring or an aliphatic ring. The term "monocyclic oxygen-containing heteroaryl group" refers to a heteroaryl group having an aromatic ring containing at least one oxygen atom, such as a furanyl or pyranyl group (which may be partially saturated). The heteroaryl group may be further fused with another aromatic ring or an aliphatic ring. The term "monocyclic nitrogen-containing oxygen-containing heteroaryl group" refers to an oxazolyl, isoxazolyl, or oxadiazolyl group (which may be further fused with another aromatic ring or an aliphatic ring). The monocyclic nitrogen-containing sulfur-containing heteroaryl group means a thiazolyl, isothiazolyl, or thiadiazolyl group, etc. This heteroaryl group may be further condensed with another aromatic ring or an aliphatic ring.

[0125] The bicyclic nitrogen-containing heteroaryl group includes indolyl, isoindolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, quinolidinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pyrrolopyridyl, imidazopyridyl, pyrazolopyridyl, pyridopyrazyl, purinyl, pteridinyl, 5,6,7,8-tetrahydrophthalazinyl, 5,6,7,8-tetrahydrocinnolinyl, 1,2,3,4-tetrahydropyrido[2,3-d]pyridazinyl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, 5,6,7,8-tetrahydropyrido[3,4-d]pyridazinyl, 5,6, means a bicyclic heteroaryl group in which the ring containing at least one nitrogen atom has aromaticity (this heteroaryl group may be partially saturated), such as 7,8-tetrahydropyrido[3,2-c]pyridazinyl, 5,6,7,8-tetrahydropyrido[4,3-c]pyridazinyl, 6,7-dihydro-5H-cyclopenta[d]pyridazinyl, 6,7-dihydro-5H-cyclopenta[c]pyridazinyl, 2,3-dihydro-1H-pyrrolo[2,3-d]pyridazinyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyridazinyl, 6,7-dihydro-5H-pyrrolo[3,2-c]pyridazinyl, 6,7-dihydro-5H-pyrrolo[3,4-c]pyridazinyl and 6,7-dihydro-5H-pyrrolo[2,3-c]pyridazinyl groups.

[0126] The bicyclic oxygen-containing heteroaryl group means a bicyclic heteroaryl group in which the ring containing at least one oxygen atom has aromaticity, such as benzofuranyl, isobenzofuranyl, and chromenyl groups (this heteroaryl group may be partially saturated).

[0127] The bicyclic nitrogen-containing and oxygen-containing heteroaryl group includes benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, dihydropyranopyridyl, dihydrodioxinopyridyl, dihydropyridoxadienyl, 3,4-dihydro-2H-pyrano[2,3-d]pyridazinyl, 7,8-dihydro-5H-pyrano[3,4-d]pyridazinyl, 7,8-dihydro-6H-pyrano[3,2-c]pyridazinyl, and 7,8-dihydro-5H-pyrano[4,3-c]pyridazinyl. and 5,6-dihydrofuro[2,3-c]pyridazinyl groups, and the like.

[0128] The heteroaliphatic ring group refers to a nitrogen-containing heteroaliphatic ring group, an oxygen-containing heteroaliphatic ring group, a sulfur-containing heteroaliphatic ring group, a nitrogen-containing oxygen-containing heteroaliphatic ring group, a nitrogen-containing sulfur-containing heteroaliphatic ring group, a heterobridged ring group, or a heterospiro ring group. The heteroaliphatic ring refers to an aliphatic ring having a heteroatom as a ring member, and preferred examples include a nitrogen-containing heteroaliphatic ring, an oxygen-containing heteroaliphatic ring, a sulfur-containing heteroaliphatic ring, a nitrogen-containing oxygen-containing heteroaliphatic ring, a nitrogen-containing sulfur-containing heteroaliphatic ring, a heterobridged ring, and a heterospiro ring.

[0129] The term "nitrogen-containing heteroaliphatic cyclic group" refers to a heteroaliphatic cyclic group in which the ring containing at least one nitrogen atom is not aromatic, such as azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, octahydroazocinyl, imidazolidinyl, pyrazolidinyl, piperazinyl, and homopiperazinyl groups. This nitrogen-containing heteroaliphatic cyclic group may be further fused with another aromatic ring or an aliphatic ring. The term "oxygen-containing heteroaliphatic cyclic group" refers to a tetrahydrofuranyl, tetrahydropyranyl, oxetanyl, or 1,3-dioxanyl group, for example. This oxygen-containing heteroaliphatic cyclic group may be further fused with another aromatic ring or an aliphatic ring. The term "nitrogen-containing oxygen-containing heteroaliphatic cyclic group" refers to a morpholinyl or 1,4-oxazepanyl group, for example. This nitrogen-containing oxygen-containing heteroaliphatic cyclic group may be further fused with another aromatic ring or an aliphatic ring.

[0130] The lipids represented by any of formulas (1) to (3) may form salts. Examples of salts of basic groups include salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid.

[0131] Salts of acidic groups include, for example, salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, ammonium salts, and salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine. Of the above-mentioned salts, preferred salts include pharmacologically acceptable salts.

[0132] In the lipid nanoparticles, the amount of the lipid represented by formula (1), formula (2), or formula (3) is preferably 20 mol% to 80 mol%, more preferably 25 mol% to 70 mol%, and even more preferably 30 mol% to 65 mol%, relative to the total lipid amount.

[0133] <Other Lipids> The second solution in step 2 may further contain at least one lipid selected from the group consisting of a neutral lipid, a lipid having a nonionic hydrophilic polymer, and a sterol. For example, the second solution in step 2 may contain all of a neutral lipid, a lipid having a nonionic hydrophilic polymer, and a sterol.

[0134] <<Neutral lipids>> The neutral lipids are not particularly limited, but include phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, etc., with phosphatidylethanolamine or phosphatidylcholine being preferred. The neutral lipids may be used alone or in combination with multiple different neutral lipids.

[0135] The phosphatidylcholine is not particularly limited, but includes soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated egg yolk lecithin (HEPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), etc. Among the above, dipalmitoylphosphatidylcholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is preferred, and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is more preferred.

[0136] The phosphatidylethanolamine is not particularly limited, and examples thereof include dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, dioctadecylphosphatidylethanolamine, diphytanylphosphatidylethanolamine, etc. Among the above, dioleoylphosphatidylethanolamine (DOPE) is particularly preferred.

[0137] Examples of sphingomyelin include, but are not limited to, egg yolk-derived sphingomyelin, milk-derived sphingomyelin, etc. Examples of ceramide include, but are not limited to, egg yolk-derived ceramide, milk-derived ceramide, etc.

