Lipid particle

Abstract

To provide lipid particles that can be produced without relying on PEG-modified lipids, that exhibit superior medicinal effects, safety, or stability compared to lipid particles that rely on PEG-modified lipids, or that have different immunodynamics from lipid particles that rely on PEG-modified lipids.SOLUTION: Provided is a lipid particle, wherein the lipid particle contains an ionized lipid, a phospholipid, and a sterol as lipids constituting the lipid particle and contains a modified polysaccharide including a hydrophobic group.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to lipid particles and the like. [Background technology]

[0002] Lipid nanoparticles (LNPs) can encapsulate various drugs and are used as drug delivery carriers. With the recent COVID-19 epidemic, vaccines using LNPs to prevent infectious diseases have come into widespread use.

[0003] LNPs currently used in vaccines contain polyethylene glycol (PEG)-modified lipids to inhibit particle aggregation (Patent Document 1). At present, LNPs cannot be obtained without using PEG-modified lipids. However, PEG is an allergen, and is contained in various products used daily or for medical purposes, so there is a high frequency of contact, raising concerns about anaphylactic reactions caused by PEG. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 8,058,069 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide lipid particles which can be produced without relying on PEG-modified lipids, or which have superior medicinal efficacy, safety, or stability to lipid particles which rely on PEG-modified lipids, or which have different immune dynamics from lipid particles which rely on PEG-modified lipids. [Means for solving the problem]

[0006] In view of the above problems, the present inventors have conducted intensive research and have surprisingly found that the above problems can be solved by lipid particles that contain ionized lipids, phospholipids, and sterols as lipid particle constituents, and also contain modified polysaccharides containing hydrophobic groups. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention includes the following aspects.

[0007] Item 1. A lipid particle, comprising an ionized lipid, a phospholipid, and a sterol as lipids constituting the lipid particle, and comprising a modified polysaccharide having a hydrophobic group.

[0008] Item 2. The content of the ionized lipid is 35 to 70 mol% relative to 100 mol% of the lipids constituting the lipid particles, The content of the phospholipid is 4 to 25 mol% relative to 100 mol% of the lipids constituting the lipid particles, and The content of the sterol is 15 to 55 mol% relative to 100 mol% of the lipids constituting the lipid particles. Item 1. The lipid particle according to item 1.

[0009] Item 3. The lipid particle according to Item 1 or 2, wherein the constituent polysaccharide of the modified polysaccharide comprises pullulan.

[0010] Item 4. The lipid particle according to any one of Items 1 to 3, wherein the hydrophobic group includes a hydrophobic group having a sterol skeleton.

[0011] Item 5. The lipid particle according to any one of Items 1 to 4, wherein the modified polysaccharide has a weight-average molecular weight of 5,000 to 2,000,000.

[0012] Item 6. The lipid particle according to any one of Items 1 to 5, which contains a drug.

[0013] Item 7. The lipid particle according to Item 6, wherein the drug is a nucleic acid.

[0014] Item 8. The lipid particle according to Item 7, wherein the nucleic acid is at least one selected from mRNA, immunostimulatory double-stranded RNA, siRNA, miRNA, ribozyme, CpG oligo DNA, antisense oligo DNA, nucleic acid aptamer, degoi, double-stranded DNA for gene expression, and dinucleotide.

[0015] Item 9. The lipid particle according to Item 7 or 8, wherein the nucleic acid is mRNA or CpG oligoDNA.

[0016] Item 10. The lipid particle according to any one of Items 7 to 9, wherein the nucleic acid is mRNA.

[0017] Item 11. The lipid particle according to any one of Items 7 to 9, wherein the nucleic acid is a CpG oligoDNA.

[0018] Item 12. The ionizable lipid is represented by the general formula (1):

[0019] [ka]

[0020] [In the formula: R 1 R represents an alkyl group. 2 R represents an alkyl group. 3 R represents an alkyl group. 4 represents an alkyl group or a hydrogen atom. 1 represents -OC(=O)- or -C(=O)-O-. 2 represents -OC(=O)- or -C(=O)-O-. m represents an integer of 1 to 8. n represents an integer of 3 to 10. p represents an integer of 3 to 10.] Amino lipids represented by general formula (2):

[0021] [ka]

[0022] [In the formula, a represents an integer of 3 to 5; b represents 0 or 1; and c represents an integer of 0 to 4. R 5and R 6 are the same or different and each represents an alkyl group or an alkenyl group. X represents an optionally substituted non-aromatic nitrogen-containing heterocyclic group or an optionally substituted amino group. and amino lipids represented by the general formula (3)

[0023] [ka]

[0024] [In the formula: R 7 and R 8 are the same or different and represent an alkyl group or an alkenyl group; Y represents an optionally substituted non-aromatic nitrogen-containing heterocyclic group or an optionally substituted amino group; and E represents a linker.] Item 12. The lipid particle according to any one of items 1 to 11, wherein the lipid particle is at least one selected from the group consisting of amino lipids represented by the following formula:

[0025] Item 13. The particle according to item 1, further comprising a conjugated lipid that inhibits particle aggregation.

[0026] Item 14. The lipid particle according to any one of Items 1 to 13, wherein the content of the complex lipid is 0 to 2 mol % relative to 100 mol % of the lipids constituting the lipid particle.

[0027] Item 15. The lipid particle according to Item 13 or 14, wherein the conjugated lipid is a polyethylene glycol-lipid conjugate.

[0028] Item 16. The lipid particles according to any one of Items 1 to 15, having a mass median particle diameter of 10 to 200 nm.

[0029] Item 17. A medicine comprising the lipid particle according to any one of items 1 to 16.

[0030] Item 18. The pharmaceutical according to Item 17, which is a therapeutic drug for cancer, immune diseases, central nervous system diseases or the like, an infectious disease vaccine, or a cancer vaccine.

[0031] Item 18A. A method for preventing or treating cancer, an immune disease, a central nervous system disease, or an infectious disease, comprising administering the lipid particle according to any one of items 1 to 16 to a subject (e.g., a patient) (preferably a subject having cancer, a subject having an immune disease, a subject having a central nervous system disease, a subject having an infectious disease, or a subject in need of cancer prevention, an immune disease prevention, a central nervous system disease prevention, or a subject in need of infectious disease prevention).

[0032] Item 18B. The lipid particle according to any one of Items 1 to 16 for use as a therapeutic agent for cancer, immune diseases, central nervous system diseases, etc., or as an infectious disease vaccine or cancer vaccine.

[0033] Item 18C. Use of the lipid particle according to any one of items 1 to 16 for producing a therapeutic drug for cancer, immune diseases, central nervous system diseases, etc., or an infectious disease vaccine or a cancer vaccine. Item 18D. Use of the lipid particle according to any one of items 1 to 16 as a therapeutic agent for cancer, immune diseases, central nervous system diseases, etc., or as an infectious disease vaccine or cancer vaccine.

