WO2017222016A1 - カチオン性脂質 - Google Patents
カチオン性脂質 Download PDFInfo
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- WO2017222016A1 WO2017222016A1 PCT/JP2017/023051 JP2017023051W WO2017222016A1 WO 2017222016 A1 WO2017222016 A1 WO 2017222016A1 JP 2017023051 W JP2017023051 W JP 2017023051W WO 2017222016 A1 WO2017222016 A1 WO 2017222016A1
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- QMQNDJUUYONERT-UHFFFAOYSA-N CC(C)(C)[Si+](C)(C)OCCC[P+](c1ccccc1)(c1ccccc1)c1ccccc1 Chemical compound CC(C)(C)[Si+](C)(C)OCCC[P+](c1ccccc1)(c1ccccc1)c1ccccc1 QMQNDJUUYONERT-UHFFFAOYSA-N 0.000 description 2
- 0 CN(CC1)CCC1C(OCC(I)IC(O*)=O)=O Chemical compound CN(CC1)CCC1C(OCC(I)IC(O*)=O)=O 0.000 description 2
- GDKBUXZTLJKRBA-UHFFFAOYSA-N CC(C)(C)OC(C(CCCCCC(OCc1ccccc1)=O)C(OC(C)(C)C)=O)=O Chemical compound CC(C)(C)OC(C(CCCCCC(OCc1ccccc1)=O)C(OC(C)(C)C)=O)=O GDKBUXZTLJKRBA-UHFFFAOYSA-N 0.000 description 1
- OALQJUSHPMEIOP-UHFFFAOYSA-N CC(C)N(C)CCCC=O Chemical compound CC(C)N(C)CCCC=O OALQJUSHPMEIOP-UHFFFAOYSA-N 0.000 description 1
- NQNZEMUGVZVBJU-DCWQJPKNSA-N CCCCCCCCCC(CCCCC[IH]CCC)C[C@@H](C)COC(CCCNC)=O Chemical compound CCCCCCCCCC(CCCCC[IH]CCC)C[C@@H](C)COC(CCCNC)=O NQNZEMUGVZVBJU-DCWQJPKNSA-N 0.000 description 1
- GHKFGCWZAJTPAD-UHFFFAOYSA-N CCCCCCCCCCC(CCCCCCCCC(O)=O)COC(C1CCN(C)CC1)=O Chemical compound CCCCCCCCCCC(CCCCCCCCC(O)=O)COC(C1CCN(C)CC1)=O GHKFGCWZAJTPAD-UHFFFAOYSA-N 0.000 description 1
- DJGRHLPMTAIACU-UHFFFAOYSA-N CCCCCCCCCCC(CCCCCCCCC(OC)=O)COC(C1CCN(C)CC1)=O Chemical compound CCCCCCCCCCC(CCCCCCCCC(OC)=O)COC(C1CCN(C)CC1)=O DJGRHLPMTAIACU-UHFFFAOYSA-N 0.000 description 1
- CRQXYWRYKDAOKA-UHFFFAOYSA-N O=C(CCCCCBr)OCc1ccccc1 Chemical compound O=C(CCCCCBr)OCc1ccccc1 CRQXYWRYKDAOKA-UHFFFAOYSA-N 0.000 description 1
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- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/52—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring condensed with a ring other than six-membered
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- C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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- C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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- C07D241/04—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
Definitions
- the present invention relates to a novel cationic lipid.
- Nucleic acids such as siRNA (small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA, or small hairpin RNA) expression vectors, antisense oligonucleotides, and the like induce sequence-specific gene expression suppression in vivo. It is a nucleic acid and is known as a nucleic acid medicine.
- siRNA is particularly attracting attention.
- siRNA is a double-stranded RNA consisting of 19 to 23 base pairs, and induces sequence-specific gene expression suppression called RNA interference (RNAi).
- RNAi RNA interference
- siRNA is chemically stable, it has problems for therapeutic use in that it is easily degraded by RNase (ribonuclease) in plasma and it is difficult to permeate the cell membrane alone.
- RNase ribonuclease
- Patent Documents 2 to 5 describe cationic lipids that are used for delivery of nucleic acid drugs such as siRNA and have improved biodegradability.
- fine particles containing cationic lipids have a problem of stability that they tend to aggregate during storage, and a method of suppressing aggregation by containing polyethylene glycol-modified lipid (PEG lipid) in the fine particles is known. ing. Further, there is a method for improving aggregation suppression and nucleic acid delivery efficiency by using PEG-DPG, which is a specific PEG lipid, as a constituent of microparticles or a preparation comprising the microparticles and a deionized solvent. Patent Document 6 describes this.
- the present invention relates to the following [1] to [15].