[0138] In lipid nanoparticles, the amount of neutral lipid is preferably 3 mol% or more and 55 mol% or less, more preferably 3 mol% or more and 45 mol% or less, and even more preferably 3 mol% or more and 30 mol% or less, based on the total amount of the constituent lipid components.

[0139] <<Lipid Having a Nonionic Hydrophilic Polymer>> By including a lipid having a nonionic hydrophilic polymer in the lipid nanoparticles, the lipid nanoparticles can have a dispersion stabilizing effect. As the lipid having a nonionic hydrophilic polymer, a lipid having a nonionic hydrophilic polymer chain is preferred.

[0140] Examples of nonionic hydrophilic polymers include, but are not limited to, nonionic vinyl polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, nonionic modified natural polymers, and block polymers or graft copolymers having two or more of these polymers as constituent units.

[0141] Of these lipids having a nonionic hydrophilic polymer, nonionic polyethers, nonionic polyesters, nonionic polyamino acids or nonionic synthetic polypeptides are preferred, nonionic polyethers or nonionic polyesters are more preferred, nonionic polyethers or nonionic monoalkoxy polyethers are even more preferred, and polyethylene glycol (polyethylene glycol will also be referred to as PEG hereinafter) is particularly preferred.

[0142] Lipids having a nonionic hydrophilic polymer include, but are not limited to, PEG-modified phosphatidylethanolamine, diacylglycerol PEG derivatives, monoacylglycerol PEG derivatives, dialkylglycerol PEG derivatives, cholesterol PEG derivatives, ceramide PEG derivatives, and polysarcosine derivatives. Among these, monoacylglycerol PEG or diacylglycerol PEG is preferred. The weight-average molecular weight of the PEG chain of the nonionic polymer derivative is preferably 500 to 5,000, more preferably 750 to 3,000. The nonionic hydrophilic polymer chain may be branched or may have a substituent such as a hydroxymethyl group.

[0143] In the lipid nanoparticles, the amount of lipid having a nonionic hydrophilic polymer is preferably 0.25 mol% to 12 mol% relative to the total lipid amount, more preferably 0.5 mol% to 10 mol%, even more preferably 0.5 mol% to 6 mol%, and particularly preferably 1 mol% to 3 mol%.

[0144] <<Sterols>> By including a sterol in lipid nanoparticles, membrane fluidity can be reduced, resulting in a stabilizing effect for the lipid nanoparticles. Examples of sterols include, but are not limited to, cholesterol, phytosterols (sitosterol, β-sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol, etc.), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, and cholesteryl-4'-hydroxybutyl ether. Among these, cholesterol is preferred. In the lipid nanoparticles of the present invention, the amount of sterol incorporated is preferably 10 mol% to 70 mol%, more preferably 15 mol% to 70 mol%, and even more preferably 20 mol% to 60 mol%, based on the total lipid amount.

[0145] <Active ingredient> The active ingredient in the present invention can be at least one selected from the group consisting of nucleic acids, proteins, peptides, and small molecules. The active ingredient is preferably a nucleic acid or a peptide. When a nucleic acid is used as the active ingredient, the method of the present invention can be used in the production of nucleic acid medicines or nucleic acid-containing formulations used in gene therapy.

[0146] Examples of nucleic acids include siRNA (small interfering RNA), miRNA (micro RNA), ASO (antisense oligonucleotide), mRNA (messenger RNA), saRNA (self-amplifying RNA), dsRNA (double-stranded RNA), sgRNA (single guide RNA), shRNA (small hairpin RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), snRNA (small nuclear RNA), circular RNA, plasmid DNA, nanoplasmid DNA, single-stranded DNA, double-stranded DNA, ribozymes, aptamers, etc., and any of these may be included. Two or more types of nucleic acids may also be used.

[0147] Chemically modified nucleic acids may also be included. For example, the use of mRNA or saRNA containing chemically modified nucleic acids has been shown to improve the expression, expression rate, half-life, and / or expressed protein concentration of proteins translated therefrom. mRNA or saRNA containing chemically modified nucleic acids has also been shown to avoid harmful biological responses, such as immune responses and / or degradation pathways. Examples of chemically modified nucleic acids include the compounds described in paragraphs 0108 to 0128 of JP-A-2022-525540. RNA is particularly preferred as the nucleic acid, and RNAs ranging from low molecular weight to high molecular weight can be preferably used, with RNAs having 5 to 20,000 bases being preferred.

[0148] Examples of proteins, peptides, and small molecules include intracellular proteins, intracellular peptides, transmembrane proteins, transmembrane peptides, secretory proteins, secretory peptides, synthetic proteins, synthetic peptides, natural low-molecular-weight compounds, synthetic low-molecular-weight compounds, and compounds with antitumor activity. Peptides that can be encapsulated are peptides with natural or unnatural amino groups and may have a linear or cyclic structure. Cyclic peptides are preferably linked via amide or thioether groups.

[0149] Examples of low molecular weight compounds include anticancer agents, antibacterial agents, and antifungal agents. These proteins, peptides, and low molecular weight compounds may or may not be physiologically active in vivo. Here, low molecular weight compounds refer to organic compounds with a molecular weight of approximately 1,000 or less.

[0150] In the lipid nanoparticles, the mass ratio of lipid to active ingredient is preferably 2 to 1000, more preferably 3 to 500, even more preferably 4 to 200, and particularly preferably 4 to 100.

[0151] Lipid nanoparticles are particles composed of lipids and have a size on the order of nanometers. Lipid nanoparticles usually have an internal aqueous phase, but the structure of lipid nanoparticles is not limited as long as they contain lipids.

[0152] The morphology of lipid nanoparticles can be confirmed by electron microscopy or structural analysis using X-rays. For example, by using a cryo-transmission electron microscope (cryo-TEM) method, it can be confirmed whether the lipid particles have a lipid bilayer structure (lamellar structure) and an inner water layer, like liposomes, or whether they have a core with high electron density inside the particle and a structure packed with lipids and other components. Small-angle X-ray scattering (SAXS) measurement can also be used to confirm the presence or absence of a lipid bilayer structure (lamellar structure) for lipid nanoparticles.

[0153] The average particle size of the lipid nanoparticles is not particularly limited, but is generally 10 nm to 1000 nm, preferably 20 nm to 500 nm. The average particle size of the lipid nanoparticles is more preferably less than 200 nm, even more preferably less than 30 nm to 150 nm, even more preferably less than 40 nm to 120 nm, and particularly preferably less than 40 nm to 100 nm. The average particle size of the lipid nanoparticles can be measured, for example, using a multi-analyte nanoparticle size measurement system, nanoSAQLA (Otsuka Electronics). The average particle size and polydispersity index (PDI) can be obtained by cumulant analysis. The polydispersity index (PDI) is preferably less than 0.20, more preferably less than 0.18, and even more preferably less than 0.10.

