WO2026004829A1 - Procédé de production de nanoparticules lipidiques - Google Patents

Procédé de production de nanoparticules lipidiques

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Publication number
WO2026004829A1
WO2026004829A1 PCT/JP2025/022598 JP2025022598W WO2026004829A1 WO 2026004829 A1 WO2026004829 A1 WO 2026004829A1 JP 2025022598 W JP2025022598 W JP 2025022598W WO 2026004829 A1 WO2026004829 A1 WO 2026004829A1
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Prior art keywords
group
carbon atoms
hydrocarbon group
lipid
independently represent
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Japanese (ja)
Inventor
佑貴 今泉
正樹 野呂
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Fujifilm Corp
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Fujifilm Corp
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Publication of WO2026004829A1 publication Critical patent/WO2026004829A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention relates to a method for producing lipid nanoparticles, which involves 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.
  • LNPs Lipid nanoparticles
  • LNPs are used to deliver nucleic acids to living organisms or cells, and have been put to practical use in disease treatments and vaccines.
  • LNPs are also known to be usable as gene transfer reagents.
  • a known method for producing nucleic acid-encapsulated LNPs is to mix a lipid solution and a nucleic acid solution in a flow channel.
  • 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 a high nucleic acid encapsulation rate and excellent nucleic acid delivery, and a method for producing the same.
  • Patent Document 3 describes that performing tangential flow filtration (TFF) after neutralization in the LNP production process improves changes in physical properties and TFF processability.
  • Patent Document 4 describes a method for producing LNPs with specified channel inner diameters and flow rates.
  • 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 desolvation, and the component composition can change during filtration.
  • the objective of the present invention is to provide a method for producing lipid nanoparticles that can produce lipid nanoparticles with a narrow particle size distribution.
  • 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 by holding the mixture for at least one minute after the completion of the mixing.
  • the present invention was completed based on the above findings.
  • the present invention provides the following:
  • Step 1 Preparing a first solution, which is an acidic aqueous solution containing an active ingredient;
  • Step 2 preparing a second solution containing 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), and an alcohol;
  • Step 3 preparing a lipid nanoparticle dispersion by mixing the first solution prepared in step 1 and the second solution prepared in step 2 in the flow channel;
  • a method for producing lipid nanoparticles comprising: 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, during the pH adjustment step, or before the pH adjustment step,
  • the lipid nanoparticle dispersion obtained in step 3 has an alcohol content of 12.5 to 35% by volume, After the completion of step 3, hold for 1 minute or more.
  • X represents —NR 1 — or —O—;
  • R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R 21 -L 1 -R 22 -, where R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or R 22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms;
  • R 2 and R 3 each independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31 -L 2 -R 32 -, where R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 represents -O(CO)O-, -O(CO)O-, -O-, or R 32 is a
  • R 1 and R 2 each independently represent a hydrocarbon group having 1 to 18 carbon atoms
  • R 3 represents a hydrocarbon group having 2 to 8 carbon atoms
  • the hydrocarbon groups represented by R 1 , R 2 , and R 3 are optionally substituted with one or more substituents selected from —OH, —NR 51 R 52 , —OC(O)O—R 53 , —C(O)O—R 54 , —OC(O)—R 55 , and —O—R 56
  • R4 represents a hydrocarbon group having 1 to 8 carbon atoms
  • R 5 and R 6 each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , provided that the case where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms is excluded
  • R 7 represents —R 10 -L 2 -R 11 -L 3 -R 12
  • R 51 and R 52 each independently represent a hydrocarbon group having 1 to 8 carbon atom
  • R 61 and R 62 each independently represent a hydrocarbon group having 1 to 8 carbon atoms;
  • R 63 , R 64 , R 65 , and R 66 each independently represent a hydrocarbon group having 1 to 24 carbon atoms;
  • the hydrocarbon group represented by R 63 , R 64 , R 65 and R 66 may be substituted with 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, —NR 61 R 62 , —OC(O)O—R 63 , —C(O)O—R 64 , —OC(O)—R 65 , —O—R 66 or —(hydrocarbon group having 1 to 12 carbon atoms)-R 67 ;
  • R 68 represents a hydrocarbon group having 1 to 12 carbon atoms;
  • L 1 , L 2 and L 3 each independently represent —OC(O)O—,
  • R8 represents a hydrocarbon group having 1 to 12 carbon atoms
  • R 9 represents a hydrocarbon group having 1 to 24 carbon atoms
  • R 10 represents a hydrocarbon group having 1 to 8 carbon atoms
  • R 11 represents a hydrocarbon group having 1 to 24 carbon atoms
  • R 12 represents a hydrocarbon group having 1 to 24 carbon atoms
  • the hydrocarbon group represented by R 9 and R 12 may be substituted with an aryl group, —OC(O)O—R 53 , —C(O)O—R 54 , —OC(O)—R 55 , or —S—R 58 , where R 53 , R 54 , R 55 , and R 58 are defined as above
  • the hydrocarbon group represented by R 11 may be substituted with —OC(O)O—R 53 , —C(O)O—R 54 , or —OC(O)—R 55 , where R 53 , R 54 , and R 55 are defined as above.
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ; R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with —S—R 17 , R 17 representing a hydrocarbon group having 1 to 12 carbon atoms; R5 and R6 each independently represent an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; Substituents on the optionally substituted hydrocarbon group having 1 to
  • ⁇ 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 from step 3 to step 4 is 1 minute or more.
  • ⁇ 3> The method for producing lipid nanoparticles according to ⁇ 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> 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.
  • the lipid nanoparticle dispersion obtained in step 3 has an alcohol content of 20 to less than 27.5 volume%, and the time from step 3 to step 4 is 1 minute to 24 hours.
  • ⁇ 6> The method for producing lipid nanoparticles according to any one of ⁇ 1> to ⁇ 5>, wherein the alcohol is ethanol.
  • 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 average particle diameter of the produced lipid nanoparticles is less than 200 nm.
  • ⁇ 9> The method for producing lipid nanoparticles according to any one of ⁇ 1> to ⁇ 8>, wherein the polydispersity index PDI of the average particle size of the produced lipid nanoparticles is less than 0.20.
  • the lipid nanoparticle manufacturing method of the present invention makes it possible to produce particles with a narrow particle size distribution.
  • the method for producing lipid nanoparticles of the present invention comprises: Step 1: preparing a first solution, which is an acidic aqueous solution containing an active ingredient; Step 2: preparing a second solution containing 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) and an alcohol; Step 3: preparing a lipid nanoparticle dispersion by mixing the first solution prepared in step 1 and the second solution prepared in step 2 in the flow channel;
  • a method for producing lipid nanoparticles comprising: 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, during the pH adjustment step, or before the pH adjustment step,
  • the lipid nanoparticle dispersion obtained in step 3 has an
  • the first solution can be obtained by dissolving an active ingredient (e.g., a nucleic acid) in water or a buffer solution.
  • concentration of the active ingredient is not particularly limited, but is preferably 1 to 1,000 ⁇ g/mL, and more preferably 10 to 500 ⁇ g/mL.
  • components such as a buffer component for pH adjustment or an antioxidant can be added.
  • 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, acetate buffer, citrate buffer, malate buffer, phosphate buffer, MES, Bis-Tris, PIPES, etc.
  • buffer component can be preferably used as a buffer component.
  • concentration of these buffer components is preferably 2 to 200 mmol/L, and more preferably 5 to 100 mmol/L.
  • salts such as sodium chloride and potassium chloride may be added as needed to adjust the salt strength
  • sugars or sugar alcohols such as sucrose, trehalose, and mannitol may be added as needed to adjust the osmotic pressure.
  • 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.
  • 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.
  • 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.
  • 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.
  • a flow path that can control the mixing ratio so that it is always constant and highly reproducible.
  • microflow paths such as branch mixers, T-shaped mixers, herringbone mixers, and baffle mixers, as 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.
  • the flow path shape is designed to achieve precise mixing suitable for the production of lipid nanoparticles.
  • impingement jet mixer IJM
  • a turbulent jet mixer that mixes using concentric jet flows, or even membrane emulsification, allowing the optimal mixer to be selected depending on the required production scale and particle properties.
  • 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 setting the shape of the microchannel, and are not particularly limited. Generally, when the first solution and the second solution are mixed, if the flow rate or flow rate of the mixed solution of the first solution and the second solution is low and the mixing efficiency is poor, the particle size obtained tends to be large.
  • the flow rate of the mixture of the first and second solutions is preferably 5.0 m/sec or more, more preferably 7.5 m/sec or more, and most preferably 10.0 m/sec or more, and the flow rate is preferably 4 to 4,000 mL/min, and more preferably 8 to 2,000 mL/min.