[0034] Item 19. The pharmaceutical agent according to Item 17 or 18, which is for use in subcutaneous administration, intravenous administration, intramuscular administration, intranasal administration, or intratracheal administration. Effect of the Invention

[0035] According to the present invention, it is possible to provide lipid particles which can be produced without relying on PEG-modified lipids, or which have superior medicinal efficacy, safety, or stability to lipid particles which rely on PEG-modified lipids, or which have different immune dynamics from lipid particles which rely on PEG-modified lipids. [Brief description of the drawings]

[0036] [Figure 1]In Test Example 5, the results are shown for the case where LNP was added and then incubated overnight at 37°C without treatment at 4°C. The results are shown for the case where LNP(EGFP) (Reference Example 2) or CHP:LNP(EGFP) (Example 2) was added, and the negative control shows the result when LNP was not added. M0 shows the result when M0 macrophages were used, and M2 shows the result when M2-like macrophages were used. [Diagram 2] This shows the results in Test Example 5, where LNP was added, followed by treatment at 4° C. for 2 hours, then the medium was replaced and further incubation was performed overnight at 37° C. The meanings of each notation in the figure are the same as in FIG. [Diagram 3] This shows the results in Test Example 5, where after addition of LNP, the cells were incubated overnight at 37° C. without treatment at 4° C. The meanings of each notation in the figure are the same as in FIG. [Figure 4] This shows the results of Test Example 8, where after addition of LNP, the cells were incubated overnight at 37° C. without treatment at 4° C. The meanings of each notation in the figure are the same as those in FIG. [Diagram 5] 1 shows the results of measuring blood PEG antibodies after administration of LNP in Test Example 9. The negative control shows the results when LNP was not administered. [Figure 6] 1 shows the results of a representative example of the administration site in Test Example 10. The negative control shows the results when LNP was not administered. [Figure 7] 1 shows the results of a representative example of nearby lymph nodes (inguinal lymph nodes) excised in Test Example 10. The negative control shows the results when LNP was not administered. [Figure 8] 1 shows the results of changes in body weight over time after administration of LNP in 12 test cases, for comparison, the results when LNP was not administered. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0038] 1. Lipid particles In one aspect, the present invention relates to a lipid particle (sometimes referred to as "lipid particle of the present invention" in this specification) that contains an ionized lipid, a phospholipid, and a sterol as lipids constituting the lipid particle, and also contains a modified polysaccharide containing a hydrophobic group. This will be described below.

[0039] The ionizable lipid is not particularly limited as long as it is capable of forming lipid particles and is a lipid that exhibits a positive charge within the lipid particles. Examples of the ionizable lipid include [4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (D-Lin-MC3-DMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-Dilinoleyl-4-(4-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-Dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol oleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy) N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl) cis,cis-9,12-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,1'-[7-[4-(dipropylamino)butyl]-7-hydroxy-1,13-tridecanediyl] di-(9Z)-9-octadecenoate (CL 4H6), 5-hydroxy-11-[[(9Z)-1-oxo-9-octadecen-1-yl]oxy]-5-[6-[[(9Z)-1-oxo-9-octadecen-1-yl]oxy]hexyl] undecyl 1-methyl-4-piperidinecarboxylate (CL 15H6), 1,1'-[7-[4-(dimethylamino)butyl]-7-hydroxy-1,13-tridecanediyl] di-(9Z)-9-octadecanoate (CL 1H6), etc.

[0040] From the viewpoint of expression of the lipid particles of the present invention in cells, preferably antigen-presenting cells, the ionizable lipid is preferably represented by the general formula (1):

[0041] [ka]

[0042] [In the formula: R 1 R represents an alkyl group. 2 R represents an alkyl group. 3 R represents an alkyl group. 4 represents an alkyl group or a hydrogen atom. 1 represents -OC(=O)- or -C(=O)-O-. 2 represents -OC(=O)- or -C(=O)-O-. m represents an integer of 1 to 8. n represents an integer of 3 to 10. p represents an integer of 3 to 10.] Amino lipids represented by the general formula (2):

[0043] [ka]

[0044] [In the formula, a represents an integer of 3 to 5; b represents 0 or 1; and c represents an integer of 0 to 4. R 5 and R6 are the same or different and each represents an alkyl group or an alkenyl group. X represents an optionally substituted non-aromatic nitrogen-containing heterocyclic group or an optionally substituted amino group. and amino lipids represented by the general formula (3)

[0045] [ka]

[0046] [In the formula: R 7 and R 8 are the same or different and represent an alkyl group or an alkenyl group; Y represents an optionally substituted non-aromatic nitrogen-containing heterocyclic group or an optionally substituted amino group; and E represents a linker.] The amino lipid is at least one selected from the group consisting of amino lipids represented by the following formula: By using the amino lipid, it is possible to more significantly exhibit the above-mentioned accumulation property.

[0047] The alkyl group and the alkenyl group are linear or branched, and are particularly preferably linear. The lower limit of the number of carbon atoms of the alkyl group and the alkenyl group can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The upper limit of the number of carbon atoms of the alkyl group and the alkenyl group can be, for example, 30, 25, 20, 18, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4. The number of carbon atoms can be within a range that is any combination of the above upper and lower limits.

[0048] R 1 The number of carbon atoms in the alkyl group represented by is not particularly limited as long as the compound of general formula (1) becomes a lipid, and is, for example, 3 to 20. From the viewpoint of the above-mentioned accumulation property, the number of carbon atoms is preferably 3 to 12, more preferably 3 to 10, even more preferably 4 to 8, still more preferably 5 to 7, and particularly preferably 6.

[0049] R 2The number of carbon atoms in the alkyl group represented by is not particularly limited as long as the compound of general formula (1) becomes a lipid, and is, for example, 3 to 20. From the viewpoint of the above-mentioned accumulation property, the number of carbon atoms is preferably 4 to 15, more preferably 5 to 12, even more preferably 6 to 10, still more preferably 7 to 9, and particularly preferably 8.

[0050] R 3 The number of carbon atoms in the alkyl group represented by is not particularly limited as long as the compound of general formula (1) becomes a lipid, and is, for example, 3 to 20. From the viewpoint of the above-mentioned accumulation property, the number of carbon atoms is preferably 3 to 12, more preferably 3 to 10, even more preferably 4 to 8, still more preferably 5 to 7, and particularly preferably 6.

[0051] R 4 From the viewpoint of the above-mentioned accumulation property, is preferably an alkyl group.

[0052] R 4 The number of carbon atoms in the alkyl group represented by is not particularly limited as long as the compound of general formula (1) becomes a lipid, and is, for example, 3 to 20. From the viewpoint of the above-mentioned accumulation property, the number of carbon atoms is preferably 4 to 15, more preferably 5 to 12, even more preferably 6 to 10, still more preferably 7 to 9, and particularly preferably 8.

[0053] L 1 From the viewpoint of the above-mentioned accumulation property, is preferably -OC(=O)- (the -O- side of the divalent group represents the hydroxy group side in general formula (1)).

[0054] L 2 From the viewpoint of the above-mentioned accumulation property, is preferably -OC(=O)- (the -O- side of the divalent group represents the hydroxy group side in general formula (1)).

[0055] From the viewpoint of the above-mentioned accumulation property, m is preferably 2 to 6, more preferably 3 to 5, and particularly preferably 4.

[0056] From the viewpoint of the above-mentioned accumulation property, n is preferably 4 to 8, more preferably 5 to 7, and particularly preferably 6.

[0057] From the viewpoint of the above-mentioned accumulation property, p is preferably 4 to 8, more preferably 5 to 7, and particularly preferably 6.

[0058] R 5 The number of carbon atoms in the alkyl group represented by the following formula is not particularly limited, so long as the compound of general formula (2) becomes a lipid, and is, for example, 12 to 24.

[0059] R 6 The number of carbon atoms in the alkyl group represented by the following formula is not particularly limited, so long as the compound of general formula (2) becomes a lipid, and is, for example, 12 to 24.

[0060] R 7 The number of carbon atoms in the alkyl group represented by the following formula is not particularly limited, so long as the compound of general formula (2) becomes a lipid, and is, for example, 12 to 24.

[0061] R 8 The number of carbon atoms in the alkyl group represented by the following formula is not particularly limited, so long as the compound of general formula (2) becomes a lipid, and is, for example, 12 to 24.

[0062] When the non-aromatic nitrogen-containing heterocyclic group or amino group is substituted, the substituent is preferably an alkyl group, for example, an alkyl group having 1 to 4 carbon atoms.