[0154] <Use of lipid nanoparticles> The lipid nanoparticles produced by the method of the present invention can be administered to cells to deliver active ingredients contained in the lipid nanoparticles to the cells. The method of delivering active ingredients to cells may exclude or include methods for treating humans.

[0155] The cells to which the active ingredient is delivered are not particularly limited, and cells can be selected according to the purpose. For example, in vivo, the cells may be cancer cells or other abnormal cells as therapeutic target cells, or normal tissue cells (such as liver cells or muscle cells) or immune cells to achieve a therapeutic effect. Ex vivo, stem cells such as induced pluripotent stem cells (iPS cells) and mesenchymal stem cells, and immune cells can be used. The cells are preferably mammalian-derived, more preferably human-derived.

[0156] Immune cells are not particularly limited, but include, for example, lymphocytes (e.g., T cells, B cells, natural killer cells (NK cells), NKT cells, iNKT cells), monocytes, macrophages, mast cells, dendritic cells, granulocytes (e.g., neutrophils, eosinophils, and basophils), hematopoietic stem / progenitor cells, primary immune cells, CD3 ＋ cells, CD4 ＋ cells, CD8 ＋ They may be selected from T cells, regulatory T cells (Treg), B cells, NK cells, innate lymphocytes, or dendritic cells (DCs).

[0157] When the lipid nanoparticles contain an active ingredient having medicinal uses, the lipid nanoparticles can be administered to a living body as a pharmaceutical composition.

[0158] When lipid nanoparticles are used as a pharmaceutical composition, they can be administered to a living body alone or mixed with a pharmaceutically acceptable administration vehicle (e.g., physiological saline or a buffer solution).

[0159] The concentration of the lipid nanoparticles in the mixture with the pharmaceutically acceptable administration vehicle is not particularly limited and can generally be 0.05% to 90% by mass. In addition, other pharmaceutically acceptable additives, such as pH adjusting buffers and osmotic pressure adjusting agents, may also be added to the pharmaceutical composition containing the lipid nanoparticles.

[0160] The route of administration of a pharmaceutical composition containing lipid nanoparticles is not particularly limited, and can be administered by any method. Administration methods include oral administration and parenteral administration (intra-articular administration, intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration, intravitreal administration, intraperitoneal administration, intramuscular administration, intravaginal administration, intravesical administration, intrathecal administration, pulmonary administration, rectal administration, colonic administration, buccal administration, nasal administration, intracisternal administration, inhalation, etc.). Parenteral administration is preferred, and preferred administration methods are intravenous injection, subcutaneous injection, intradermal injection, or intramuscular injection. A pharmaceutical composition containing lipid nanoparticles can also be administered by direct injection into the diseased site.

[0161] The dosage form of a pharmaceutical composition containing lipid nanoparticles is not particularly limited, but when administered orally, the lipid nanoparticles can be combined with an appropriate excipient and used in the form of tablets, troches, capsules, pills, suspensions, syrups, etc. Furthermore, formulations suitable for parenteral administration can contain additives such as antioxidants, buffers, bacteriostatic agents, and isotonic sterile injections, suspending agents, solubilizing agents, thickening agents, stabilizers, or preservatives, as appropriate.

[0162] The present invention will now be described with reference to examples, but the present invention is not limited to these examples.

[0163] The structures of Compound A, Compound B and Compound C used in the Comparative Examples and Examples are shown below.

[0164] Compound A (described in WO2021 / 095876)

[0165] Compound B (described in WO2022 / 230964)

[0166] Compound C

[0167] [Synthesis Example of Compound C] (1)

[0168] To a mixture of 2-hexyl-1-octanol (5.0 g), 5-bromovaleric acid (4.6 g) and toluene (25 mL) was added sulfuric acid (0.5 mL), and the mixture was stirred for 5 hours at 110° C. After cooling to room temperature, the reaction mixture was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2-hexyloctyl 5-bromopentanoate (8.2 g) as a colorless oil. 1 H-NMR(CDCl3)δ: 3.98 (2H, d, J=5.7Hz), 3.42 (2H, t, J=6.5Hz), 2.34 (2H, t, 7.2Hz), 1.94-1.87 (2H, m), 1.82-1.74 (2H, m), 1.65-1.57 (1H, m), 1.32-1.23 (20H, m), 0.90-0.86 (6H, m).

[0169] (2)

[0170] To a mixture of 2-hexyloctyl 5-bromopentanoate (1.2 g), n-octylamine (1.2 g), and 1-methyl-2-pyrrolidone (6 mL), potassium carbonate (1.3 g) was added and stirred at 60°C for 5 hours. After the reaction mixture was cooled to room temperature, ethyl acetate (12 mL) and water (6 mL) were added, and the organic layer was separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (methanol-ethyl acetate-hexane) to yield 2-hexyloctyl 5-(octylamino)pentanoate (1.25 g) as a pale yellow oil. 1 H-NMR(CDCl3)δ: 3.96 (2H, d, J=5.8Hz), 2.63-2.52 (4H, m), 2.32 (2H, t, J=7.4Hz), 2.06-1.98 (1H, m), 1.70-1.40 (7H, m), 1.34-1.20 (30H, m), 0.90-0.87 (9H, m).

[0171] (3)

[0172] A mixture of 2-hexyloctyl 5-(octylamino)pentanoate (1.25 g), acetonitrile (4 mL), 2,2-diethoxyethyl(4-nitrophenyl)carbonate (0.49 g), and triethylamine (0.46 mL) was stirred at 60°C for 4 hours. Ethyl acetate (4 mL) and water (4 mL) were added to the reaction mixture, which was cooled to room temperature, and the organic layer was separated. The resulting organic layer was washed with water and saturated brine, then dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to yield 2-hexyloctyl 5-(((2,2-diethoxyethoxy)carbonyl)(octyl)amino)pentanoate (0.83 g) as a pale yellow oil. 1 H-NMR(CDCl3)δ: 4.69 (1H, t, J=5.4Hz), 4.08 (2H, d, J=5.5Hz), 3.97 (2H, d, J=5.7Hz), 3.74-3.62 (2H, m), 3.60-3.52 (2H, m), 3.26-3.14 (4H, m), 2.36-2.30 (2H, m), 1.65-1.46 (7H, m), 1.33-1.19 (36H, m), 0.90-0.85 (9H, m).