  • the present invention may further include a step 4 of mixing the mixture obtained in the step 3 with a third solution to prepare a lipid nanoparticle dispersion.
  • step 4 it is preferable that the time from step 3 to step 4 is 1 minute or more.
  • step 4 it is preferable that the time interval before moving to the step which is performed first, either the pH adjustment step or the alcohol removal step, is 1 minute or more.
  • the method for producing lipid nanoparticles of the present invention includes: Step 1: preparing a first solution, which is an acidic aqueous solution containing an active ingredient; Step 2: preparing a second solution containing 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) and an alcohol; Step 3: preparing a lipid nanoparticle dispersion by mixing the acidic aqueous solution prepared in step 1 and the alcohol solution prepared in step 2 in a flow channel; Step 4: mixing the mixture obtained in step 3 with a third solution to prepare a lipid nanoparticle dispersion B; A method for producing lipid nanoparticles, comprising: a pH adjustment step of adjusting the pH of the 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
  • Step 1 preparing a first solution, which is an acidic aqueous solution containing an active ingredient
  • Step 2 preparing a second solution containing 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) and an alcohol
  • Step 3 preparing a lipid nanoparticle dispersion by mixing the acidic aqueous solution prepared in step 1 and the alcohol solution prepared in step 2 in a flow channel
  • a method for producing lipid nanoparticles comprising: a pH adjustment step after step 3, in which the pH of the lipid nanoparticle dispersion is adjusted to a pH higher than the pKa of the lipid nanoparticles; and an alcohol removal step after step 3, in which alcohol is removed from the lipid nanoparticle dispersion after, during, or before the pH adjustment step,
  • the lipid nanoparticle dispersion obtained in step 3 has an
  • Step 4 may be carried out continuously after step 3, or may be carried out again after temporarily storing the lipid nanoparticle dispersion obtained in step 3 in a container.
  • 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. If the lipid nanoparticle dispersion obtained in step 3 is stored in a container and then re-processed, step 4 can be started after at least one minute has elapsed since the lipid nanoparticle dispersion was prepared in step 3.
  • the third solution is added for the purpose of reducing the alcohol content to an appropriate range.
  • the main component is water, and if necessary, it may contain other additives such as a buffer component to adjust the pH, and/or salts such as sodium chloride or potassium chloride to adjust the salt strength, and/or sugars or sugar alcohols (sucrose, trehalose, mannitol, etc.) to adjust the osmotic pressure.
  • step 4 the mixing ratio of the lipid nanoparticle dispersion A obtained in step 3 and the third solution described above affects particle formation, so it is preferable to mix them so that the mixing ratio can be controlled to be constant and reproducible.
  • 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.
  • 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. Furthermore, 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 lipid nanoparticle dispersion A obtained in step 3 is lower than its pKa.
  • 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.
  • 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.
  • the lipid nanoparticle dispersion obtained in step 3 has an alcohol content of less than 20 to 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.
  • the lipid nanoparticle dispersion A obtained in step 3 has an alcohol content of 12.5 to less than 27.5% by volume, and can be held for 1 minute to 24 hours after the completion of step 3.
  • the lipid nanoparticle dispersion A obtained in step 3 has an alcohol content of 27.5 to less than 30.0% by volume, and can be held for 1 to 150 minutes after completion of step 3.
  • the lipid nanoparticle dispersion A obtained in step 3 has an alcohol content of 30.0 to less than 35.0% by volume, and can be held for 1 to 10 minutes after completion of step 3.
  • the method for producing lipid nanoparticles of the present invention includes 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 a step of removing the alcohol contained in the lipid nanoparticle dispersion.
  • 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 mixing may be carried out multiple times. If carried out multiple times, the lipid nanoparticle dispersion may be stored between 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.
  • the pH may be adjusted during dialysis using a dialysis solution with a higher pH than that of the lipid nanoparticle dispersion.
  • the timing of removing alcohol may be any of before, during, 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.
  • 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.
  • alcohol removal and pH adjustment may be carried out continuously by the TFF method.
  • 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).
  • TMS 2-(p-toluidino)-6-naphthalenesulfonic acid
  • the surface of the lipid nanoparticles becomes cationic, and TNS interacts with the lipid nanoparticles through electrostatic interactions, causing TNS to emit fluorescence.
  • the fluorescence intensity of TNS is plotted on the vertical axis, a plot similar to a titration curve is obtained.
  • 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 when lipid nanoparticles become cationic at physiological pH, their safety decreases, and when the pH is too low, the efficiency of intracellular payload delivery decreases.
  • the constituent 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 more and 7.0 or less.
  • the pKa is preferably 5.5 or more and 7.0 or less.
  • the pH of the lipid nanoparticle dispersion during the production process is adjusted across pKa.At around pKa, the charge of the lipid nanoparticles becomes close to neutral, and the dispersion stability decreases, so the lipid nanoparticles are likely to become coarse or aggregate, which may affect the post-treatment process, so pH control in the production process is important.
  • the concentration can be adjusted in the post-treatment process of the lipid nanoparticle dispersion.
  • phosphate buffered saline, normal saline, or a solution containing the above-mentioned additives for example, those mentioned above for adjusting pH, salt strength, and osmotic pressure
  • a diluent for example, those mentioned above for adjusting pH, salt strength, and osmotic pressure
  • concentrating it can be concentrated by ultrafiltration using an ultrafiltration membrane.
  • the concentrated dispersion can be used as is, or it can be concentrated and then adjusted to the desired concentration using the above-mentioned diluent.
  • the TFF method can be used to continuously remove alcohol, adjust pH, and concentrate.
  • the organic solvent removal process and the concentration adjustment process may be performed in any order. If necessary, the organic solvent removal process and the concentration adjustment process may each be performed multiple times.
  • Solutions that can be used for alcohol removal, pH adjustment, and concentration adjustment in post-treatment processes of lipid nanoparticle dispersions may contain excipients, cryoprotectants, buffers, and antioxidants.
  • excipients and cryoprotectants include, but are not limited to, sugars and sugar alcohols.
  • sugars include sucrose, trehalose, maltose, glucose, lactose, and fructose
  • sugar alcohols include mannitol, sorbitol, inositol, and xylitol.
  • 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.
  • antioxidants include EDTA, ascorbic acid, and tocopherol.
  • a lipid nanoparticle dispersion As a pharmaceutical composition, it is preferable to carry out sterile filtration.
  • a filtration method a hollow fiber membrane, a reverse osmosis membrane, a membrane filter, etc. can be used to remove insoluble materials from the lipid particle dispersion.
  • a sterilizable pore size preferably a 0.2 ⁇ m filtration sterilization filter
  • 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.
  • the above steps 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 to these.
  • a second solution is prepared, which is a solution containing 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), and an alcohol.
  • the lipids represented by formula (1), formula (2), and formula (3) used in the present invention are described below.
  • X represents —NR 1 — or —O—;
  • R 1 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R 21 -L 1 -R 22 -, where R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or R 22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms;
  • R 2 and R 3 each independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31 -L 2 -R 32 -, where R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 represents -O(CO)O-, -O(CO)O-, -O-, or R 32 is
  • the hydrocarbon group having 6 to 24 carbon atoms in R1 and the hydrocarbon group having 3 to 24 carbon atoms in R2 and R3 are preferably alkyl groups, alkenyl groups, or alkynyl groups, and more preferably alkyl groups or alkenyl groups.
  • the alkyl groups having 6 to 24 carbon atoms and the alkyl groups having 3 to 24 carbon atoms may be linear or branched, and may be linear or cyclic.
  • the alkyl groups having 6 to 24 carbon atoms are preferably alkyl groups having 6 to 20 carbon atoms, and more preferably the alkyl groups having 3 to 24 carbon atoms are alkyl groups 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.
  • 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.
  • 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.
  • the hydrocarbon group having 1 to 24 carbon atoms for R 21 and R 31 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.
  • 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-trimethyloct
  • 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 (
  • 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.
  • the divalent linking group for R 22 and R 32 which is a 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.
  • 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.
  • L1 is —O(CO)O—, —O(CO)—, or —(CO)O—, and —O(CO)— or —(CO)O— is more preferred.
  • L2 is —O(CO)O—, —O(CO)—, or —(CO)O—, and —O(CO)— or —(CO)O— is more preferred.
  • the alkyl group having 1 to 18 carbon atoms in the optionally substituted alkyl group having 1 to 18 carbon atoms for R 4 , R 6 , R 9 , R 10 , R 11 , and R 12 may be linear or branched, chain 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.