[0063] A non-aromatic nitrogen-containing heterocyclic group is a monovalent group obtained by removing one hydrogen atom from a non-aromatic nitrogen-containing heterocycle. Examples of non-aromatic nitrogen-containing heterocycles include a pyrrolidinyl group, a piperidinyl group, a morpholinyl group, and a piperazinyl group.

[0064] The linker represented by E may be a linker having, for example, 1 to 10, and preferably 3 to 8, atoms constituting the chain. The linker is preferably an alkyl group optionally having -OC(=O)- or -C(=O)-O- in the chain.

[0065] The ionic lipid may be one type alone or two or more types in combination.

[0066] The content of the ionic lipid is preferably 35 to 70 mol %, more preferably 40 to 65 mol %, even more preferably 45 to 60 mol %, and even more preferably 45 to 55 mol %, relative to 100 mol % of the lipids constituting the lipid particles, from the viewpoints of lipid particle formability, the above-mentioned accumulation property, stability, and the like.

[0067] The ionic lipid can be prepared by purchasing a commercially available product, or can be produced according to or in accordance with a known method. For the production, for example, WO2018 / 230710 and WO2010 / 129687 can be referred to.

[0068] The phospholipid is not particularly limited as long as it is capable of forming lipid particles, and examples thereof include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, and the like. More specific examples include distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and the like.

[0069] From the viewpoints of lipid particle formability, stability, and the like, the phospholipid is preferably phosphatidylcholine, more preferably DSPC, DPPC, and the like, and even more preferably DSPC.

[0070] The phospholipids may be one type alone or two or more types in combination.

[0071] From the viewpoints of lipid particle formability, stability, etc., the content of phospholipids is preferably 4 to 25 mol%, more preferably 4 to 20 mol%, even more preferably 4 to 15 mol%, even more preferably 6 to 12 mol%, and particularly preferably 8 to 11 mol%, relative to 100 mol% of the lipids constituting the lipid particles.

[0072] The sterol is not particularly limited as long as it has a sterol skeleton and can constitute lipid particles, and examples thereof include cholesterol, cholesteryl hemisuccinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, phytosterol, phytosterol, stigmasterol, zymosterol, ergosterol, sitosterol, campesterol, brassicasterol, etc. In addition to these, cholesterol derivatives such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, and cholesteryl-4'-hydroxybutyl ether are also included.

[0073] From the viewpoints of lipid particle forming ability, stability, and the like, the sterol is preferably cholesterol.

[0074] The sterol may be one type alone or two or more types in combination.

[0075] From the viewpoints of lipid particle formability, stability, etc., the sterol content is preferably 15 to 55 mol %, more preferably 25 to 50 mol %, even more preferably 30 to 45 mol %, and even more preferably 35 to 45 mol %, relative to 100 mol % of the lipids constituting the lipid particles.

[0076] Modified polysaccharide is a compound that polysaccharide is modified, and as long as it contains hydrophobic group as modification group, it is not particularly limited.In the present invention, it has been found that by using modified polysaccharide, it is possible to form lipid particles (particularly, lipid particles that contain each lipid in the above content) without relying on PEG-modified lipid (preferably, it does not use the lipid, but it may contain some PEG as long as it avoids side reactions caused by PEG).

[0077] The polysaccharide constituting the modified polysaccharide (i.e., the polysaccharide before modification) is not particularly limited as long as it is a polymer in which the sugar residue is glycosidicly bonded. For example, the sugar residue constituting the polysaccharide may be a residue derived from a monosaccharide such as glucose, mannose, galactose, fucose, or a sugar such as a disaccharide or oligosaccharide. The sugar residue may be 1,2-, 1,3-, 1,4-, or 1,6-glycosidic bond, and the bond may be either α- or β-type bond. The polysaccharide may be either linear or branched. The sugar residue is preferably a glucose residue, and examples of the polysaccharide include natural or synthetic pullulan, mannan, dextran, amylose, amylopectin, etc. Among these, from the viewpoint of lipid particle forming property, stability, etc., preferably pullulan, mannan, dextran, etc., more preferably pullulan, mannan, etc., and particularly preferably pullulan, etc. are used.

[0078] The weight-average molecular weight of the polysaccharide is not particularly limited as long as the modified polysaccharide can form gelling particles, but is, for example, 5,000 to 2,000,000. The weight-average molecular weight is preferably 10,000 to 1,000,000, more preferably 20,000 to 500,000, even more preferably 40,000 to 250,000, and still more preferably 80,000 to 125,000.

[0079] As the polysaccharide, commercially available products can be used, or those obtained according to known production methods can be used.

[0080] The hydrophobic group is not particularly limited as long as it is a group having hydrophobicity. From the viewpoint of lipid particle formability, stability, etc., the hydrophobic group is preferably a hydrophobic group having a sterol skeleton, a hydrocarbon group, etc., and more preferably a hydrophobic group having a sterol skeleton.

[0081] The sterol skeleton is an alcohol having a hydroxy group bonded to a cyclopentahydrophenanthrene ring as shown in formula (I). The symbols A to D in formula (I) represent the individual rings constituting the cyclopentahydrophenanthrene ring.

[0082] [ka]

[0083] In the sterol skeleton, the cyclopentahydrophenanthrene ring may have a double bond, and the position of the hydroxyl group is not particularly limited. Preferably, the sterol has a hydroxyl group bonded to the C-3 position and a double bond to the B ring, or the stanol has a hydroxyl group bonded to the C-3 position and is composed of a saturated ring. Examples of the hydrophobic group having a sterol skeleton include a group derived from a compound in which the sterol skeleton is modified, for example, the ring carbon is substituted with a hydrocarbon group (e.g., a linear or branched alkyl group having 1 to 20 carbon atoms). Here, the "group derived from" refers to a group obtained by removing a functional group such as a hydrogen atom or a hydroxyl group from a certain compound.

[0084] Examples of hydrophobic groups having a sterol skeleton include a cholesterol-derived group, a cholestanol-derived group, a lanosterol-derived group, an ergosterol-derived group, a β-sitosterol-derived group, a campesterol-derived group, a stigmasterol-derived group, a brassicasterol-derived group, etc. Among these, preferred are sterol-derived groups such as a cholesterol-derived group, a cholestanol-derived group, a lanosterol-derived group, an ergosterol-derived group, etc., and more preferred are cholesterol-derived groups.

[0085] The hydrocarbon group as the hydrophobic group is not particularly limited, and examples thereof include chain (preferably linear) hydrocarbon groups (preferably alkyl groups) having 8 to 50 carbon atoms (preferably 10 to 30, more preferably 12 to 20).

[0086] The weight-average molecular weight of the modified polysaccharide is not particularly limited as long as the modified polysaccharide can form gelling particles, but is, for example, 5,000 to 2,000,000. The weight-average molecular weight is preferably 10,000 to 1,000,000, more preferably 20,000 to 500,000, even more preferably 40,000 to 250,000, and still more preferably 80,000 to 125,000.

[0087] The number of hydrophobic groups contained in the modified polysaccharide is not particularly limited as long as the modified polysaccharide can form gelling particles, and is, for example, 1 to 10, preferably 1 to 5, per 100 sugar residues that form the polysaccharide.

[0088] The hydrophobic group can be linked to the polysaccharide directly or indirectly (eg, via a linker).

[0089] The modified polysaccharide may, for example, be one in which the primary hydroxyl groups of, for example, 1 to 10 (preferably 1 to 5) saccharide units per 100 saccharide residues constituting the polysaccharide are represented by the formula (II): -O-(CH2) x CONH(CH2) y Preferred is one represented by NH-CO-OR (II) (wherein R represents a hydrophobic group or a hydrocarbon group having a sterol skeleton; x represents 0 or 1; and y represents any positive integer). y is preferably 1 to 8.