- a compound represented by the following formula (1a) or a pharmaceutically acceptable salt thereof [Wherein, L 1 and L 2 each independently represent an alkylene group having 3 to 10 carbon atoms, and R 1 and R 2 each independently represent an alkyl group having 4 to 22 carbon atoms or an alkenyl group having 4 to 22 carbon atoms. , X 1 represents a single bond or —CO—O—, and ring P represents any of the following formulas (P-1) to (P-5). ] [Wherein R 3 represents an alkyl group having 1 to 3 carbon atoms. ] [2] The compound according to [1] or a pharmaceutically acceptable salt thereof represented by the following formula (1).
- L 1 and L 2 each independently represent an alkylene group having 3 to 10 carbon atoms
- R 1 and R 2 each independently represent an alkyl group having 4 to 22 carbon atoms or an alkenyl group having 4 to 22 carbon atoms.
- X 1 represents a single bond or —CO—O—.
- [3a] The above formulas (A1), (A2), (A3), (A4), (A5), (A9), (A12), (A15), (A16), (A17), (A19), ( The compound according to [3] or a pharmaceutically acceptable salt thereof selected from the group consisting of compounds represented by A20) and (A22).
- [3b] Selected from the group consisting of compounds represented by the above formulas (A1), (A2), (A3), (A4), (A5), (A9), (A12), (A15), and (A20) Or a pharmaceutically acceptable salt thereof.
- [3c] The compound according to [3] or a pharmaceutically acceptable salt thereof selected from the group consisting of compounds represented by the above formulas (A1) to (A5).
- [3d] The compound according to [3] or a pharmaceutically acceptable salt thereof selected from the group consisting of compounds represented by the above formulas (A6) to (A8).
- [14] The group consisting of (I) the compound according to any one of [1] to [12] or a pharmaceutically acceptable salt thereof, and (II) a neutral lipid, a polyethylene glycol-modified lipid, and a sterol.
- [15] The group consisting of the compound according to any one of [I] [1] to [12] or a pharmaceutically acceptable salt thereof, and (II) a neutral lipid, a polyethylene glycol-modified lipid, and a sterol.
- the cationic lipid of the present invention has one or more of the following effects. (1) According to the cationic lipid of the present invention, nucleic acid can be efficiently released into the cytoplasm. (2) The cationic lipid of the present invention makes it possible to suppress an increase in the particle size of the lipid complex when stored for a certain period. Therefore, the cationic lipid of the present invention has applicability as a lipid for nucleic acid delivery to the cytoplasm.
- 6 is a graph showing the results of Test Example 1. 6 is a graph showing the results of Test Example 2.
- the present invention is a compound represented by the following formula (1a) or a pharmaceutically acceptable salt thereof, and can be used as a cationic lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- the ring P represents any one of the following formulas (P-1) to (P-5).
- R 3 represents an alkyl group having 1 to 3 carbon atoms.
- the ring P represents any one of the following formulas (P-1), (P-2), (P-4), and (P-5). In one embodiment, ring P represents the following formula (P-1).
- the present invention is a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, and can be used as a cationic lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- L 1 and L 2 are each independently an alkylene group having 3 to 10 carbon atoms
- R 1 and R 2 are each independently an alkyl group having 4 to 22 carbon atoms or 4 carbon atoms Represents an alkenyl group of ⁇ 22
- X 1 represents a single bond or —CO—O—.
- alkyl refers to a linear, cyclic or branched saturated aliphatic hydrocarbon group having the specified number of carbon atoms.
- alkenyl refers to a straight or branched hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond. Examples include, but are not limited to, monoene, diene, triene, and tetraene.
- alkylene means a linear, cyclic or branched divalent saturated aliphatic hydrocarbon group having a specified number of carbon atoms.
- halogen means F, Cl, Br, or I.
- L 1 and L 2 are each independently 3 to 10 carbon atoms (for example, 5 to 10 carbon atoms or 3 to 8 carbon atoms).
- An alkylene group, R 1 and R 2 are each independently an alkyl group having 4 to 18 carbon atoms or an alkenyl group having 4 to 18 carbon atoms,
- X 1 is a compound represented by a single bond or —CO—O—, or a pharmaceutical thereof It is an acceptable salt and can be used as a cationic lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- L 1 and L 2 are each independently 3 to 10 carbon atoms (for example, 5 to 10 carbon atoms or 3 to 8 carbon atoms).
- a linear alkylene group, R 1 and R 2 are each independently a linear or branched alkyl group having 4 to 18 carbon atoms or a linear alkenyl group having 4 to 18 carbon atoms; 1 is a compound which is —CO—O— or a pharmaceutically acceptable salt thereof, and can be used as a cationic lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- one embodiment of the present invention is a compound represented by the following formula (1b) or a pharmaceutically acceptable salt thereof.