  • the substituent is preferably a hydroxyl group, a carboxyl group, or a group represented by —O(CO)O—R 41 , —O(CO)—R 42 , —(CO)O—R 43 or —O—R 44 , and more preferably a group represented by —O(CO)—R 42 or —(CO)O—R 43 .
  • the alkyl group having 1 to 18 carbon atoms in the alkyl group having 1 to 18 carbon atoms which may be substituted for R 5 , R 7 , and R 8 may be linear or branched, open-chain 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.
  • the substituent is preferably a hydroxyl group, a carboxyl group, or a group represented by —O(CO)O—R 41 , —O(CO)—R 42 , —(CO)O—R 43 or —O—R 44 , and more preferably a group represented by —O(CO)—R 42 or —(CO)O—R 43 .
  • Examples of 4- to 7-membered rings that may contain an O atom include an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring, and an azepane ring. Six-membered rings are preferred, with piperidine and morpholine rings being more preferred.
  • the aryl group when 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, 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.
  • Preferred substituents on the aryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 , and a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 , or -O-R 44 , with a hydroxyl group or a carboxyl group being more preferred.
  • Specific examples of the substituted aryl group include a hydroxyphenyl group and a carboxyphenyl group.
  • the heteroaryl group when 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, and 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.
  • Preferred substituents on the heteroaryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 , or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 , or -O-R 44 , with a hydroxyl group or a carboxyl group being more preferred.
  • Specific examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxypyridyl group, and a pyridonyl group.
  • the hydrocarbon group having 1 to 18 carbon atoms for R 41 , R 42 , R 43 , R 44 , R 45 and R 46 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.
  • 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.
  • R1 represents a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R21 - L1 - R22- .
  • one of R2 and R3 is a hydrogen atom, and the other of R2 and R3 represents a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R31 - L2 - R32- .
  • R 2 and R 3 each independently represent a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R 31 -L 2 -R 32 —.
  • R 4 , R 6 , R 9 , R 10 , R 11 and R 12 are preferably hydrogen atoms.
  • R 5 is preferably a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R 42 or -(CO)O-R 43 , 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, and when it is an alkyl group, it may be linked to R 4 , R 6 , R 10 , and R 12 to form a ring which may contain an O atom.
  • an alkyl group having 1 to 18 carbon atoms an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R 42 or -(CO)O-R 43 , an alkyl group having 1 to 12 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 is preferred, and an alkyl group having 1 to 18 carbon atoms, or an alkyl group having 1 to 18 carbon atoms which may be substituted with -O(CO)-R 42 or -(CO)O-R 43 is more preferred.
  • R 7 and R 8 are each independently a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted with —O(CO)—R 42 or —(CO)O—R 43 , 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 that R 7 and R 8 are linked to each other to form a 4- to 7-membered ring which may contain an O atom.
  • R5 and R7 or R8 are not linked to each other to form a ring.
  • a+b is preferably 1 or 2, more preferably 1.
  • c+d is preferably 1 or 2, more preferably 1.
  • the lipid represented by formula (1) is preferably a lipid represented by the following formula (1-1):
  • R 24 represents a hydrogen atom, a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R 21 -L 1 -R 22 -, where R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 1 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or and R 22 is a divalent linking group and a hydrocarbon linking group having 1 to 18 carbon atoms.
  • R 25 represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31 -L 2 -R 32 -, where R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 represents -O(CO)O-, -O(CO)-, -(CO)O-, -O-, or and R 32 is 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 10 , and R 12 each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms; Any one or more pairs of R4 and R5 , R10 and R5 , R5 and R12 , R4 and R6 , R5 and R6 , R6 and R7 , R6 and R10 , R12 and R7, and R7 and R8 may be linked to each other to form a 4- to 7-membered ring optionally containing an O atom. However, preferably, R5 and R7 or R8 are not linked 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, an amino group represented by -NR 45 R 46 , a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 or -O-R 44 , wherein R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms; Substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are alkyl groups having 1 to 18 carbon atoms, hydroxyl groups, carboxyl groups, amino groups represented by -NR 45 R 46 , groups represented by -O(CO)O-R 41 ,
  • R 4 , R 5 , R 6 , R 7 , R 8 , R 10 and R 12 in formula (1-1) are the same as those in formula (1).
  • R 24 in formula (1-1) is 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, 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably, 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.
  • Specific examples include an octenyl group, a nonenyl group, a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl 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-enyl group), and a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8-enyl group).
  • each of the above alkenyl groups has one or two double bonds.
  • R 25 in formula (1-1) is 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.
  • 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.
  • Specific examples include an octenyl group, a nonenyl group, a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl 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-enyl group), and a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8-enyl group).
  • each of the above alkenyl groups has one or two double bonds.
  • X represents —O—;
  • R 2 , R 3 , R 31 , L 2 , and R 32 are defined as in formula (1);
  • R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms;
  • the definitions of the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms, the substituent on the substituted or unsubstituted aryl group, and the substituent on the substituted or unsubstituted heteroaryl group are the same as those defined in formula (1), a+b is 1, and c+d is 1 or 2.
  • the lipid represented by formula (1) is a lipid represented by the following formula (1-2):
  • R 2 and R 3 each independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31 -L 2 -R 32 -;
  • 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, which is a hydrocarbon linking group having 1 to 18 carbon atoms;
  • R5 represents a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms;
  • R 7 and R 8 each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 18 carbon atoms;
  • the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 , a substituted or unsub
  • R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms; the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms for R 5 is a hydroxyl group, a substituted or unsubstituted aryl group, or a group represented by -O(CO)O-R 41 , -O(CO)-R 42 , -(CO)O-R 43 , or -O-R 44 ; R 41 , R 42 , R 43 , R 44 , R 45 , and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms; 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, an amino group represented by -NR 45 R 46 , -O(CO)O-R 41 , -O(CO)-R 42
  • R 2 and R 3 each independently represent a hydrocarbon group having 3 to 24 carbon atoms or a group represented by R 31 -L 2 -R 32 -, L 2 represents -O(CO)- or -(CO)O-, R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and for R 5 , the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is an unsubstituted aryl group, -O(CO)-R 42 or -(CO)O-R 43 , and R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
  • R 2 and R 3 each independently represent a hydrogen atom or a hydrocarbon group having 3 to 24 carbon atoms
  • R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms
  • the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms for R 5 is an unsubstituted aryl group, a group represented by -O(CO)-R 42 , or -(CO)O-R 43
  • R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
  • R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -
  • L 2 represents -O(CO)- or -(CO)O-
  • R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms
  • R 5 the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is an unsubstituted aryl group, or a group represented by -O(CO)-R 42 or -(CO)O-R 43
  • R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
  • R 2 and R 3 each independently represent a group represented by R 31 -L 2 -R 32 -
  • L 2 represents -O(CO)- or -(CO)O-
  • R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms
  • the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is an unsubstituted aryl group, or a group represented by -O(CO)-R 42 or -(CO)O-R 43
  • R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
  • R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -, the other of R 2 and R 3 represents a hydrocarbon group having 3 to 24 carbon atoms, L 2 represents -O(CO)- or -(CO)O-, R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and for R 5 , the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is an unsubstituted aryl group, or a group represented by -O(CO)-R 42 or -(CO)O-R 43 , and R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
  • R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -, the other of R 2 and R 3 represents a hydrocarbon group having 6 carbon atoms, L 2 represents -O(CO)- or -(CO)O-, R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and for R 5 , the substituent on the optionally substituted alkyl group having 1 to 18 carbon atoms is a group represented by -O(CO)-R 42 or -(CO)O-R 43 , and R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
  • one of R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -, the other of R 2 and R 3 represents a hydrocarbon group having 6 carbon atoms, L 2 represents -O(CO)- or -(CO)O-, R 5 represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms.
  • one of R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -, the other of R 2 and R 3 represents a hydrocarbon group having 6 carbon atoms, L 2 represents -O(CO)- or -(CO)O-, R 5 represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and e represents 2.
  • one of R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -, the other of R 2 and R 3 represents a hydrocarbon group having 3 to 5 carbon atoms, L 2 represents -O(CO)- or -(CO)O-, R 5 represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms.
  • one of R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -, the other of R 2 and R 3 represents a hydrocarbon group having 3 to 5 carbon atoms, L 2 represents -O(CO)- or -(CO)O-, R 5 represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and e represents 2.