[0090] The modified polysaccharides can be synthesized according to or in accordance with known methods (e.g., International Publication No. WO00 / 12564). One example is the following method: First, a hydroxyl-containing hydrocarbon or sterol having 12 to 50 carbon atoms and OCN-R A NCO (in the formula, R Ais a hydrocarbon group having 1 to 50 carbon atoms.) is reacted with a diisocyanate compound represented by the formula (I) to produce an isocyanate group-containing hydrophobic compound in which one molecule of a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms has reacted. Next, the obtained isocyanate group-containing hydrophobic compound is further reacted with a polysaccharide to produce a hydrophobic group-containing polysaccharide containing a hydrocarbon group having 12 to 50 carbon atoms or a steryl group as the hydrophobic group. The obtained reaction product is purified with a ketone solvent to produce a high-purity hydrophobic group-containing polysaccharide.

[0091] The modified polysaccharides may be one type alone or two or more types in combination.

[0092] The content of the modified polysaccharide is, for example, 0.5 to 15 mol %, and preferably 1 to 10 mol %, relative to 100 mol % of the lipids constituting the lipid particles, from the viewpoints of lipid particle formability, stability, and the like.

[0093] The lipid particles of the present invention can contain a complexed lipid that inhibits aggregation of particles (hereinafter, simply referred to as "complexed lipid"). The complexed lipid is not particularly limited as long as it can inhibit aggregation of lipid particles and can constitute lipid particles, and examples thereof include polyethylene glycol (PEG)-lipid conjugates, polyamide (ATTA)-lipid conjugates, and cationic polymer-lipid conjugates (CPL). More specifically, the complexed lipid can contain, for example, PEG-lipids including PEG-diacylglycerol (DAG), PEG dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The molecular weight of the PEG portion of the PEG-lipid conjugate is not particularly limited, and is, for example, 200 to 10,000, preferably 500 to 7,000, more preferably 500 to 4,000, even more preferably 1,000 to 3,000, and even more preferably 1,500 to 2,500.

[0094] The conjugated lipid may be one type alone or a combination of two or more types.

[0095] The content of the complex lipid is, for example, 0 to 2 mol% relative to 100 mol% of the lipids constituting the lipid particles, from the viewpoints of lipid particle formability, stability, and avoidance of anaphylactic shock, etc. The content is preferably 0 to 1 mol%, more preferably 0 to 0.5 mol%, even more preferably 0 to 0.2 mol%, still more preferably 0 to 0.1 mol%, particularly preferably 0 to 0.01 mol%, and particularly preferably 0 mol% (no complex lipid is contained).

[0096] The lipid particles of the present invention preferably contain a drug. The drug is not particularly limited, but examples thereof include nucleic acid adjuvants and anticancer drugs, and they may be contained in combination.

[0097] In a preferred embodiment of the present invention, the drug is disposed inside the lipid particle of the present invention (separated from the outside of the lipid particle). Preferably, the drug (particularly, a nucleic acid) is disposed inside the lipid particle of the present invention in a complexed state with an ionized lipid.

[0098] The nucleic acid of the present invention is not particularly limited, and examples thereof include mRNA, immunostimulatory double-stranded RNA (poly-IC RNA, etc.), siRNA, miRNA, ribozyme, CpG oligo DNA, antisense oligo DNA, nucleic acid aptamer, degoy, double-stranded DNA for gene expression, and dinucleotides. The nucleic acid may be not only DNA and RNA, but also anything that can be a pharmaceutical active ingredient, and includes those that have been subjected to known chemical modifications, as exemplified below. In order to prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide can be replaced with chemically modified phosphate residues, such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or by replacing the carbonyl group at the 2-position with a thiocarbonyl. Furthermore, examples include those in which the phosphate portion or the hydroxyl portion is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc., but are not limited thereto. In addition, the term "nucleic acid" includes any of BNA (bridged nucleic acid), LNA (locked nucleic acid), PNA (peptide nucleic acid), etc.

[0099] The nucleic acid of the present invention can be preferably CpG oligo DNA or mRNA, particularly an mRNA encoding a microbial antigen or a cancer antigen described below.

[0100] Specific examples of CpG oligo DNA are as follows: Class A: D35-CpG, ODN1585, ODN2216, ODN2336, etc. Class B: K3-CpG, ODNBW006, ODN D-SL01, ODN1668, OND1826, OND2006 (CpG7909, PF-3512676), ODN2007, ODN684, etc. Class C: ODN D-SL03, ODN 2395, ODN M362, etc. Examples include:

[0101] Other examples include CpG-28, CpG-685 (GNKG-168), CpG-ODN C274, KSK-13 (KSK-CpG), CpG ODN 10104 (CpG-10104), CpG ODN-1585, ODN-5890, 1018-ISS, EMD-1201081 (HYB-2055, IMO-2055), and the like.

[0102] As the CpG oligo DNA, a commercially available product can be used, or one obtained according to a known manufacturing method can be used. For example, based on the contents of US2022 / 0111061 A1, it may be modified to include a hydrophobic group A having a sterol skeleton, but preferably is not modified to include a hydrophobic group A having a sterol skeleton.

[0103] In the case of mRNA, examples of the microbial antigens encoded by it include viral antigens, fungal antigens, etc. Examples of viruses from which viral antigens are derived include, but are not limited to, enveloped viruses (viruses having an envelope) such as influenza viruses (e.g., types A and B), rubella viruses, Ebola viruses, coronaviruses, measles viruses, varicella-zoster viruses, herpes simplex viruses, mumps viruses, arboviruses, RS viruses, SARS viruses, hepatitis viruses (e.g., hepatitis B viruses, hepatitis C viruses, etc.), yellow fever viruses, AIDS viruses, rabies viruses, hantaviruses, dengue viruses, Nipah viruses, and lyssaviruses; and non-enveloped viruses (viruses not having an envelope) such as adenoviruses, noroviruses, rotaviruses, human papillomaviruses, polioviruses, enteroviruses, coxsackieviruses, human parvoviruses, encephalomyocarditis viruses, and rhinoviruses. The bacteria from which the bacterial antigen is derived are not particularly limited, but examples include Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Salmonella, Helicobacter pylori, Clostridium perfringens, Clostridium botulinum, Campylobacter, Escherichia coli, Staphylococcus aureus, Streptococcus cereus, Vibrio parahaemolyticus, Propionibacterium acnes, Clostridium faecalis, Clostridium difficile, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella, Klebsiella pneumoniae, Koinebacterium, Streptococcus hemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma, Candida, and Aspergillus.

[0104] In the case of mRNA, the cancer antigens it encodes include, but are not limited to, ERK1, ERK2, MART-1 / Melan-A, gp100, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), P SA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-X p3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, GAGE-1, GAGE-2, GAGE -3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8 and GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, Tyrocina p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100, Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside The antibody may be selected from the group consisting of GD2 ganglioside, human papillomavirus proteins, the Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, c-erbB-2, and partial peptides thereof.

[0105] Target cancers are not particularly limited, and examples thereof include leukemia (including chronic lymphocytic leukemia and acute lymphocytic leukemia), lymphoma (including non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoma, B-cell lymphoma, Burkitt's lymphoma, malignant lymphoma, diffuse lymphoma, and follicular lymphoma), myeloma (including multiple myeloma), breast cancer, colon cancer, kidney cancer, stomach cancer, ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer, and esophageal cancer. , liver cancer, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, thecoma, male germinoma, endometrial hyperplasia, endometriosis, embryonal tumor, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, osteoblastoma, leiomyosarcoma, thyroid sarcoma, and Wilms' tumor.