[0173] (4)

[0174] A mixture of 2-hexyloctyl 5-(((2,2-diethoxyethoxy)carbonyl)(octyl)amino)pentanoic acid (0.83 g), formic acid (6 mL), and water (1.5 mL) was stirred at 50°C for 3 hours, after which toluene was added and the solvent was evaporated under reduced pressure. This procedure of adding toluene again and evaporating under reduced pressure was repeated twice to obtain a pale yellow oily substance, 2-hexyloctyl 5-(octyl((2-oxoethoxy)carbonyl)amino)pentanoic acid (0.94 g), as a crude product. 1H-NMR(CDCl3)δ: 4.10 (4H, t, J=6.3Hz), 3.97 (4H, d, J=5.7Hz), 3.25-3.11 (8H, m), 2.79 (4H, t, J=6.4Hz), 2.69-2.63 (2H, m), 2.56-2.47 (6H, m), 2.36-2.29 (4H, m), 1.64-1.49 (14H, m), 1.32-1.21 (60H, m), 1.01 (6H, t, J=7.0Hz), 0.90-0.85 (18H, m).

[0175] (5)

[0176] To a solution of 2-hexyloctyl 5-(octyl((2-oxoethoxy)carbonyl)amino)pentanoate (0.73 g) in ethyl acetate (8 mL), N,N-diethylethylenediamine (0.083 g), acetic acid (43 mg), and sodium triacetoxyborohydride (0.91 g) were added at room temperature, and the mixture was stirred at room temperature for 5 hours. After adding 20% ​​aqueous potassium carbonate solution (10 mL) to the reaction mixture, the organic layer was separated and washed with water and saturated brine. After drying over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (methanol-ethyl acetate-hexane) to give bis(2-hexyloctyl) 11-(2-(diethylamino)ethyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate (0.39 g) as a pale yellow oil. 1 H-NMR(CDCl3)δ: 4.10 (4H, t, J=6.3Hz), 3.97 (4H, d, J=5.7Hz), 3.25-3.11 (8H, m), 2.79 (4H, t, J=6.4Hz), 2.69-2.63 (2H, m), 2.56-2.47 (6H, m), 2.36-2.29 (4H, m), 1.64-1.49 (14H, m), 1.32-1.21 (60H, m), 1.01 (6H, t, J=7.0Hz), 0.90-0.85 (18H, m). MS m / z(M+H):1108.

[0177] Comparative Example 1 and Examples 1 to 9 <Step 1: Preparation of First Solution (Aqueous Phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 82.5 μg / mL to obtain an aqueous phase.

[0178] <Step 2: Preparation of second solution (oil phase)> Compound A (lipid represented by formula (1) of the present invention; 2-pentylheptyl 6-(2-(decanoyloxy)ethyl)-3-ethyl-12-hexyl-10-oxo-9,11-dioxa-3,6-diazahexadecan-16-oate), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, product name: COATSOME ME-8181; NOF Corporation), cholesterol (product name: Cholesterol HP; Nippon Fine Chemical Co., Ltd.), DMG-PEG2000 (product name: SUNBRIGHT® GM-020; NOF Corporation), Corporation) was dissolved in ethanol at a mixing ratio (molar ratio) of 40 / 10 / 47 / 3 to a total lipid concentration of 12.5 mmol / L to obtain an oil phase.

[0179] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 3:1 using a NanoAssembler Ignite and a microchannel cartridge NexGen without a dilution channel (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, as described above, the FLuc mRNA concentration of the first solution and the total lipid concentration of the second solution were set so that the weight concentration ratio of lipid to nucleic acid in LNP dispersion A was 32.

[0180] <Step 4: Mixing of LNP Dispersion A and Third Solution> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 1, and then mixed with a third solution (20 mmol / L Tris-HCl buffer, pH 7.4) to obtain LNP Dispersion B. The above mixing was performed at a mixing ratio (volume ratio) of LNP Dispersion A to the third solution of 1:3.

[0181] <Step 5: pH Adjustment and Post-Treatment Step> LNP Dispersion B obtained in step (4) above was allowed to stand at room temperature for approximately 150 minutes, and then mixed with 20 mmol / L Tris-HCl buffer containing 8% sucrose at pH 8.4 using a T-mixer to adjust the pH to 7.5 to 7.7, which is higher than the pKa.

[0182] The pH-adjusted LNP dispersion was concentrated approximately 10-fold using ultrafiltration with a centrifugal filter (Amicon Ultra-15 100 kDa), then transferred to a dialysis unit (Slide-Alyzer G3 Dialisis Cassettes, 10 k MWCO) and dialyzed against a dialysate (20 mmol / L Tris-HCl buffer containing 8% sucrose, pH 7.4). The resulting dialyzed sample was adjusted to a nucleic acid concentration of 30 μg / mL using the dialysate, and filtered through a 0.22 μm filter to obtain FLuc mRNA-encapsulating lipid particles.

[0183] Comparative Example 2 and Examples 10 to 15 <Step 1: Preparation of First Solution (Aqueous Phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 77.4 μg / mL to obtain an aqueous phase.

[0184] <Step 2: Preparation of second solution (oil phase)> Compound A, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40 / 10 / 47 / 3 in the same manner as in Examples 1 to 9, except that the total lipid concentration was adjusted to 15.6 mmol / L, to obtain an oil phase.

[0185] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 4:1 using a NanoAssembler Ignite and a microchannel cartridge NexGen (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, as described above, the FLuc mRNA concentration of the first solution and the total lipid concentration of the second solution were set so that the weight concentration ratio of lipid to nucleic acid in LNP dispersion A was 32.

[0186] <Step 4: Mixing LNP Dispersion A with the Third Solution> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 2, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B. The above mixing was performed with a mixing ratio (volume ratio) of LNP Dispersion A to the third solution of 1:3.

[0187] The subsequent steps were carried out in the same manner as in Examples 1 to 9 and Comparative Example 1 described above.

[0188] Comparative Example 3 and Examples 16 to 18 <Step 1: Preparation of First Solution (Aqueous Phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 72.2 μg / mL to obtain an aqueous phase.

[0189] <Step 2: Preparation of second solution (oil phase)> Compound A, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40 / 10 / 47 / 3 in the same manner as in Examples 1 to 9, except that the total lipid concentration was adjusted to 21.9 mmol / L, to obtain an oil phase.