  • R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -, the other of R 2 and R 3 represents a hydrocarbon group having 6 carbon atoms, L 2 represents -O(CO)- or -(CO)O-, R 5 represents a hydrogen atom or a substituted alkyl group having 1 to 18 carbon atoms, R 7 and R 8 each independently represent 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 a group represented by -O(CO)-R 42 or -(CO)O-R 43 , and R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
  • R 2 and R 3 represents a group represented by R 31 -L 2 -R 32 -, the other of R 2 and R 3 represents a hydrocarbon group having 6 carbon atoms, L 2 represents -O(CO)- or -(CO)O-, R 5 represents a hydrogen atom or a substituted alkyl group having 1 to 18 carbon atoms, R 7 and R 8 each independently represent 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 a group represented by -O(CO)-R 42 or -(CO)O-R 43 , R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms, and e represents 2.
  • lipids represented by formula (1) Specific examples of lipids represented by formula (1) and methods for producing them are described in WO2019/235635. The entire contents of WO2019/235635 are incorporated herein by reference.
  • R 1 and R 2 each independently represent a hydrocarbon group having 1 to 18 carbon atoms
  • R 3 represents a hydrocarbon group having 2 to 8 carbon atoms
  • the hydrocarbon groups represented by R 1 , R 2 , and R 3 are optionally substituted with one or more substituents selected from —OH, —NR 51 R 52 , —OC(O)O—R 53 , —C(O)O—R 54 , —OC(O)—R 55 , and —O—R 56
  • R4 represents a hydrocarbon group having 1 to 8 carbon atoms
  • R 5 and R 6 each independently represent a hydrocarbon group having 1 to 8 carbon atoms or -R 8 -L 1 -R 9 , provided that the case where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms is excluded
  • R 7 represents —R 10 -L 2 -R 11 -L 3 -R 12
  • R 51 and R 52 each independently represent a hydrocarbon group having 1 to 18 carbon atom
  • R 61 and R 62 each independently represent a hydrocarbon group having 1 to 8 carbon atoms;
  • R 63 , R 64 , R 65 , and R 66 each independently represent a hydrocarbon group having 1 to 24 carbon atoms;
  • the hydrocarbon group represented by R 63 , R 64 , R 65 and R 66 may be substituted with 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, —NR 61 R 62 , —OC(O)O—R 63 , —C(O)O—R 64 , —OC(O)—R 65 , —O—R 66 or —(hydrocarbon group having 1 to 12 carbon atoms)-R 67 ;
  • R 68 represents a hydrocarbon group having 1 to 12 carbon atoms;
  • L 1 , L 2 and L 3 each independently represent —OC(O)O—,
  • R8 represents a hydrocarbon group having 1 to 12 carbon atoms
  • R 9 represents a hydrocarbon group having 1 to 24 carbon atoms
  • R 10 represents a hydrocarbon group having 1 to 8 carbon atoms
  • R 11 represents a hydrocarbon group having 1 to 24 carbon atoms
  • R 12 represents a hydrocarbon group having 1 to 24 carbon atoms
  • the hydrocarbon group represented by R 9 and R 12 may be substituted with an aryl group, —OC(O)O—R 53 , —C(O)O—R 54 , —OC(O)—R 55 , or —S—R 58 , where R 53 , R 54 , R 55 , and R 58 are defined as above
  • the hydrocarbon group represented by R 11 may be substituted with —OC(O)O—R 53 , —C(O)O—R 54 , or —OC(O)—R 55 , where R 53 , R 54 , and R 55 are defined as above.
  • the hydrocarbon group having 1 to 12 carbon atoms in -(hydrocarbon group having 1 to 12 carbon atoms)-R 67 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.
  • the aryl group preferably has 6 to 20 carbon atoms, more preferably 6 to 18, and even more preferably 6 to 10. Specific examples include a phenyl group, a naphthyl group, an anthracenyl group, and a phenanthrenyl group.
  • R 1 and R 2 each independently represent a hydrocarbon group preferably 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.
  • R3 preferably represents a hydrocarbon group having 2 to 6 carbon atoms, and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.
  • the hydrocarbon groups represented by R 1 , R 2 and R 3 may preferably be substituted with —OH.
  • L 1 and L 3 each independently preferably represent —C(O)O— or —OC(O)—.
  • L2 preferably represents —OC(O)O—, —C(O)O—, or —OC(O)—.
  • R 8 preferably represents a hydrocarbon group having 1 to 10 carbon atoms, and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.
  • R 9 preferably represents a hydrocarbon group having 1 to 20 carbon atoms, and more preferably represents a hydrocarbon group having 1 to 16 carbon atoms.
  • R 11 preferably represents a hydrocarbon group having 1 to 16 carbon atoms, and more preferably represents a hydrocarbon group having 1 to 9 carbon atoms.
  • R 12 preferably represents a hydrocarbon group having 1 to 20 carbon atoms, and more preferably represents a hydrocarbon group having 1 to 16 carbon atoms.
  • the hydrocarbon groups represented by R 9 and R 12 may be substituted with preferably an aryl group or S—R 58 , where R 58 preferably represents a hydrocarbon group having 1 to 8 carbon atoms.
  • the hydrocarbon group represented by R 11 may be preferably substituted with —C(O)O—R 55 or —OC(O)—R 56 , where R 55 and R 56 each independently represent a hydrocarbon group having 1 to 16 carbon atoms.
  • the hydrocarbon groups represented by R 55 and R 56 may be substituted with an aryl group having 6 to 20 carbon atoms or —S—R 58 , where R 58 is defined as above.
  • the lipid represented by formula (2) is preferably, as a first example, a compound represented by the following formula (2-1):
  • R 1 and R 2 each independently represent a hydrocarbon group having 1 to 18 carbon atoms
  • R 3 represents a hydrocarbon group having 2 to 8 carbon atoms
  • the hydrocarbon groups represented by R 1 , R 2 and R 3 may be substituted with —OH, —COOH, —NR 51 R 52 , —OC(O)O—R 53 , —C(O)O—R 54 , —OC(O)—R 55 or —O—R 56
  • R4 represents a hydrocarbon group having 1 to 8 carbon atoms
  • R 5 and R 6 each independently represent a hydrocarbon group having 1 to 8 carbon atoms or R 8 -L 1 -R 9 , excluding the case where R 5 and R 6 are both hydrocarbon groups having 1 to 8 carbon atoms
  • L1 represents —OC(O)O—, —C(O)O—, —OC(O
  • R 13 represents a hydrocarbon group having 1 to 8 carbon atoms
  • R 14 represents -R 15 -L 5 -R 16
  • R 15 represents a hydrocarbon group having 1 to 24 carbon atoms
  • L 5 represents -OC(O)O-, -C(O)O-, -OC(O)- or -O-
  • R 16 represents a hydrocarbon group having 1 to 24 carbon atoms
  • the hydrocarbon group having 1 to 24 carbon atoms represented by R 15 may be substituted with —OC(O)O—R 53 , —C(O)O—R 54 , or OC(O)—R 55 , where R 53 , R 54 , and R 55 are defined as above
  • the hydrocarbon group having 1 to 24 carbon atoms represented by R 16 may be substituted with 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 1 and R 2 each independently represent a hydrocarbon group preferably 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.
  • R 3 preferably represents a hydrocarbon group having 2 to 6 carbon atoms, and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.
  • the hydrocarbon groups represented by R 1 , R 2 and R 3 may preferably be substituted with —OH.
  • L 1 preferably represents —C(O)O— or —OC(O)—.
  • R 8 preferably represents a hydrocarbon group having 1 to 10 carbon atoms, and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.
  • R 9 preferably represents a hydrocarbon group having 1 to 18 carbon atoms, and the hydrocarbon group represented by R 9 may be substituted with an aryl group having 6 to 20 carbon atoms or S—R 58 .
  • R 14 preferably represents —R 15 -L 5 —R 16 , where 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.
  • the hydrocarbon group having 1 to 18 carbon atoms represented by R 15 may preferably be substituted with —C(O)O—R 55 or OC(O)—R 56.
  • R 55 and R 56 each independently represent a hydrocarbon group having 1 to 16 carbon atoms, and the hydrocarbon group represented by R 55 and R 56 may be substituted with an aryl group having 6 to 20 carbon atoms or —S—R 58 , where R 58 is defined as above.
  • the hydrocarbon group having 1 to 18 carbon atoms represented by R 16 may be substituted with preferably an aryl group or S—R 58 , where R 58 is defined as above.