[0106] The adjuvant is not particularly limited as long as it exhibits an adjuvant function that supports or enhances the original function of the active ingredient by being used in combination with the active ingredient of the main agent, but specifically, it can be selected from nucleic acids, lipopolysaccharides, lipopeptides, synthetic low molecular weight compounds, etc., and preferably CpG oligo DNA, poly IC RNA, imidazoquinoline (e.g., R848, imiquimod, etc.), STING agonists (e.g., cyclic di-GMP, etc.), monophosphoryl lipids, lipopeptides, etc. are used. In addition to the above, examples of adjuvants include taxane drugs, anthracycline drugs, JAK / STAT inhibitors, indole deoxygenase (IDO) inhibitors, tryptophan deoxygenase (TDO) inhibitors, etc. These inhibitors include compounds that have an antagonistic effect against the factor, as well as neutralizing antibodies, siRNA, and antisense DNA of the factor.

[0107] Anticancer agents include, for example, alkylating agents, metabolic antagonists, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum agents, molecular targeted drugs, hormone agents, and biological agents.

[0108] The drug may be a single type or a combination of two or more types.

[0109] In the lipid particles of the present invention, the mass ratio of the drug to CHP, a lipid particle component (CHP / mRNA mass), is not more than 200, more preferably not more than 100, and even more preferably not more than 90. The upper limit of this mass ratio can be, for example, 70, 50, 40, 30, 20, 10, or 8, and the lower limit of this mass ratio can be, for example, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1. The above upper and lower limits can be combined in any manner.

[0110] In one embodiment of the present invention, a liquid containing the lipid particles of the present invention can contain a drug (preferably a nucleic acid, more preferably an mRNA) at a concentration of, for example, 1 to 1000 μg / mL, preferably 5 to 500 μg / mL, more preferably 15 to 400 μg / mL, even more preferably 30 to 300 μg / mL, and even more preferably 50 to 200 μg / mL.

[0111] The lipid particles of the present invention may contain other components (other than the solvent) in addition to the above. Examples of other components include various components known to be incorporated into lipid particles, specifically, for example, antioxidants, membrane proteins, etc.

[0112] The antioxidant can be contained to prevent oxidation of the membrane, and is used as a component of the membrane as necessary.The antioxidant used as a component of the membrane can be exemplified by butylated hydroxytoluene, propyl gallate, tocopherol, tocopherol acetate, concentrated mixed tocopherol, vitamin E, ascorbic acid, L-ascorbic acid stearate, ascorbic palmitate, sodium hydrogen sulfite, sodium sulfite, sodium edetate, erythorbic acid, citric acid, etc.

[0113] Membrane proteins can be contained for the purpose of adding functions to the membrane or stabilizing the membrane structure, and are used as membrane components as necessary. Examples of membrane proteins include surface membrane proteins, integral membrane proteins, albumin, recombinant albumin, etc.

[0114] The content of other components is, for example, 10% by mass or less, preferably 5% by mass or less, more preferably 2% by mass or less, and even more preferably 1% by mass or less, relative to 100% by mass of the lipid particles of the present invention.

[0115] The mass median particle diameter of the lipid particles of the present invention is, for example, 200 nm or less, preferably 10 to 200 nm, more preferably 20 to 200 nm, even more preferably 40 to 190 nm, still more preferably 40 to 180 nm, and particularly preferably 40 to 160 nm. The particle diameter can be measured by a dynamic light scattering method.

[0116] The lipid particles of the present invention are usually formed in an aqueous solution, such as various buffer solutions (e.g., acetate buffer, phosphate buffer, formate buffer, histidine buffer, etc.).

[0117] The lipid particles of the present invention can be preferably produced by a method including a step of mixing an alcohol solution containing lipids constituting the lipid particles with an aqueous solution containing modified polysaccharides (preferably further containing a drug), or a step (step 1) of mixing an alcohol solution containing lipids constituting the lipid particles with an aqueous solution (preferably further containing a drug) and an aqueous solution containing modified polysaccharides. By employing this method, the lipid particles of the present invention can be obtained simply and efficiently without relying on PEG-modified lipids or avoiding side reactions caused by PEG.

[0118] The alcohol that is the solvent of the alcohol solution is not particularly limited as long as it is an alcohol that can dissolve lipids. A preferred example of the alcohol is ethanol.

[0119] The alcohol solution preferably contains the lipids constituting the lipid particles in the molar ratio described above, thereby making it possible to obtain lipid particles containing the lipids constituting the lipid particles in the molar ratio described above.

[0120] The lipid concentration in the alcohol solution is, for example, 2 to 20 mM, and preferably 4 to 12M.

[0121] An aqueous solution is a solution in which the solvent contains water (preferably the water content is 80% or more, 90% or more, 95% or more, or 100%).

[0122] The concentration of the modified polysaccharide in the aqueous solution is, for example, 0.2 to 5 mg / mL, preferably 0.4 to 3 mg / mL, and more preferably 0.6 to 1.5 mg / mL.

[0123] The mixing ratio of the aqueous solution to the alcohol solution (aqueous solution / alcohol solution, v / v) is, for example, 1-20, and preferably 2-4.

[0124] The mixing mode is not particularly limited as long as it allows the formation of lipid particles, but is usually performed by vigorously stirring with a vortex mixer, etc. Alternatively, when the reaction is performed in a reaction system using a microchannel, the mixture is mixed within the reaction system.

[0125] Step 1 is usually carried out at room temperature or with heating.

[0126] The lipid particles of the present invention are useful in that they can be produced without relying on PEG-modified lipids, and / or have superior efficacy, safety, or stability to lipid particles that rely on PEG-modified lipids, or have different immune dynamics from lipid particles that rely on PEG-modified lipids.

[0127] In another embodiment of the lipid particles of the present invention, a conjugated lipid that inhibits particle aggregation, such as a PEG-modified lipid, may be further included, and is useful in that it has superior efficacy, safety, or stability to lipid particles based on PEG-modified lipids, or has different immune dynamics from lipid particles based on PEG-modified lipids.

[0128] The safety can be, for example, reduced anti-PEG antibody induction, reduced inflammation at the administration site, reduced fever after administration, and the like.

[0129] The stability is the stability as a formulation, and for example, the increase in particle size due to freezing and thawing is suppressed. Specifically, lipid particles containing conventional PEG-modified lipids, such as COVID-19 vaccines, cannot withstand freezing and thawing, and the particle size increases with repeated freezing and thawing regardless of the presence or absence of an isotonic agent (Sucrose), but the lipid particles of the present invention can improve the stability of the lipid particle formulation by containing modified polysaccharides such as CHP, and the lipid particles of the present invention that do not contain PEG-modified lipids produced by the production method of Example 4 (Method 2) described below have further improved stability against freezing and thawing, and can suppress the increase in particle size even without Sucrose.

[0130] The immune dynamics can be, for example, differences in the quality of induced cellular immunity.

[0131] 2.Applications The lipid particles of the present invention can encapsulate a drug and selectively accumulate in DC-SIGN (CD209)-expressing cells (e.g., M2-like macrophages and antigen-presenting cells). For this reason, the lipid particles of the present invention can be used, for example, as medicines, reagents, etc. (sometimes referred to as "drugs of the present invention" in the present specification), more specifically, as therapeutic drugs for cancer, immune diseases, diabetes-induced diseases, central nervous system diseases, etc., as well as infectious disease vaccines or cancer vaccines. Infectious disease vaccines are not particularly limited as long as they are vaccines used against infectious diseases, and include not only preventive vaccines but also therapeutic vaccines.