[0190] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 6:1 using a NanoAssembler Ignite and a microchannel cartridge NexGen (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, as described above, the FLuc mRNA concentration of the first solution and the total lipid concentration of the second solution were set so that the weight concentration ratio of lipid to nucleic acid in LNP dispersion A was 32.

[0191] <Step 4: Mixing LNP Dispersion A with the Third Solution> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 3, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B. The above mixing was performed with a mixing ratio (volume ratio) of LNP Dispersion A to the third solution of 1:3.

[0192] The subsequent steps were carried out in the same manner as in Examples 1 to 9 and Comparative Example 1 described above.

[0193] Comparative Example 4 and Examples 19 to 21 <Step 1: Preparation of First Solution (Aqueous Phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 74.3 μg / mL to obtain an aqueous phase.

[0194] <Step 2: Preparation of second solution (oil phase)> Compound A, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40 / 10 / 47 / 3 in the same manner as in Examples 1 to 9, except that the total lipid concentration was adjusted to 18.8 mmol / L, to obtain an oil phase.

[0195] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 5:1 using a NanoAssembler Ignite and a microchannel cartridge NexGen (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, as described above, the FLuc mRNA concentration of the first solution and the total lipid concentration of the second solution were set so that the weight concentration ratio of lipid to nucleic acid in LNP dispersion A was 32.

[0196] <Step 4: Mixing LNP Dispersion A with the Third Solution> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 3, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B. The above mixing was performed with a mixing ratio (volume ratio) of LNP Dispersion A to the third solution of 1:3.

[0197] The subsequent steps were carried out in the same manner as in Examples 1 to 9 and Comparative Example 1 described above.

[0198] Comparative Example 5 and Examples 22 to 24 <Step 1: Preparation of First Solution (Aqueous Phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 87.7 μg / mL to obtain an aqueous phase.

[0199] <Step 2: Preparation of second solution (oil phase)> Compound A, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40 / 10 / 47 / 3 in the same manner as in Examples 1 to 9, except that the total lipid concentration was adjusted to 10.6 mmol / L, to obtain an oil phase.

[0200] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 2.4:1 using a NanoAssembler Ignite and a microchannel cartridge NexGen (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, in LNP dispersion A, the FLuc mRNA concentration of the first solution and the total lipid concentration of the second solution were set as described above so that the weight concentration ratio of lipid to nucleic acid was 32.

[0201] <Step 4: Mixing LNP Dispersion A with the Third Solution> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 4, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B. The above mixing was performed at a mixing ratio (volume ratio) of LNP Dispersion A to the third solution of 1:3.

[0202] The subsequent steps were carried out in the same manner as in Examples 1 to 9 and Comparative Example 1 described above.

[0203] Comparative Example 6 and Examples 25 and 26 <Step 1: Preparation of First Solution (Aqueous Phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 92.9 μg / mL to obtain an aqueous phase.

[0204] <Step 2: Preparation of second solution (oil phase)> Compound A, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40 / 10 / 47 / 3 in the same manner as in Examples 1 to 9, except that the total lipid concentration was adjusted to 9.4 mmol / L, to obtain an oil phase.

[0205] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 2:1 using a NanoAssembler Ignite and a microchannel cartridge NexGen (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, as described above, the FLuc mRNA concentration of the first solution and the total lipid concentration of the second solution were set so that the weight concentration ratio of lipid to nucleic acid in LNP dispersion A was 32.

[0206] <Step 4: Mixing LNP Dispersion A with the Third Solution> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 5, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B. The above mixing was performed at a mixing ratio (volume ratio) of LNP Dispersion A to the third solution of 1:3.

[0207] The subsequent steps were carried out in the same manner as in Examples 1 to 9 and Comparative Example 1 described above.

[0208] Comparative Examples 7 and 8 and Examples 27 and 28 <Step 1: Preparation of First Solution (Aqueous Phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 82.5 μg / mL to obtain an aqueous phase.

[0209] <Step 2: Preparation of second solution (oil phase)> In the same manner as in Examples 1 to 9, Compound A, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40 / 10 / 47 / 3 to give a total lipid concentration of 12.5 mmol / L, to obtain an oil phase.

[0210] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 3:1 using a NanoAssembler Ignite and a microchannel cartridge NexGen without a dilution channel (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, as described above, the FLuc mRNA concentration of the first solution and the total lipid concentration of the second solution were set so that the weight concentration ratio of lipid to nucleic acid in LNP dispersion A was 32.

[0211] <Step 4: Mixing of LNP Dispersion A and Third Solution> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 5, and then mixed with the third solution to obtain LNP Dispersion B. The above mixing was performed at a mixing ratio (volume ratio) of LNP Dispersion A to the third solution of 1:3.

[0212] <Step 5: pH Adjustment and Post-Treatment Step> In Comparative Example 7 and Example 27, the LNP dispersion B obtained in step (4) above was allowed to stand at room temperature for approximately 150 minutes, then concentrated approximately 5-fold using a tangential flow filtration system (KR2i TFF system) manufactured by Repligen, and then dialyzed against a dialysate (20 mmol / L Tris-HCl buffer containing 8% sucrose, pH 7.4) to adjust the pH to 7.3-7.4. The resulting pH-adjusted sample was filtered through a Sartoguard PES filter and then adjusted to a nucleic acid concentration of 36 μg / mL using the dialysate. The concentration-adjusted sample was filtered through a Millipore 0.5 / 0.2 μm Express SHC filter to obtain lipid particles encapsulating FLuc mRNA.

[0213] In Comparative Example 8 and Example 28, LNP dispersion B obtained in step (4) above was allowed to stand at room temperature for approximately 150 minutes, then transferred to a dialysis unit (Slide-Alyzer G3 Dialisis Cassettes, 10k MWCO) and dialyzed against a dialysate (20 mmol / L Tris-HCl buffer containing 8% sucrose, pH 7.4) to adjust the pH to 7.3-7.4. The resulting pH-adjusted sample was filtered through a 0.22 μm filter to obtain lipid particles encapsulating FLuc mRNA.

[0214] Examples 29 and 30 <Step 1: Preparation of First Solution (Aqueous Phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 82.5 μg / mL to obtain an aqueous phase.

[0215] <Step 2: Preparation of second solution (oil phase)> In the same manner as in Examples 1 to 9, Compound A, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40 / 10 / 47 / 3 to give a total lipid concentration of 12.5 mmol / L, to obtain an oil phase.

[0216] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 3:1 using a NanoAssembler Ignite and a microchannel cartridge NexGen without a dilution channel (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, as described above, the FLuc mRNA concentration of the first solution and the total lipid concentration of the second solution were set so that the weight concentration ratio of lipid to nucleic acid in LNP dispersion A was 32.