  • a second example of the lipid represented by formula (2) is preferably a compound represented by the following formula (2-2):
  • R 1 and R 2 each independently represent a hydrocarbon group having 1 to 18 carbon atoms
  • R 3 represents a hydrocarbon group having 2 to 8 carbon atoms
  • the hydrocarbon groups represented by R 1 , R 2 , and R 3 may be substituted with —OH, —COOH, —NR 51 R 52 , —OC(O)O—R 53 , —C(O)O—R 54 , —OC(O)—R 55 , or —O—R 56
  • R 4 and R 8 each independently represent a hydrocarbon having 1 to 8 carbon atoms
  • R 21 and R 22 each independently represent a hydrocarbon group having 1 to 18 carbon atoms
  • R 23 and R 24 each independently represent a hydrocarbon group having 1 to 12 carbon atoms
  • R 25 and R 26 each independently represent a hydrocarbon group having 1 to 24 carbon atoms
  • L 21 and L 22 each independently represent —OC
  • R1 and R2 each independently represent a hydrocarbon group preferably 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.
  • the hydrocarbon groups represented by R1 and R2 may preferably be substituted with —OH, but are more preferably unsubstituted hydrocarbon groups.
  • R 3 preferably represents a hydrocarbon group having 2 to 6 carbon atoms, and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.
  • R 21 and R 22 each independently represent a hydrocarbon group preferably 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.
  • R 23 and R 24 each independently represent a hydrocarbon group preferably having 1 to 10 carbon atoms, more preferably a hydrocarbon group having 1 to 8 carbon atoms.
  • R 25 and R 26 each independently represent a hydrocarbon group preferably having 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.
  • L 21 and L 22 each independently preferably represent —C(O)O— or —OC(O)—.
  • the lipid represented by formula (2) is preferably a compound represented by the following formula (2-3):
  • R 1 and R 2 each independently represent a hydrocarbon group having 1 to 18 carbon atoms
  • R 3 represents a hydrocarbon group having 2 to 8 carbon atoms
  • the hydrocarbon groups represented by R 1 , R 2 , and R 3 may be substituted with —OH, —COOH, —NR 51 R 52 , —OC(O)O—R 53 , —C(O)O—R 54 , —OC(O)—R 55 , or —O—R 56
  • R4 and R8 each independently represent a hydrocarbon group having 1 to 8 carbon atoms
  • R 31 , R 32 , R 33 , and R 34 each independently represent a hydrocarbon group having 1 to 12 carbon atoms
  • R 35 , R 36 , R 37 , and R 38 each independently represent a hydrocarbon group having 1 to 24 carbon atoms
  • R1 and R2 each independently represent a hydrocarbon group preferably 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.
  • the hydrocarbon groups represented by R1 and R2 may preferably be substituted with —OH, but are more preferably unsubstituted hydrocarbon groups.
  • R 3 preferably represents a hydrocarbon group having 2 to 6 carbon atoms, and more preferably represents a hydrocarbon group having 2 to 4 carbon atoms.
  • R 31 , R 32 , R 33 , and R 34 each independently represent a hydrocarbon group preferably 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.
  • R 35 , R 36 , R 37 , and R 38 each independently represent a hydrocarbon group preferably having 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.
  • the hydrocarbon groups represented by R 35 , R 36 , R 37 , and R 38 may be substituted with preferably an aryl group having 6 to 20 carbon atoms or -S-R 58. More preferably, they may be substituted with -S-R 58 .
  • R 35 , R 36 , R 37 , and R 38 each independently preferably represent a hydrocarbon group having 1 to 12 carbon atoms substituted with —S—R 58 , or a hydrocarbon group having 1 to 12 carbon atoms.
  • L 31 , L 32 , L 33 and L 34 each independently preferably represent —C(O)O— or —OC(O)—.
  • R 58 preferably represents a hydrocarbon group having 1 to 10 carbon atoms, and more preferably represents a hydrocarbon group having 1 to 8 carbon atoms.
  • lipids represented by formula (2) and methods for producing them are described in WO2022/230964. The entire contents of WO2022/230964 are incorporated herein by reference.
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or an optionally substituted hydrocarbon group having 1 to 24 carbon atoms; Substituents on the optionally substituted hydrocarbon group having 1 to 24 carbon atoms represented by R 1 , R 2 , R 3 and R 4 each independently represent -C(O)O-R 11 , -OC(O)-R 12 , -O-R 13 , -CO-R 14 , -OC(O)O-R 15 or -S-S-R 16 ; R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently represent a hydrocarbon group having 1 to 24 carbon atoms which may be substituted with —S—R 17 , R 17 representing a hydrocarbon group having 1 to 12 carbon atoms; R5 and R6 each independently represent an optionally substituted hydrocarbon group having 1 to 18 carbon atoms; Substituents on
  • R 1 represents -R 1a -L 1 -R 1b
  • 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-
  • R 1b represents a hydrocarbon group having 1 to 18 carbon atoms
  • R 3 represents -R 3a -L 3 -R 3b
  • R 3a represents a hydrocarbon group having 1 to 18 carbon atoms
  • L 3 represents -C(O)O-, -OC(O)-, -OC(O)O-, or -S-S-
  • R 3b represents a hydrocarbon group having 1 to 18 carbon atoms
  • R 2 and R 4 each independently represent an optionally substituted hydrocarbon group having 1 to 18 carbon atoms, and substituents on the optionally substituted hydrocarbon group having 1 to 18 carbon atoms represented by R 2 and R 4 each independently represent -C(O)
  • R 1 represents -R 1a -L 1 -R 1b
  • R 1a represents a hydrocarbon group having 1 to 18 carbon atoms
  • L 1 represents -C(O)O- or -OC(O)-
  • R 1b represents a hydrocarbon group having 1 to 18 carbon atoms
  • R 3 represents -R 3a -L 3 -R 3b
  • R 3a represents a hydrocarbon group having 1 to 18 carbon atoms
  • L 3 represents -C(O)O- or -OC(O)-
  • R 3b represents a hydrocarbon group having 1 to 18 carbon atoms
  • R 2 and R 4 each independently represent a hydrocarbon group having 1 to 10 carbon atoms
  • R 5 and R 6 each independently represent an optionally substituted hydrocarbon group having 1 to 6 carbon atoms
  • Substituents on the optionally substituted hydrocarbon group having 1 to 6 carbon atoms represented by R 5 and R 6 each independently represent —OH, —O—R 26 , —C
  • R 1 represents -R 1a -L 1 -R 1b
  • R 1a represents a hydrocarbon group having 1 to 5 carbon atoms
  • L 1 represents -C(O)O-
  • R 1b represents a hydrocarbon group having 7 to 14 carbon atoms
  • R 3 represents -R 3a -L 3 -R 3b
  • R 3a represents a hydrocarbon group having 1 to 5 carbon atoms
  • L 3 represents -C(O)O-
  • R 3b represents a hydrocarbon group having 7 to 14 carbon atoms
  • R 2 and R 4 each independently represent a hydrocarbon group having 3 to 8 carbon atoms
  • R5 and R6 each independently represent a hydrocarbon group having 2 carbon atoms
  • R 7 , R 8 and R 9 each independently represent —(CH 2 ) n —, where n is an integer of 2 to 4.
  • the lipid represented by formula (3) can be produced by combining known methods, for example, according to the production method shown below.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 have the same meanings as above;
  • R 8a , R 9a and R A represent a hydrocarbon group having 1 to 7 carbon atoms.
  • the compound of formula [3A] can be produced by reacting the compound of formula [2] in the presence of water and an acid, in the presence or absence of a solvent.
  • the acid used in this reaction may be an inorganic acid or an organic acid, preferably an organic acid, such as formic acid, acetic acid, trifluoroacetic acid, 4-toluenesulfonic acid, or methanesulfonic acid, with formic acid being more preferred.
  • the amount of the acid used may be 1 to 100 times (v/w), preferably 1 to 10 times (v/w), relative to the amount of the compound of formula [2].
  • the amount of water used may be 0.1 to 100 times (v/w), preferably 0.1 to 10 times (v/w), relative to the amount of the compound of formula [2].
  • the solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons, and 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 a temperature of from -30 to 150°C, preferably from 0 to 100°C, for 5 minutes to 48 hours.
  • 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.
  • a reducing agent examples 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 of the solvent include halogenated hydrocarbons, alcohols, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons, and these solvents may be used in combination. Preferred solvents include esters, with ethyl acetate being more preferred.
  • the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v/w) the amount of the compound of formula [3A].
  • the reducing agent used in this reaction includes sodium borohydride, sodium cyanoborohydride, pyridine borane, 2-picoline borane, and sodium triacetoxyborohydride, with sodium triacetoxyborohydride being more preferred.
  • the amount of the reducing agent used may be 1 to 100 times, preferably 1 to 10 times, the molar amount of the compound of the formula [3A].