[0132] The drug of the present invention is not particularly limited as long as it contains the lipid particles of the present invention, and may further contain additives as necessary. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0133] The mode of use of the drug of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type of drug. The drug of the present invention can be used, for example, in vitro (e.g., added to a culture medium for cultured cells) or in vivo (e.g., administered to an animal) depending on the purpose.

[0134] The subject to which the agent of the present invention is applied is not particularly limited, and examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. Examples of cells include animal cells. The types of cells are also not particularly limited, and examples of the cells include blood cells, hematopoietic stem cells and progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0135] The drug of the present invention may take any dosage form, for example, oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies, or parenteral preparations such as injectable preparations (e.g., drip injections (e.g., intravenous drip preparations, etc.), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, and lotions), suppositories, inhalants, eye drops, eye ointments, nasal drops, ear drops, and liposomes.

[0136] The route of administration of the drug of the present invention is not particularly limited as long as the desired effect can be obtained, and examples of such administration include oral administration; enteral administration such as tube feeding and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, nasal administration, intratracheal administration, and eye drop administration.

[0137] The content of the lipid particles of the present invention in the drug of the present invention depends on the mode of use, the subject of application, the condition of the subject of application, etc., and is not limited, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0138] The dosage of the agent of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exerts a pharmacological effect, and is usually 0.1 to 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day in the case of oral administration, and 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight per day in the case of parenteral administration. The dosage can be appropriately increased or decreased depending on age, pathological condition, symptoms, etc. EXAMPLES

[0139] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0140] Preparation Example 1. Preparation of pullulan nanogel According to the previous report (Macromolecules 1993, 23, 3062-3068), cholesterol-modified pullulan (CHP) was prepared by introducing 1.2 cholesterol molecules per 100 monosaccharides into pullulan with a weight-average molecular weight of 100,000. A pullulan nanogel solution was prepared using the prepared CHP by a known method.

[0141] Reference Example 1. Preparation of LNP 1 (mRNA: ovalbumin (OVA) mRNA) An aqueous solution of OVA mRNA (mRNA concentration: 70 μg / mL, solvent: 25 mM NaAc, 154 mM NaCl, pH = 4.5) was prepared (Solution A). An ethanol solution of lipids (lipid concentration: 8 mM) mixed in the ratio (molar ratio) of ALC-0315 ([(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate)):DSPC (Distearoylphosphatidylcholine):DMG-PEG200 (1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol):Cholesterol = 50:10:1.5:38.5 was prepared (Solution B). The prepared solutions were mixed using an iLiNP microchannel chip (Lilac Pharma) or NanoAssemblr (Precision NanoSystems) at a flow rate of 500 μL / min (iLiNP microchannel chip) or 12 mL / min (NanoAssemblr) (Flow Rate Ratio; A:B=3). The mixed solution was solvent-substituted with PBS using a dialysis membrane (MWCO; 3.5K). The collected solution was concentrated 5 times using an Amicon tube (MWCO; 30K) and passed through a 0.45 μm filter to obtain an LNP (OVA) solution (mass average particle size: 81 nm, ELSZ-2000ZS Otsuka Electronics Co., Ltd.). Ribo Green Assay (Invitrogen) was performed, and the mRNA concentration was calculated to be 185 μg / mL.

[0142] Reference Example 2. Preparation of LNP 2 (mRNA: EGFP mRNA) An LNP(EGFP) solution was obtained in the same manner as in Reference Example 1, except that mRNA for EGFP, a fluorescent protein, was used instead of OVA mRNA (mass average particle size: 75 nm, mRNA concentration: 186 μg / mL).

[0143] Reference Example 3. Preparation of LNP 3 (mRNA: HPV E7 mRNA) An LNP (HPV E7) solution is obtained in the same manner as in Reference Example 1, except that mRNA of the E7 protein of human papillomavirus is used instead of OVA mRNA.

[0144] Example 1. Preparation of CHP:LNP 1 (mRNA: OVA mRNA) To 1 mL of the LNP(OVA) solution (Reference Example 1), the same amount of 10 mg / mL CHP aqueous solution was added and stirred for 30 minutes. The mixture was passed through a 0.45 μm filter to obtain a CHP:LNP(OVA) solution (mass average particle size: 122 nm, mRNA concentration: 93 μg / mL) (Method 1).

[0145] Example 2. Preparation of CHP:LNP 2 (mRNA: EGFP mRNA) A CHP:LNP(EGFP) solution was obtained in the same manner as in Example 1, except that the LNP(EGFP) solution (Reference Example 2) was used instead of the LNP(OVA) solution (Reference Example 1) (mass average particle size: 117 nm, mRNA concentration: 95 μg / mL) (Method 1).

[0146] Example 3. Preparation of CHP:LNP 3 (mRNA: HPV E7 mRNA) A CHP:LNP(HPV E7) solution is obtained in the same manner as in Example 1, except that the LNP(HPV E7) solution (Reference Example 3) is used instead of the LNP(OVA) solution (Reference Example 1) (Method 1).

[0147] Example 4. Preparation of CHP:LNP 4 (mRNA: OVA mRNA) An aqueous solution of OVA mRNA (mRNA concentration: 70 μg / mL, solvent: 1 mg / mL CHP in 25 mM NaAc, 154 mM NaCl, pH = 4.0) was prepared (Solution A). An ethanol solution of lipids mixed at a ratio (molar ratio) of MC3:DSPC:Cholesterol = 50:10:40 (lipid concentration: 8 mM) was prepared (Solution B). Each of the prepared solutions was mixed using an iLiNP microchannel chip or NanoAssemblr at a flow rate of 500 μL / min (iLiNP microchannel chip) or 12 mL / min (NanoAssemblr) (Flow Rate Ratio; A:B = 3). The mixed solution was solvent-substituted with PBS using a dialysis membrane (MWCO; 3.5K). The collected solution was concentrated 5-fold using an Amicon tube (MWCO; 30K) and passed through a 0.45 μm filter to obtain a CHP:LNP(OVA) solution (mass average particle size: 155 nm, mRNA concentration: 70 μg / mL) (Method 2).

[0148] Example 5. Preparation of CHP:LNP 5 (mRNA: EGFP mRNA) An EGFP mRNA aqueous solution (mRNA concentration: 70 μg / mL, solvent: 2 mg / mL CHP in 25 mM NaAc, 154 mM NaCl, pH = 4.0) was prepared (Solution A). An ethanol solution (lipid concentration: 8 mM) of lipids mixed at a ratio (molar ratio) of ALC-0315:DSPC:Cholesterol = 45.5:9:45.5 was prepared (Solution B). Each of the prepared solutions was mixed using an iLiNP microchannel chip or NanoAssemblr at a flow rate of 500 μL / min (iLiNP microchannel chip) or 12 mL / min (NanoAssemblr) (Flow Rate Ratio; A:B = 3). The mixed solution was solvent-substituted with PBS using a dialysis membrane (MWCO; 3.5K). The collected solution was concentrated 5-fold using an Amicon tube (MWCO; 30K) and passed through a 0.45 μm filter to obtain a CHP:LNP(EGFP) solution (weight average particle size: 135 nm, mRNA concentration: 82 μg / mL) (Method 2).

[0149] Example 6. Preparation of CHP:LNP 6 (mRNA: HPV E7 mRNA) A CHP:LNP(HPV E7) solution is obtained in the same manner as in Example 4, except that HPV E7 mRNA is used instead of OVA mRNA (Method 2).

[0150] Examples 7 to 23. CHP: Preparation of LNPs 7 CHP:LNP was prepared using the methods (Method 1, Method 2), nucleic acid, ionized lipid, and lipid ratio shown in Table 1. The preparation compositions and physical properties of the CHP:LNP prepared in Examples 1 to 23 are shown in the table below.