[0217] <Step 5: pH Adjustment and Post-Treatment Step> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 6, and then mixed with 20 mmol / L Tris-HCl buffer containing 8% sucrose at pH 8.4 using a T-mixer to adjust the pH to 7.5 to 7.7, which is higher than the pKa.

[0218] The pH-adjusted LNP dispersion was concentrated approximately 10-fold using ultrafiltration with a centrifugal filter (Amicon Ultra-15 100 kDa), then transferred to a dialysis unit (Slide-Alyzer G3 Dialisis Cassettes, 10 k MWCO) and dialyzed against a dialysate (20 mmol / L Tris-HCl buffer containing 8% sucrose, pH 7.4). The resulting dialyzed sample was adjusted to a nucleic acid concentration of 30 μg / mL using the dialysate, and filtered through a 0.22 μm filter to obtain FLuc mRNA-encapsulating lipid particles.

[0219] Comparative Example 9 and Examples 31 to 33 <Step 1: Preparation of first solution (aqueous phase)> saRNA (saRNA containing the base sequence of mRNA encoding the spike protein and RNA replication protein of SARS-CoV-2 described in Nature Communications, Vol. 11, 3523 (2020)) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 82.5 μg / mL to obtain an aqueous phase.

[0220] <Step 2: Preparation of second solution (oil phase)> In the same manner as in Examples 1 to 9, Compound A, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40 / 10 / 47 / 3 to give a total lipid concentration of 12.5 mmol / L, to obtain an oil phase.

[0221] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 3:1 using NanoAssembler Ignite and a microchannel cartridge NexGen (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, in LNP dispersion A, the saRNA concentration of the first solution and the total lipid concentration of the second solution were set as described above so that the weight concentration ratio of lipid to nucleic acid was 32.

[0222] <Step 4: Mixing LNP Dispersion A with the Third Solution> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 7, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B. The above mixing was performed at a mixing ratio (volume ratio) of LNP Dispersion A to the third solution of 1:3.

[0223] The subsequent steps were carried out in the same manner as in Examples 1 to 9 and Comparative Example 1 described above.

[0224] Examples 34 to 37 <Step 1: Preparation of First Solution (Aqueous Phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer adjusted to pH 4 to a concentration of 82.5 μg / mL to obtain an aqueous phase.

[0225] <Step 2: Preparation of second solution (oil phase)> In Examples 34 and 35, compound B (lipid represented by formula (2)) bis(2-pentylheptyl) 11-(3-(diethylamino)propyl)-5,17-dihexyl-7,15-dioxo-6,8,14,16-tetraoxa-11-azahenicosanedioate was used instead of compound A as the pH-responsive lipid. Compound B, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40 / 10 / 47 / 3 to give a total lipid concentration of 12.5 mmol / L, in the same manner as in Examples 1 to 9, to obtain an oil phase.

[0226] In Examples 36 and 37, as the pH-responsive lipid, compound C (lipid represented by formula (3)) bis(2-hexyloctyl) 11-(2-(diethylamino)ethyl)-6,16-dioctyl-7,15-dioxo-8,14-dioxa-6,11,16-triazahenicosanedioate was used instead of compound A. In the same manner as in Examples 1 to 9, except that compound C, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a ratio of 40 / 10 / 47 / 3 (molar ratio) so that the total lipid concentration was 12.5 mmol / L, an oil phase was obtained.

[0227] <Step 3: Mixing the first solution and the second solution> The first solution and the second solution were mixed at a mixing ratio (volume ratio) of 3:1 using a NanoAssembler Ignite and a microchannel cartridge NexGen (Precision NanoSystems) to obtain LNP dispersion A. At this time, the flow rate of LNP dispersion A, which is a mixture of the first and second solutions, was mixed to be 14 mL / min. In addition, as described above, the FLuc mRNA concentration of the first solution and the total lipid concentration of the second solution were set so that the weight concentration ratio of lipid to nucleic acid in LNP dispersion A was 32.

[0228] <Step 4: Mixing LNP Dispersion A with the Third Solution> LNP Dispersion A obtained in Step 3 was allowed to stand at room temperature for the time shown in Table 8, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B. The above mixing was performed at a mixing ratio (volume ratio) of LNP Dispersion A to the third solution of 1:3.

[0229] The subsequent steps were carried out in the same manner as in Examples 1 to 9 and Comparative Example 1 described above.

[0230] <Particle size measurement> The particle size of mRNA-encapsulated lipid particles was measured using a nanoSAQLA multi-analyte nanoparticle size measurement system (Otsuka Electronics) after diluting 5-fold or 40-fold with phosphate-buffered saline (PBS). Data analysis was performed using the Marquardt method, and the average particle size and polydispersity index (PDI) were obtained by cumulant analysis.

[0231] <Evaluation of mRNA encapsulation rate> (Quantification of total mRNA concentration) 450 μL of methanol was added to 50 μL of mRNA and lipid particle samples encapsulating mRNA to dissolve the lipids, and the total mRNA concentration was quantified by measuring the absorbance at 260 nm using an absorption spectrometer (Thermo Fisher Scientific).

[0232] (Quantification of mRNA concentration in the external aqueous phase) Using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific), the external aqueous phase mRNA concentration was quantified by the standard addition method of mRNA solution. A nucleic acid dilution series was prepared by diluting the mRNA with 1x TE buffer to a final concentration of 20-400 ng / mL. TE stands for Tris / EDTA (ethylenediaminetetraacetic acid). The lipid particle sample encapsulating mRNA was either diluted undiluted or diluted 5-fold with 1x TE buffer to prepare a measurement sample solution. 10 μL of the measurement sample solution and 90 μL of the nucleic acid dilution series were added to a 96-well plate, and 100 μL of RiboGreen reagent (contained in the above-mentioned Quanti-iT Ribogreen RNA Assay Kit) diluted 200-fold with 1×TE buffer was added to each well. The external aqueous phase mRNA concentration of each sample was calculated from the fluorescence intensity (excitation wavelength: 485 nm, fluorescence wavelength: 535 nm) obtained using a fluorometer (Infinite 200 ProM Nano+) according to the method of standard addition.