  • the amount of the compound of formula [4] used may be 0.1 to 1 mole per mole of the compound of formula [3A]. This reaction may be carried out at a temperature of from -30 to 150°C, preferably from 0 to 100°C, for 5 minutes to 48 hours.
  • 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 may be carried out in accordance with the production method (1-2), and the compound of formula [4] may be used in an amount of 1 to 10 times by mole relative to the compound of formula [3A].
  • the compound of formula [1] can be produced by reacting a compound of formula [3B] with a compound of formula [5] in the presence of a reducing agent. This reaction may be carried out in accordance with the production method (1-2), and the compound of formula [3B] may be used in an amount of 1 to 10 times by mole relative to the compound of formula [5].
  • compounds having an amino group, a hydroxyl group, a carboxyl group, or the like 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 reactions known per se, such as condensation, addition, oxidation, reduction, rearrangement, substitution, halogenation, dehydration, or hydrolysis, or by appropriately combining these reactions.
  • the hydrocarbon groups are preferably alkyl groups, alkenyl groups, or alkynyl groups.
  • the alkyl group may be straight-chained or branched, and may be linear or cyclic. Specific examples of the alkyl group include methyl, ethyl, 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, tetramethylhexadecyl (preferably 3,7,11,15-tetramethylhexadecyl), heptadecyl, octadecyl, 2-but
  • the alkenyl group may be linear or branched, and may be 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.
  • the alkynyl group may be straight-chain or branched, and may be linear or cyclic. Specific examples include propargyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, and octadecynyl groups.
  • each of the above alkenyl groups has one or two double bonds, and it is preferable that each of the alkynyl groups has one or two triple bonds.
  • the hydrocarbon group having 2 to 8 carbon atoms represented by R 7 , R 8 and R 9 in formula (3) 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 straight-chain 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.
  • aryl groups having 6 to 20 carbon atoms aryl groups having 6 to 18 carbon atoms are preferred, and aryl groups having 6 to 10 carbon atoms are even more preferred.
  • Specific examples include phenyl groups, naphthyl groups, anthracenyl groups, and phenanthrenyl groups.
  • a heterocyclic group means a heteroaryl group or a heteroaliphatic cyclic group.
  • heteroaryl group refers to an aromatic heterocyclic group, and may be an aromatic heterocyclic group fused with an aromatic hydrocarbon ring, an aromatic aliphatic ring, or an aliphatic hydrocarbon ring. It 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.
  • a five-membered heteroaryl group is a monocyclic heteroaryl group having five atoms constituting the ring.
  • an aromatic heterocycle refers to an aromatic ring having a heteroatom as a ring member, and may be a condensed aromatic heterocycle, aromatic hydrocarbon ring, heteroaliphatic ring, or aliphatic hydrocarbon ring. It 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.
  • the monocyclic nitrogen-containing heteroaryl group refers to a heteroaryl group (which may be partially saturated) in which the ring containing at least one nitrogen atom has aromaticity, such as pyrrolinyl, pyrrolyl, tetrahydropyridyl, pyridyl, imidazolinyl, imidazolyl, pyrazolinyl, pyrazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazolyl, and tetrazolyl groups.
  • This 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 (which may be partially saturated) containing at least one oxygen atom in a ring that has aromaticity, such as a furanyl or pyranyl group, and which may be further fused with another aromatic ring or an aliphatic ring.
  • the monocyclic nitrogen-containing and oxygen-containing heteroaryl group means an oxazolyl, isoxazolyl, or oxadiazolyl group, etc. This heteroaryl group may be further condensed 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.
  • Bicyclic nitrogen-containing heteroaryl groups include 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,
  • a bicyclic oxygen-containing heteroaryl group refers to 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).
  • Bicyclic nitrogen-containing and oxygen-containing heteroaryl groups include 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.
  • a bicyclic heteroaryl group containing at least one nitrogen atom and at least one oxygen atom in an aromatic ring such as 2,3-dihydrofuro[2,3-d]pyridazinyl, 5,7-dihydrofuro[3,4-d]pyridazinyl, 6,7-dihydrofuro[3,2-c]pyridazinyl, 5,7-dihydrofuro[3,4-c]pyridazinyl, and 5,6-dihydrofuro[2,3-c]pyridazinyl (this heteroaryl group may be partially saturated).
  • the heteroaliphatic cyclic group means a nitrogen-containing heteroaliphatic cyclic group, an oxygen-containing heteroaliphatic cyclic group, a sulfur-containing heteroaliphatic cyclic group, a nitrogen-containing and oxygen-containing heteroaliphatic cyclic group, a nitrogen-containing and sulfur-containing heteroaliphatic cyclic group, a heterobridged cyclic group, or a heterospiro cyclic group.
  • the heteroaliphatic ring means an aliphatic ring having a heteroatom as a ring member, and preferred examples thereof include a nitrogen-containing heteroaliphatic ring, an oxygen-containing heteroaliphatic ring, a sulfur-containing heteroaliphatic ring, a nitrogen-containing and oxygen-containing heteroaliphatic ring, a nitrogen-containing and sulfur-containing heteroaliphatic ring, a heterobridged ring, and a heterospiro ring.
  • the nitrogen-containing heteroaliphatic cyclic group refers to a heteroaliphatic cyclic group in which the ring containing at least one nitrogen atom does not have aromaticity, such as azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, octahydroazocinyl, imidazolidinyl, pyrazolidinyl, piperazinyl, and homopiperazinyl groups.
  • This nitrogen-containing heteroaliphatic cyclic group may further be condensed with another aromatic ring or an aliphatic ring.
  • the oxygen-containing heteroaliphatic cyclic group means a tetrahydrofuranyl, tetrahydropyranyl, oxetanyl, 1,3-dioxanyl group, etc. This oxygen-containing heteroaliphatic cyclic group may be further condensed with another aromatic ring or an aliphatic ring.
  • the nitrogen- and oxygen-containing heteroaliphatic cyclic group means a morpholinyl or 1,4-oxazepanyl group, etc. This nitrogen- and oxygen-containing heteroaliphatic cyclic group may be further condensed with another aromatic ring or an aliphatic ring.
  • the lipid represented by any one of formulas (1) to (3) may form a salt.
  • 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.
  • mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid
  • organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic
  • 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- ⁇ -phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine.
  • preferred salts include pharmacologically acceptable salts.
  • the amount of 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.
  • 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.
  • the second solution in step 2 may contain all of a neutral lipid, a lipid having a nonionic hydrophilic polymer, and a sterol.
  • the neutral lipid is not particularly limited, but includes phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, etc., and phosphatidylethanolamine or phosphatidylcholine is preferred.
  • the neutral lipid may be used alone or in combination with multiple different neutral lipids.
  • Phosphatidylcholines include, but are not limited to, 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), and dioleoylphosphatidylcholine (DOPC).
  • SPC soybean lecithin
  • HSPC hydrogenated soybean lecithin
  • EPC egg yolk lecithin
  • HEPC hydrogenated egg yolk lecithin
  • DMPC dimyristoylphosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • POPC 1-palmitoyl-2-oleo
  • dipalmitoylphosphatidylcholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is preferred, with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) being more preferred.
  • Phosphatidylethanolamines are not particularly limited, but include dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, dioctadecylphosphatidylethanolamine, and diphytanylphosphatidylethanolamine.
  • DMPE dimyristoylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DSPE distea
  • sphingomyelin examples include, but are not limited to, egg yolk-derived sphingomyelin and milk-derived sphingomyelin.
  • the ceramide is not particularly limited, but examples thereof include egg yolk-derived ceramide and milk-derived ceramide.
  • the amount of neutral lipids is preferably 3 mol% or more and 55 mol% or less, more preferably 3 mol% or more and 45 mol%, and even more preferably 3 mol% or more and 30 mol% of the total amount of constituent lipid components.
  • lipid having a nonionic hydrophilic polymer By including a lipid having a nonionic hydrophilic polymer in the lipid nanoparticles, the dispersion stabilization effect of the lipid nanoparticles can be obtained.
  • a lipid having a nonionic hydrophilic polymer a lipid having a nonionic hydrophilic polymer chain is preferred.
  • nonionic hydrophilic polymers include, but are not limited to, nonionic vinyl polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, modified nonionic natural polymers, and block polymers or graft copolymers containing two or more of these polymers as building blocks.
  • nonionic polyethers are preferred, 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.