[0151] [Table 1]

[0152] Example 58. Preparation of CHP:LNP 8 (mRNA: mCherry mRNA) An mCherry mRNA aqueous solution (mRNA concentration: 70 μg / mL, solvent 25 mM NaAc, 154 mM NaCl, pH = 4.0) was prepared (Solution A). An ethanol solution (lipid concentration: 8 mM) of lipids mixed at a ratio (molar ratio) of SM-102: DSPC: Cholesterol = 45.5: 9: 45.5 was prepared (Solution B). Furthermore, a pullulan nanogel solution (CHP concentration: 2 mg / mL, solvent PBS) was prepared by the method shown in Preparation Example 1 (Solution C). Each of the prepared solutions was mixed at a flow rate of 12 mL / min (Flow Rate Ratio; A: B = 1: 3: 2.46) using NanoAssemblr (Precision NanoSystems). The mixed solution was solvent-substituted with PBS using a dialysis membrane (MWCO; 10K). The collected solution was concentrated 7-fold using an Amicon tube (MWCO; 100K) and passed through a 0.45 μm filter to obtain a CHP:LNP(mCherry) solution (weight average particle size: 131 nm, mRNA concentration: 135 μg / mL) (Method 3).

[0153] Examples 24 to 57 and 59 to 62. Preparation of CHP:LNP 9 CHP:LNP was prepared using the preparation methods (Method 1, Method 2, Method 3), nucleic acid, ionized lipid, phospholipid, PEG-modified lipid, lipid ratio, and CHP concentration shown in Tables 2 and 3. The preparation compositions and physical properties of CHP:LNP prepared in Examples 24 to 62 are shown in the following table together with Examples 1 to 23.

[0154] [Table 2]

[0155] [Table 3] Furthermore, when PEG-modified lipids are not added or only a small amount is added, LNPs tend to precipitate and cannot be produced, or the particles tend to be very large, with a particle size of 200 nm or more, making it impossible to produce LNPs suitable for administration to the body. However, unexpectedly, by adding CHP, LNPs of ideal particle sizes could be produced in all of Examples 1 to 62 of the present invention.

[0156] Test example 1. Antigenicity evaluation test by immunization of mice CHP:LNP(OVA) (Example 1, Example 4) or LNP(OVA) (Reference Example 1) was administered subcutaneously at 10 μg mRNA amount to C57BL / 6 mice (Japan SLC). In addition, the same dose was administered intramuscularly to other mice for comparison. After 3 weeks, the same dose and administration method were administered again, and after another week, blood was collected and spleen cells and lymph nodes were collected.

[0157] Spleen cells are promptly harvested and stimulated with OVA CD4 epitope peptide (ROCOLAND, Ovalbumin 323-339-OH) and OVA CD8 epitope peptide (ROCOLAND, OVA 257-264). Secreted IFN-γ is measured using ELISA, and intracellular IFN-γ, IL-2, and TNFα are measured using intracellular cytokine staining. In addition, expression analysis of multiple cell surface markers (CD3, CD4, CD8, CD127, CD62L, CD44, KLRG1, Tim-3, Lag-3, PD-1) will be performed on cells derived from the regional lymph nodes and collected spleen cells to analyze the status of T cells.

[0158] The frequency of antigen-presenting cells and the amount of presented OVA antigen are measured using an antibody (Biolegend) that recognizes the OVA antigen and MHC complex. At this time, antigen-presenting cells are identified by co-staining with cell surface markers such as CD11b, CD11c, F4 / 80, B220, and CD8a. In addition, an antibody against SIGN-R1 is used to evaluate whether uptake into antigen-presenting cells is receptor-dependent.

[0159] OVA-specific antibodies in serum will be measured by ELISA.

[0160] Test Example 2. Comparative toxicity test of CHP:LNP(OVA) and LNP(OVA) (1) Cytokine release syndrome CHP:LNP(OVA) (Examples 1 and 4) or LNP(OVA) (Reference Example 1) was subcutaneously administered at 10 μg per mRNA dose to C57BL / 6 mice. Blood samples were collected over time, and the blood concentrations of IL-6, IL-1β, etc. were measured by ELISA or a multiplex bead-based assay panel (BioLegend).

[0161] (2) Local skin inflammation at the injection site CHP:LNP(OVA) (Example 1, Example 4) or LNP(OVA) (Reference Example 1) is subcutaneously administered at 10 μg of mRNA to C57BL / 6 mice. After 24 hours, the skin at the administration site is excised. After observing the flushing of the skin, the number of infiltrating lymphocytes is measured.

[0162] (3) Weight Fluctuation CHP:LNP(OVA) (Example 1, Example 4) or LNP(OVA) (Reference Example 1) was subcutaneously administered at 10 μg as mRNA amount to C57BL / 6 mice, after which the change in body weight was measured over time.

[0163] (4) Body temperature fluctuations CHP:LNP(OVA) (Example 1, Example 4) or LNP(OVA) (Reference Example 1) was subcutaneously administered at 10 μg as mRNA amount to C57BL / 6 mice, after which rectal body temperature was measured over time.

[0164] Test example 3. Vaccine evaluation test by immunization of mice C57BL / 6 mice were immunized subcutaneously with CHP:LNP(HPV E7) (Example 6) or LNP(HPV E7) (Reference Example 3), each at 10 μg in terms of mRNA. Two or three weeks later, a second immunization was performed with the same dose, and on the same day, 1x10 5 A mouse lung cancer cell line (TC-1 cells) stably expressing HPV E7 protein is subcutaneously implanted. Then, the size of the tumor is measured over time.

[0165] Test Example 4. Immunogenicity test CHP:LNP(OVA) (Example 1, Example 4) or LNP(OVA) (Reference Example 1) is subcutaneously administered at 10 μg as mRNA to C57BL / 6 mice. The same dose is then subcutaneously administered multiple times (2 to 5 times) at 2- or 3-week intervals. The amount of anti-PEG antibody and anti-CHP antibody in the blood is then measured by ELISA.

[0166] Test Example 5. Functional evaluation test using human peripheral blood lymphocyte-derived macrophages CD14 positive monocytes were isolated from human peripheral blood and cultured at 2 x 10 5The cells were seeded at 100 cells / well in 500 μL of basal medium (RPMI, 10% fetal bovine serum, 50 ng / mL M-CSF) and cultured for 3 days in an incubator at 37°C with 5% CO2. The medium was then replaced with 500 μL of basal medium and cultured for another 3 days. For differentiation induction into M0 macrophages, the medium was replaced with 500 μL of basal medium, while for differentiation induction into M2-like macrophages, the medium was replaced with 500 μL of differentiation induction medium supplemented with 50 ng / mL IL-4, 50 ng / mL IL-6, 50 ng / mL IL-10, and 50 ng / mL IL-13, and cultured for two days. CHP:LNP(EGFP) (Example 2, Example 5) or LNP(EGFP) (Reference Example 2) containing 0.5 μg of mRNA was added to the basal medium, and after 2 hours of culture at 4° C., the medium was replaced with 500 μL and incubated overnight at 37° C. and 5% CO2. After washing with PBS, 200 μL of Cell Dissociation solution (Biological Industries) was added, and the cells were incubated at 37° C. for 20 minutes, 200 μL of Wash solution (Miltenyi) was added, the cells were detached by pipetting, and the cells were collected by centrifugation at 500 g for 5 minutes. Then, the cells were stained with PB450-labeled anti-CD206 antibody (BD Biosciences) and APC-labeled anti-DC-SIGN antibody (R&D Systems) and analyzed by a flow cytometer.