[0233] (Calculation of Encapsulation Rate) Using the quantitative results of the total mRNA concentration and the mRNA concentration in the external aqueous phase obtained in the above steps, the mRNA encapsulation rate of the mRNA-encapsulating lipid particles was calculated according to the following formula: mRNA encapsulation rate (%) = (total mRNA concentration - mRNA concentration in the external aqueous phase) ÷ total mRNA concentration × 100

[0234] <Lipid quantification> The amount of lipid in the mRNA-encapsulated lipid particle sample was calculated by measuring it using an HPLC equipped with a CAD detector. HPLC apparatus: Thermo Ultimate 3000 (Thermo Fisher Scientific) Column: Triart C18 manufactured by YMC, inner diameter 3 mm, length 150 mm, particle diameter 1.9 μm Detector: CAD

[0235] The change in particle size before and after pH adjustment and the lipid residual rate after filtration were used to evaluate the variation in physical properties between processes. The change in particle size before and after pH adjustment was obtained by measuring the particle size of the mRNA-encapsulated lipid particle sample before and after adjusting the pH to a pH higher than the pKa of the LNP, and calculating the difference. The lipid residual rate after filtration was calculated by quantifying the lipid amount in the mRNA-encapsulated lipid particle sample before filtration and the mRNA-encapsulated lipid particle sample after filtration, and dividing the lipid amount after filtration by the lipid amount before filtration.

[0236] <pKa Measurement> The pKa of the mRNA-encapsulated lipid particle sample was calculated by TNS assay. To the LNP sample diluted with Milli-Q water, TNS reagent dissolved in DMSO and various buffers with pH values ​​varying in 0.5 increments in the pH range of 3 to 8.5 were added and mixed. The fluorescence intensity of the above measurement solution was observed using a fluorescent plate reader at an excitation wavelength of 337 nm and a wavelength of 435 nm, and the pKa was calculated from the change in fluorescence intensity with respect to pH. The pKa of the LNP of Example 1 was 6.58, the pKa of the LNP of Example 4 was 6.57, the pKa of the LNP of Example 6 was 6.50, the pKa of the LNP of Example 33 was 6.51, the pKa of the LNP of Comparative Example 1 was 6.56, and the pKa of the LNP of Comparative Example 9 was 6.54.

[0237] <Luciferase Luminescence Measurement> The LNPs encapsulating FLuc mRNA prepared in Examples 1, 4, 6, and Comparative Example 1 were administered intravenously once to ICR mice at an mRNA dose of 0.2 mg / kg. Five hours and 45 minutes after administration, 150 mg / kg of D-luciferin potassium (Fujifilm Wako Pure Chemical Industries) was administered intraperitoneally. Six hours after administration, the liver was removed under isoflurane gas anesthesia, and the luminescence (Photones / Sec) was quantified ex vivo using an IVIS Lumina III (PerkinElmer) to confirm luciferase expression. The luminescence (Total Flux (P / S)) in Table 1 indicates Photos / Sec (light intensity).

[0238]

[0239] It was confirmed that if the time between step (3) and step (4) is set to 1 minute or more, the particle size polydispersity index (PDI) of the LNP particle size after pH adjustment is small, and particles with a narrow particle size distribution are obtained. Furthermore, it was confirmed that the LNPs that underwent the above manufacturing process also showed small changes in particle size before and after pH adjustment and in lipid content before and after filtration, and that stable LNPs could be formed with little variation between steps. It was also found that the expression efficiency of luciferase protein was improved by increasing the holding time between step (3) and step (4).

[0240]

[0241]

[0242]

[0243]

[0244] As with the results in Table 1, it was confirmed that even in LNPs whose pH had been adjusted by the TFF method or dialysis, if the time between step (3) and step (4) was set to 1 minute or longer, the particle size polydispersity index PDI of the LNPs after pH adjustment was small, and particles with a narrow particle size distribution were obtained.

[0245]

[0246]

[0247] For LNPs encapsulating a nucleic acid species (saRNA) different from the RNA (FLuc mRNA) shown in Table 1, similar to the results in Table 1, it was confirmed that if the time between step (3) and step (4) is set to 1 minute or longer, the polydispersity index PDI of the particle size of the LNPs after pH adjustment is small, and stable LNPs with little change in physical properties due to the pH adjustment and filtration steps can be formed.

[0248]

[0249] Even in LNPs using pH-responsive lipids different from those used in Table 1, it was confirmed that LNPs in which the time between step (3) and step (4) was 1 minute or longer had a small particle size polydispersity index (PDI) in the formulation after pH adjustment, and that the change in particle size before and after pH adjustment was small, making them stable LNPs.