  • PEG polyethylene glycol
  • the lipid having a nonionic hydrophilic polymer is not particularly limited, but includes PEG-modified phosphatidylethanolamine, diacylglycerol PEG derivative, monoacylglycerol PEG derivative, dialkylglycerol PEG derivative, cholesterol PEG derivative, ceramide PEG derivative, polysarcosine derivative, etc. 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 and may have a substituent such as a hydroxymethyl group.
  • the amount of lipid having a nonionic hydrophilic polymer is preferably 0.25 mol% to 12 mol% of 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%.
  • sterols>> By including sterol in lipid nanoparticles, it is possible to reduce membrane fluidity and obtain a stabilizing effect on the lipid nanoparticles.
  • the sterol is not particularly limited, but examples thereof include cholesterol, phytosterols (sitosterol, ⁇ -sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol, etc.), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, etc.
  • cholesterol is preferred.
  • 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% relative to the total lipid amount.
  • 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.
  • 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.
  • 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.
  • Chemically modified nucleic acids may also be included.
  • 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, 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.
  • proteins, peptides, and small molecules include intracellular proteins, intracellular peptides, transmembrane proteins, transmembrane peptides, secreted proteins, secreted peptides, synthetic proteins, synthetic peptides, natural small molecular weight compounds, synthetic small molecular weight compounds, and compounds having antitumor activity.
  • the peptides that can be encapsulated are peptides that have natural or unnatural amino groups and may have a linear or cyclic structure. Cyclic peptides are preferably linked by amide or thioether groups.
  • 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.
  • low molecular weight compounds refer to organic compounds with a molecular weight of approximately 1,000 or less.
  • 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.
  • 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.
  • the morphology of lipid nanoparticles can be confirmed by electron microscopy or structural analysis using X-rays. For example, using cryo-transmission electron microscopy (cryo-TEM), it can be determined whether the lipid particles have a lipid bilayer structure (lamellar structure) and an inner water layer, like liposomes, or whether they have an electron-dense core inside the particle packed with lipids and other components. Small-angle X-ray scattering (SAXS) measurements can also be used to confirm whether lipid nanoparticles have a lipid bilayer structure (lamellar structure).
  • SAXS Small-angle X-ray scattering
  • 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, more preferably less than 200 nm, even more preferably 30 nm to less than 150 nm, even more preferably 40 nm to less than 120 nm, and particularly preferably 40 nm to less than 100 nm.
  • the average particle size of lipid nanoparticles can be measured using, for example, a multi-analyte nanoparticle size measurement system nanoSAQLA (Otsuka Electronics Co., Ltd.).
  • 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.
  • the lipid nanoparticles produced by the method of the present invention can be administered to cells to deliver the active ingredient contained in the lipid nanoparticles to the cells.
  • the method of delivering the active ingredient to cells may exclude or include a method of treating humans.
  • the cells to which the active ingredient is delivered are not particularly limited, and cells can be selected according to the purpose.
  • 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.
  • normal tissue cells such as liver cells or muscle cells
  • immune cells to achieve a therapeutic effect.
  • examples of cells include stem cells such as induced pluripotent stem cells (iPS cells) and mesenchymal stem cells, and immune cells.
  • iPS cells induced pluripotent stem cells
  • mesenchymal stem cells and immune cells.
  • Cells are preferably derived from mammals, and more preferably from humans.
  • the immune cells are not particularly limited, and can be selected from, 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 + T cells, regulatory T cells (Tregs), B cells, NK cells, innate lymphocytes, or dendritic cells (DCs).
  • lymphocytes e.g., T cells, B cells, natural killer cells (NK cells), NKT cells, iNKT cells
  • monocytes e.g., macrophages, mast cells
  • dendritic cells granulocytes (e.g., neutrophils, eosinophils, and basophils)
  • the lipid nanoparticles contain an active ingredient with medicinal uses, the lipid nanoparticles can be administered to a living body as a pharmaceutical composition.
  • lipid nanoparticles When lipid nanoparticles are used as pharmaceutical compositions, they can be administered to a living body either alone or mixed with a pharmaceutically acceptable administration vehicle (e.g., physiological saline or a buffer solution).
  • a pharmaceutically acceptable administration vehicle e.g., physiological saline or a buffer solution.
  • the concentration of lipid nanoparticles in a mixture with a pharmaceutically acceptable administration vehicle is not particularly limited, and can generally be 0.05% to 90% by mass.
  • Pharmaceutical compositions containing lipid nanoparticles may also contain other pharmaceutically acceptable additives, such as pH-adjusting buffers and osmotic pressure adjusters.
  • the route of administration for pharmaceutical compositions containing lipid nanoparticles is not particularly limited, and any method can be used.
  • 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.
  • Pharmaceutical compositions containing lipid nanoparticles can also be administered by direct injection into the diseased site.
  • the dosage form of a pharmaceutical composition containing lipid nanoparticles is not particularly limited, but for oral administration, the lipid nanoparticles can be combined with an appropriate excipient and used in the form of tablets, troches, capsules, pills, suspensions, syrups, etc.
  • formulations suitable for parenteral administration can contain additives such as antioxidants, buffers, bacteriostats, and isotonic sterile injections, suspending agents, solubilizers, thickeners, stabilizers, or preservatives, as appropriate.
  • 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 ⁇ g/mL to obtain an aqueous phase.
  • FLuc mRNA product name: CleanCap FLuc mRNA (5 moU); TriLink
  • 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) 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.
  • Step 4 Mixing 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 mixing ratio (volume ratio) of LNP dispersion A to the third solution was 1:3.
  • Step 5 pH adjustment and post-treatment step>
  • the 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.
  • the LNP dispersion after the pH adjustment was concentrated approximately 10 times by ultrafiltration using a centrifugal filter (Amicon Ultra-15 100 kDa), and 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 ⁇ g/mL using the above dialysate, and filtered through a 0.22 ⁇ m filter to obtain FLuc mRNA-encapsulating lipid particles.
  • 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.
  • 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.
  • the first and second solutions were mixed so that the flow rate of LNP dispersion A, which was a mixture of the first and second solutions, was 14 mL/min.
  • 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 in LNP dispersion A was 32.
  • Step 4 Mixing 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 2, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B.
  • the mixing ratio (volume ratio) of LNP Dispersion A to the third solution was 1:3.
  • 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.
  • 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.
  • the first and second solutions were mixed so that the flow rate of LNP dispersion A, which was a mixture of the first and second solutions, was 14 mL/min.
  • 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 in LNP dispersion A was 32.
  • Step 4 Mixing 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 3, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B.
  • the mixing ratio (volume ratio) of LNP Dispersion A to the third solution was 1:3.
  • 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.
  • 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.
  • the first and second solutions were mixed so that the flow rate of LNP dispersion A, which was a mixture of the first and second solutions, was 14 mL/min.
  • 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 in LNP dispersion A was 32.
  • Step 4 Mixing 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 3, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B.
  • the mixing ratio (volume ratio) of LNP Dispersion A to the third solution was 1:3.
  • 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 ⁇ g/mL to obtain an aqueous phase.
  • FLuc mRNA product name: CleanCap FLuc mRNA (5 moU); TriLink
  • 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.
  • 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.
  • the first and second solutions were mixed so that the flow rate of LNP dispersion A, which was a mixture of the first and second solutions, was 14 mL/min.
  • 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 in LNP dispersion A was 32.
  • Step 4 Mixing 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 4, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B.
  • the mixing ratio (volume ratio) of LNP Dispersion A to the third solution was 1:3.
  • Step 1 Preparation of first solution (aqueous phase)> FLuc mRNA (product name: CleanCap FLuc mRNA (5 moU); TriLink) was diluted to 92.9 ⁇ g/mL with 50 mmol/L citrate buffer adjusted to pH 4 to obtain an aqueous phase.
  • FLuc mRNA product name: CleanCap FLuc mRNA (5 moU); TriLink
  • 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.
  • 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.
  • the first and second solutions were mixed so that the flow rate of LNP dispersion A, which was a mixture of the first and second solutions, was 14 mL/min.
  • 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 in LNP dispersion A was 32.
  • Step 4 Mixing 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 in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B.
  • the mixing ratio (volume ratio) of LNP Dispersion A to the third solution was 1:3.
  • 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 ⁇ g/mL to obtain an aqueous phase.
  • FLuc mRNA product name: CleanCap FLuc mRNA (5 moU); TriLink
  • 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 to a total lipid concentration of 12.5 mmol/L to obtain an oil phase.
  • 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.
  • the first and second solutions were mixed so that the flow rate of LNP dispersion A, which was a mixture of the first and second solutions, was 14 mL/min.
  • 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 in LNP dispersion A was 32.