[0167] <Test Results> The results of incubation at 37°C overnight after addition of CHP:LNP(EGFP) (Example 2) or LNP(EGFP) without treatment at 4°C are shown in Figure 1. The results of incubation at 37°C overnight after addition of CHP:LNP(EGFP) (Example 2) or LNP(EGFP), treatment at 4°C for 2 hours, replacement of the medium, and incubation at 37°C overnight are shown in Figure 2. In another experiment, the results of incubation at 37°C overnight after addition of CHP:LNP(EGFP) (Example 2), CHP:LNP(EGFP) without PEG (Example 5), or LNP(EGFP) are shown in Figure 3.

[0168] Test Example 6: Experiment to confirm expression in antigen-presenting cells after subcutaneous administration to mice CHP:LNP(EGFP) (Example 5) or LNP(EGFP) (Reference Example 2) was subcutaneously administered at 10 μg each in terms of mRNA amount to C57BL / 6 mice. After 24 hours, the inguinal lymph nodes were excised, and the cells were collected and analyzed using a flow cytometer.

[0169] Test Example 7. Measurement of particle size before and after freezing and thawing CHP:LNP (Examples 19, 20, 23, 26-54, 57-62) was frozen once at -30°C and then thawed, and the particle size was measured before and after freezing and thawing. Similarly, LNP with the same composition but not containing CHP (Reference Examples 19, 26, 32, 36, 39, 42, 44-50, 54, 60) was frozen once at -30°C and then thawed, and the particle size was measured before and after freezing and thawing.

[0170] <Test Results> The results are shown in Table 4 below. The particle size of the comparative LNP that does not contain CHP increases after just one freeze-thaw cycle, but unexpectedly, the increase in particle size of CHP:LNP was suppressed before and after freeze-thawing. From the above, it can be said that CHP has the effect of improving the stability of LNP.

[0171] [Table 4]

[0172] Test Example 8. Functional evaluation test using human peripheral blood lymphocyte-derived macrophages-2 M0 macrophages and M2-like macrophages were prepared in the same manner as in Test Example 5, and CHP:LNP(mCherry) (Examples 51, 58, and 61) or LNP(mCherry) (Reference Example 50) containing 0.5 μg of mRNA was added to the basal medium and incubated overnight at 37° C. and 5% CO2. After washing with PBS, 200 μL of Cell Dissociation solution (Biological Industries) was added, and the mixture was incubated at 37° C. for 20 minutes, 200 μL of Wash solution (Miltenyi) was added, the cells were detached by pipetting, and the cells were collected by centrifugation at 500 g for 5 minutes. After that, the mixture was stained with PB450-labeled anti-CD206 antibody (BD Biosciences) and APC-labeled anti-DC-SIGN antibody (R&D Systems), and analyzed by a flow cytometer.

[0173] <Test Results> The results are shown in Figure 4. Only in CHP-added LNPs (CHP:LNPs), expression was higher in DC-SIGN-positive M2-like macrophages than in DC-SIGN-negative M0 macrophages. From the above, it was found that CHP-added LNPs can selectively deliver mRNA to DC-SIGN-positive cells without relying on ionized lipids, and have the ability to express proteins from the loaded mRNA.

[0174] Test Example 9. Immunogenicity test-2 C57BL / 6 mice were subcutaneously administered 10 μg of CHP:LNP(mCherry) (Example 40), CHP:LNP(mCherry) without PEG-modified lipid (Example 57), or LNP(mCherry) (Reference Example 39) in terms of mRNA. Each test group consisted of n=5. After another 3 weeks, the same dose was subcutaneously administered again. One week later, the amount of anti-PEG antibodies in the blood was measured by ELISA (Life Diagnostics). As a negative control, only the solvent (PBS) was administered.

[0175] <Test Results> The results are shown in Figure 5. Compared to the negative control, LNP with CHP (CHP:LNP(mCherry)), and LNP with CHP without PEG-modified lipids (CHP:LNP(mCherry)PEG Free), the LNP(mCherry) group showed significantly higher levels of anti-PEG antibodies (IgM) (*; p < 0.05, unpaired t-test). These results suggest that CHP-modified LNP, even though it contains PEG-modified lipids, may be able to avoid side effects caused by anti-PEG antibodies, such as anaphylaxis and the accelerated blood clearance (ABC) phenomenon, in which the efficacy of the drug is weakened by repeated administration.

[0176] Test Example 10. Experiment to confirm expression in nearby lymph nodes after subcutaneous administration to mice - 1 C57BL / 6 mice were intradermally administered 5 μg of CHP:LNP(mCherry) (Example 40) or LNP(mCherry) (Reference Example 39) as mRNA around the right dorsal buttock. 17 hours after administration, the fluorescence intensity of mCherry protein was detected using an in vivo imaging device (Vilber Bio Imaging) and expression at the administration site and nearby lymph nodes (inguinal lymph nodes) was measured. In addition, fluorescent protein was detected at the same wavelength in the excised nearby lymph nodes (inguinal lymph nodes) using the same device.

[0177] <Test Results> The results of a representative example of the administration site are shown in Figure 6. Furthermore, the results of a representative example of an excised nearby lymph node (inguinal lymph node) are shown in Figure 7. At the administration site, expression of LNP(mCherry) was confirmed widely, but expression of CHP:LNP(mCherry) was low, while in the nearby lymph node, expression of CHP:LNP(mCherry) was higher than that of LNP(mCherry). Furthermore, in the excised nearby lymph node, higher expression was confirmed for CHP:LNP(mCherry) compared to LNP(mCherry). These results indicate that when administered intradermally, LNP with CHP has superior delivery ability to nearby lymph nodes compared to LNP.

[0178] Test Example 11. Evaluation of local skin inflammation at the administration site - 2 After shaving the hair of C57BL / 6 mice, 9.8 μg of CHP:LNP(EGFP) (Example 2) or LNP(EGFP) (Reference Example 2) was administered intradermally around the back and buttocks as mRNA. Three and five days after administration, skin tissues were collected and fixed with 4% paraformaldehyde for 48 hours, and then replaced with a 70% ethanol solution. Then, HE staining and immunostaining with anti-IBA1 antibody were performed, and pathological observation was performed.

[0179] <Test Results> The results are shown in Table 5. In the LNP(EGFP) administration group, the infiltration of inflammatory cells and the increase in IBA1-positive cells, which are indicators of activated macrophages, were higher than in the CHP:LNP(EGFP) administration group. These results indicate that inflammation induced by local administration of lipid nanoparticles is suppressed in CHP-added LNPs (CHP:LNPs) compared to LNPs.

[0180] [Table 5] Test Example 12. Evaluation of effects on body weight C57BL / 6 mice were subcutaneously administered 10 μg of CHP:LNP(OVA) (Example 1) or LNP(OVA) (Reference Example 1) as mRNA (day 0). Furthermore, 3 weeks after the first administration (day 21), the same dose was subcutaneously administered again. Body weight was measured on each administration day and for two consecutive days immediately thereafter, and further once a week from the first administration until day 28. Each test group consisted of n=3. The comparison group was not administered any drug solution.

[0181] <Test Results> The results are shown in Figure 8. The body weight of each group on the first day of administration (day 0) was converted to 100%, and the average value of each group was used to graph the results. The arrows in the figure indicate the days on which LNP was administered. As a result, weight loss was observed in the LNP-administered group the day after each administration, whereas the change was minor in the LNP-administered group to which CHP was added, and the weight transition was similar to that of the drug-unadministered group. From the above, it was shown that the side effect of weight loss caused by LNP can be reduced by adding CHP to LNP.

Claims

[Claim 1] The invention described in the specification.

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