Claims

1. A method for producing lipid nanoparticles, comprising: step 1 preparing a first solution, which is an acidic aqueous solution containing an active ingredient; step 2 preparing a second solution, which is a solution containing an alcohol and at least one lipid selected from the group consisting of a lipid represented by formula (1), a lipid represented by formula (2), and a lipid represented by formula (3); step 3 preparing a lipid nanoparticle dispersion by mixing the first solution prepared in step 1 with the second solution prepared in step 2 in a flow path; a pH adjustment step after step 3, adjusting the pH of the lipid nanoparticle dispersion to a pH higher than the pKa of the lipid nanoparticles; and an alcohol removal step after step 3, which is after, during, or before the pH adjustment step, removing alcohol from the lipid nanoparticle dispersion, wherein the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is 12.5 to 35% by volume, and the lipid nanoparticle dispersion is held for 1 minute or more after completion of step 3. In formula (1), X is -NR １ - or -O-, R １ is a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or R ２１ -L １ -R ２２ represents a group represented by -, and R ２１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L １ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or indicates R ２２ represents a divalent linking group and a hydrocarbon linking group having 1 to 18 carbon atoms; R ２ and R ３ are each independently a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or R ３１ -L ２ -R ３２ represents a group represented by -, and R ３１ represents a hydrocarbon group having 1 to 24 carbon atoms, and L ２ is —O(CO)O—, —O(CO)—, —(CO)O—, —O—, or indicates R ３２ represents a divalent linking group and a hydrocarbon linking group having 1 to 18 carbon atoms; R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , and R １２ each independently represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms; R ４ and R ５ , R １０ and R ５ , R ５ and R １２ , R ４ and R ６ , R ５ and R ６ , R ６ and R ７ , R ６ and R １０ , R １２ and R ７ , and R ７ and R ８ may be bonded to each other to form a 4- to 7-membered ring which may contain an O atom, and the substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is a hydroxyl group, a carboxyl group, -NR ４５ R ４６ an amino group represented by the formula: ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms, and the substituent on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group is an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, —NR ４５ R ４６ an amino group represented by —O(CO)O—R ４１ , —O(CO)—R ４２ , -(CO)O-R ４３ , or -O-R ４４ and R ４１ , R ４２ , R ４３ , R ４４ , R ４５ and R ４６ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; a, b, c, and d each independently represent an integer of 0 to 3, with the proviso that a+b is 1 or more and c+d is 1 or more. In formula (2), R １ and R ２ each independently represents a hydrocarbon group having 1 to 18 carbon atoms; R ３ represents a hydrocarbon group having 2 to 8 carbon atoms, and R １ , R ２ and R ３ The hydrocarbon group represented by is —OH, COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , and -O-R ５６ and R ４ represents a hydrocarbon group having 1 to 8 carbon atoms, and R ５ and R ６ are each independently a hydrocarbon group having 1 to 8 carbon atoms, or -R ８ -L １ -R ９ where R ５ and R ６ and R are both hydrocarbon groups having 1 to 8 carbon atoms, ７ is -R １０ -L ２ -R １１ -L ３ -R １２ indicates, R ５１ and R ５２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ５３ , R ５４ , R ５５ , and R ５６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms; R ５３ , R ５４ , R ５５ , and R ５６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ５８ The aryl group having 6 to 20 carbon atoms may be substituted with —OH, —COOH, —NR ５１ R ５２ , -OC(O)OR ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , -O-R ５６ or -(C1-C12 hydrocarbon group)-R ５７ and R ５８ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ５７ is -OH, COOH, -NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , -O-R ６６ Indicates. ６１ and R ６２ each independently represents a hydrocarbon group having 1 to 8 carbon atoms; R ６３ , R ６４ , R ６５ , and R ６６ each independently represents a hydrocarbon group having 1 to 24 carbon atoms; R ６３ , R ６４ , R ６５ , and R ６６ The hydrocarbon group represented by is an aryl group having 6 to 20 carbon atoms or -S-R ６８ The aryl group having 6 to 20 carbon atoms may be substituted with —OH, —COOH, —NR ６１ R ６２ , -OC(O)OR ６３ , -C(O)O-R ６４ , —OC(O)—R ６５ , -O-R ６６ or -(C1-C12 hydrocarbon group)-R ６７ and R ６８ represents a hydrocarbon group having 1 to 12 carbon atoms; L １ , L ２ , and L ３ R each independently represents —OC(O)O—, —C(O)O—, —OC(O)—, or —O—. ８ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ９ represents a hydrocarbon group having 1 to 24 carbon atoms, R １０ represents a hydrocarbon group having 1 to 8 carbon atoms, and R １１ represents a hydrocarbon group having 1 to 24 carbon atoms, R １２ represents a hydrocarbon group having 1 to 24 carbon atoms, R ９ , and R １２ The hydrocarbon group represented by is an aryl group, —OC(O)O—R ５３ , -C(O)O-R ５４ , —OC(O)—R ５５ , or -S-R ５８ and R ５３ , R ５４ , R ５５ , and R ５８ is defined as above, and R １１ The hydrocarbon group represented by is —OC(O)O—R ５３ , -C(O)O-R ５４ or —OC(O)—R ５５ and R ５３ , R ５４ , and R ５５ The definition of is as above. In formula (3), R １ , R ２ , R ３ and R ４ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R １ , R ２ , R ３ and R ４ The substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by the formula (I) are each independently —C(O)O—R １１ , —OC(O)—R １２ , -O-R １３ , -CO-R １４ , -OC(O)OR １５ , or -S-S-R １６ indicates, R １１ , R １２ , R １３ , R １４ , R １５ and R １６ are each independently -S-R １７ represents a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with １７ represents a hydrocarbon group having 1 to 12 carbon atoms, and R ５ and R ６ each independently represents an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; R ５ and R ６ The substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by the formula (I) are each independently —OH, —COOH, —NR ２１ R ２２ , -OC(O)OR ２３ , -C(O)O-R ２４ , —OC(O)—R ２５ , -O-R ２６ , —C(O)NR ２７ R ２８ , -NR ２９ C(O)R ３０ , -N(R ３１ ) S (O) ２ R ３２ , -N(R ３３ )C(O)N(R ３４ ) R ３５ , -N(R ３６ ) C(S)N(R ３７ ) R ３８ , -OC(O)N(R ３９ ) R ４０ , or -N(R ４１ )C(O)OR ４２ indicates, R ２１ and R ２２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; R ２３ , R ２４ , R ２５ , R ２６ , R ２７ , R ２８ , R ２９ , R ３０ , R ３１ , R ３２ , R ３３ , R ３４ , R ３５ , R ３６ , R ３７ , R ３８ , R ３９ , R ４０ , R ４１ , and R ４２ each independently represents a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; R ２３ , R ２４ , R ２５ , R ２６ , R ２７ , R ２８ , R ２９ , R ３０ , R ３１ , R ３２ , R ３３ , R ３４ , R ３５ , R ３６ , R ３７ , R ３８ , R ３９ , R ４０ , R ４１ , and R ４２ The substituent on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by is an aryl group having 6 to 20 carbon atoms, a heterocyclic group, —OH, —COOH, or NR ５１ R ５２ indicates R ５１ and R ５２ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms; R ７ , R ８ , and R ９ each independently represents a hydrocarbon group having 2 to 8 carbon atoms; R ５ and R ６ , or R ５ and R ７ may be joined together to form a 4- to 7-membered ring.

2. The method for producing lipid nanoparticles described in claim 1, further comprising step 4 of mixing the mixture obtained in step 3 with a third solution to prepare a lipid nanoparticle dispersion, wherein the time from step 3 to step 4 is 1 minute or more.

3. The method for producing lipid nanoparticles described in claim 1, wherein the time from step 3 to the step which is carried out earlier among the pH adjustment step and the alcohol removal step is 1 minute or more.

4. A method for producing lipid nanoparticles described in any one of claims 1 to 3, wherein the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is less than 20 to 30 volume %.

5. The method for producing lipid nanoparticles according to claim 2, wherein the alcohol content of the lipid nanoparticle dispersion obtained in step 3 is 20 to less than 27.5 volume %, and the time from step 3 to step 4 is 1 minute or more to 24 hours or less.

6. A method for producing lipid nanoparticles described in any one of claims 1 to 3, wherein the alcohol is ethanol.

7. A method for producing lipid nanoparticles described in any one of claims 1 to 3, wherein the second solution in step 2 further contains at least one lipid selected from the group consisting of neutral lipids, lipids having nonionic hydrophilic polymers, and sterols.

8. A method for producing lipid nanoparticles according to any one of claims 1 to 3, wherein the average particle size of the produced lipid nanoparticles is less than 200 nm.

9. A method for producing lipid nanoparticles described in any one of claims 1 to 3, wherein the polydispersity index PDI of the average particle size of the produced lipid nanoparticles is less than 0.20.

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