  • Step 4 Mixing 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 mixing ratio (volume ratio) of LNP Dispersion A to the third solution was 1:3.
  • Step 5 pH adjustment and post-treatment step>
  • 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 ⁇ g/mL using the dialysate.
  • the concentration-adjusted sample was filtered through a Millipore 0.5/0.2 ⁇ m Express SHC filter to obtain lipid particles encapsulating FLuc mRNA.
  • 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 ⁇ m filter to obtain lipid particles encapsulating FLuc mRNA.
  • 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 ⁇ g/mL to obtain an aqueous phase.
  • FLuc mRNA product name: CleanCap FLuc mRNA (5 moU); TriLink
  • 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 to a total lipid concentration of 12.5 mmol/L to obtain an oil phase.
  • 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.
  • the first and second solutions were mixed so that the flow rate of LNP dispersion A, which was a mixture of the first and second solutions, was 14 mL/min.
  • 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 in LNP dispersion A was 32.
  • Step 5 pH adjustment and post-treatment step>
  • the 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.
  • the LNP dispersion after the pH adjustment was concentrated approximately 10 times by ultrafiltration using a centrifugal filter (Amicon Ultra-15 100 kDa), and 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 ⁇ g/mL using the above dialysate, and filtered through a 0.22 ⁇ m filter to obtain lipid particles encapsulating FLuc mRNA.
  • 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)
  • 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)
  • 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 to a total lipid concentration of 12.5 mmol/L to obtain an oil phase.
  • 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.
  • the first and second solutions were mixed so that the flow rate of LNP dispersion A, which was a mixture of the first and second solutions, was 14 mL/min.
  • 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 in LNP dispersion A was 32.
  • Step 4 Mixing 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 7, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B.
  • the mixing ratio (volume ratio) of LNP Dispersion A to the third solution was 1:3.
  • 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 ⁇ g/mL to obtain an aqueous phase.
  • FLuc mRNA product name: CleanCap FLuc mRNA (5 moU); TriLink
  • Step 2 Preparation of second solution (oil phase)>
  • 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, except that the compound B, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40/10/47/3 to give an oil phase.
  • 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 as the pH-responsive lipid.
  • Compound C, DOPE, cholesterol, and DMG-PEG2000 were dissolved in ethanol in a molar ratio of 40/10/47/3 to a total lipid concentration of 12.5 mmol/L to obtain an oil phase, similar to Examples 1 to 9.
  • 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.
  • the first and second solutions were mixed so that the flow rate of LNP dispersion A, which was a mixture of the first and second solutions, was 14 mL/min.
  • 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 in LNP dispersion A was 32.
  • Step 4 Mixing 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 8, and then mixed with the third solution in the same manner as in Examples 1 to 9 to obtain LNP Dispersion B.
  • the mixing ratio (volume ratio) of LNP Dispersion A to the third solution was 1:3.
  • PDI polydispersity index
  • ⁇ Evaluation of mRNA Encapsulation Rate> (Quantification of total mRNA concentration) 450 ⁇ L of methanol was added to 50 ⁇ L of mRNA and mRNA-encapsulating lipid particle samples 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).
  • the external aqueous phase mRNA concentration was quantified using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific) 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).
  • Lipid particle samples encapsulating mRNA were prepared as undiluted solutions or diluted 5-fold with 1x TE buffer to prepare measurement samples.
  • mRNA encapsulation rate (%) (total mRNA concentration - mRNA concentration in the external aqueous phase) ⁇ total mRNA concentration ⁇ 100
  • ⁇ Lipid determination> The amount of lipid in the mRNA-encapsulating lipid particle sample was calculated by measuring it using an HPLC equipped with a CAD detector.
  • the change in particle size before and after pH adjustment and the residual lipid content 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 mRNA-encapsulated lipid particle samples before and after adjusting the pH to a pH higher than the pKa of the LNP, and calculating the difference.
  • the residual lipid content after filtration was calculated by quantifying the lipid content of the mRNA-encapsulated lipid particle samples before and after filtration, and dividing the lipid content after filtration by the lipid content before filtration.
  • pKa measurement> The pKa of the mRNA-encapsulated lipid particle sample was calculated by TNS assay.
  • the LNP sample was diluted with Milli-Q water, and then mixed with TNS reagent dissolved in DMSO and various buffers with pH values ranging from 3 to 8.5, each with a pH difference of 0.5.
  • the fluorescence intensity of the above measurement solution was measured using a fluorescent plate reader at excitation wavelengths of 337 nm and 435 nm, and the pKa was calculated from the change in fluorescence intensity with respect to pH.
  • the pKa of the LNP in Example 1 was 6.58
  • the pKa of the LNP in Example 4 was 6.57
  • the pKa of the LNP in Example 6 was 6.50
  • the pKa of the LNP in Example 33 was 6.51
  • the pKa of the LNP in Comparative Example 1 was 6.56
  • the pKa of the LNP in Comparative Example 9 was 6.54.
  • ⁇ Luciferase luminescence measurement> The LNPs containing FLuc mRNA prepared in Examples 1, 4, 6, and Comparative Example 1 were administered intravenously once to ICR mice at a dose of 0.2 mg/kg mRNA. Five hours and 45 minutes after administration, 150 mg/kg of D-luciferin potassium (Fujifilm Wako Pure Chemical Industries, Ltd.) was administered intraperitoneally. Six hours after administration, the liver was excised 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 Photons/Sec (light intensity).
  • step (3) and step (4) 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).
  • PDI particle size polydispersity index
  • LNPs using pH-responsive lipids different from those used in Table 1 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.
  • PDI polydispersity index

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Abstract

L'objet de la présente invention est de mettre à disposition un procédé de production de nanoparticules lipidiques, qui permet la production de nanoparticules lipidiques ayant une distribution étroite de taille de particule. À cet effet, la présente invention concerne un procédé de production de nanoparticules lipidiques, le procédé comprenant une étape 1 consistant à préparer une première solution qui est une solution aqueuse acide contenant un ingrédient actif, une étape 2 consistant à préparer une seconde solution qui est une solution contenant un lipide spécifique et un alcool ; une étape 3 consistant à préparer une dispersion de nanoparticules lipidiques par mélange de la première solution avec la seconde solution dans un canal d'écoulement, une étape de correction de pH consistant à corriger le pH de la dispersion de nanoparticules lipidiques, et une étape d'élimination d'alcool, la teneur en alcool dans la dispersion de nanoparticules lipidiques obtenue à l'étape 3 étant de 12,5 à 33 % en volume, et la dispersion de nanoparticules lipidiques étant maintenue pendant 1 minute ou plus après l'achèvement de l'étape 3.
PCT/JP2025/022598 2024-06-24 2025-06-24 Procédé de production de nanoparticules lipidiques Pending WO2026004829A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019235635A1 (fr) * 2018-06-08 2019-12-12 富士フイルム株式会社 Composé, sel de celui-ci et particules lipidiques
WO2021095876A1 (fr) * 2019-11-15 2021-05-20 富士フイルム株式会社 Composition lipidique
WO2022230964A1 (fr) * 2021-04-28 2022-11-03 富士フイルム株式会社 Composé ou sel de celui-ci, particules lipidiques et composition pharmaceutique
CN117645549A (zh) * 2023-11-28 2024-03-05 东南大学 一种可离子化脂质或其药学上可接受的盐、组合物和应用
WO2024085190A1 (fr) * 2022-10-19 2024-04-25 Fujifilm Corporation Composition lipidique et méthode d'administration d'un agent thérapeutique
WO2024158042A1 (fr) * 2023-01-27 2024-08-02 富士フイルム株式会社 Composé ou sel de celui-ci, composition lipidique, composition pharmaceutique et support d'administration

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019235635A1 (fr) * 2018-06-08 2019-12-12 富士フイルム株式会社 Composé, sel de celui-ci et particules lipidiques
WO2021095876A1 (fr) * 2019-11-15 2021-05-20 富士フイルム株式会社 Composition lipidique
WO2022230964A1 (fr) * 2021-04-28 2022-11-03 富士フイルム株式会社 Composé ou sel de celui-ci, particules lipidiques et composition pharmaceutique
WO2024085190A1 (fr) * 2022-10-19 2024-04-25 Fujifilm Corporation Composition lipidique et méthode d'administration d'un agent thérapeutique
WO2024158042A1 (fr) * 2023-01-27 2024-08-02 富士フイルム株式会社 Composé ou sel de celui-ci, composition lipidique, composition pharmaceutique et support d'administration
CN117645549A (zh) * 2023-11-28 2024-03-05 东南大学 一种可离子化脂质或其药学上可接受的盐、组合物和应用

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