- R 1 and R 2 are each independently an alkyl group having 4 to 22 carbon atoms or an alkenyl group having 4 to 22 carbon atoms, preferably a linear or branched alkyl group having 4 to 18 carbon atoms. Or a linear alkenyl group having 4 to 18 carbon atoms, and n1 and n2 each independently represents an integer of 3 to 10 (for example, 5 to 10 or 3 to 8).
- the present invention provides the compound of the above formula (1a) or formula (1), wherein X 1 is —CO—O—, L 1 is the same as L 2 , and R 1 is R 2 Or a pharmaceutically acceptable salt thereof, and can be used as a cationic lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- the present invention relates to the above formula (1a) or formula (1), wherein L 1 and L 2 are each independently a linear alkylene group having 5 to 10 carbon atoms, and R 1 Is a linear or branched alkyl group having 4 to 18 carbon atoms or a linear alkenyl group having 4 to 18 carbon atoms, R 2 is a linear alkyl group having 4 to 18 carbon atoms, X 1 is a compound which is a single bond or a pharmaceutically acceptable salt thereof.
- the total number of carbon atoms of L 2 and R 2 is preferably 9-12.
- the compound of this embodiment can be used as a cationic lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt. Therefore, one embodiment of the present invention is a compound represented by the following formula (1c) or a pharmaceutically acceptable salt thereof.
- R 1 represents an alkyl group having 4 to 22 carbon atoms or an alkenyl group having 4 to 22 carbon atoms, preferably a linear or branched alkyl group having 4 to 18 carbon atoms, or 4 to 18 carbon atoms.
- N1 represents an integer of 3 to 10 (for example, 5 to 10 or 3 to 8), and n2 represents an integer of 8 to 25, preferably 8 to 11.
- One embodiment of the present invention is a compound represented by any one of the above formulas (A1) to (A22) or a pharmaceutically acceptable salt thereof, and can be used as a cationic lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- One embodiment of the present invention is a compound represented by any one of the above formulas (A1) to (A5) and (A9) to (A22) or a pharmaceutically acceptable salt thereof, which is used as a cationic lipid. be able to.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- One embodiment of the present invention includes the above formulas (A1), (A2), (A3), (A4), (A5), (A9), (A12), (A15), (A16), (A17), The compound represented by any one of (A19), (A20), and (A22) or a pharmaceutically acceptable salt thereof, and can be used as a cationic lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- One embodiment of the present invention is represented by any one of the above formulas (A1), (A2), (A3), (A4), (A5), (A9), (A12), (A15), and (A20).
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- One embodiment of the present invention is a compound represented by any one of the above formulas (A1), (A2), (A3), (A4), and (A5) or a pharmaceutically acceptable salt thereof, and a cation It can be used as a sex lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- One embodiment of the present invention is a compound represented by any one of the above formulas (A6), (A7), and (A8) or a pharmaceutically acceptable salt thereof, and can be used as a cationic lipid.
- the cationic lipid may be a hydrate of the salt or a solvate of the salt.
- a cationic lipid is an amphiphilic molecule having a lipophilic region containing one or more hydrocarbon groups and a hydrophilic region containing a polar group that is protonated at physiological pH. That is, the cationic lipid of the present invention can be protonated to form a cation.
- the compound represented by the above formula (1) includes a compound (cation) represented by the following formula (1) ′ in which a hydrogen ion is coordinated to a lone electron pair on the nitrogen atom of the piperidine ring.
- the anion that can be contained in the cationic lipid of this embodiment in combination with the cation is not particularly limited as long as it is pharmaceutically acceptable.
- chloride ion, bromide ion, nitric acid Inorganic ions such as ions, sulfate ions and phosphate ions
- organic acid ions such as acetate ions, oxalate ions, maleate ions, fumarate ions, citrate ions, benzoate ions and methanesulfonate ions.
- the cationic lipid of the present invention may exist as stereoisomers such as geometric isomers and optical isomers, tautomers and the like, but the cationic lipid of the present invention includes all possible isomers including these. And mixtures thereof.
- the cationic lipid of the formula (1) (compound in which X 1 is a single bond) can be synthesized, for example, according to Scheme 1 shown in the above formula (10).
- Step 1-1 esterification
- Examples of the condensing agent include 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDC) hydrochloride, N, N′-dicyclohexylcarbodiimide (DCC), and the like.
- a base may be added as necessary.
- Examples of the base include NMM, TEA, DIPEA, DMAP, pyridine, picoline, lutidine and the like.
- Examples of the solvent include tetrahydrofuran (THF), methylene chloride, chloroform, benzene, hexane, ethyl acetate and the like.
- Step 1-2 introduction of alkyl chain
- the ester halide (a2) is reacted with di-tert-butyl malonate in the presence of a base.
- a base is NaH.
- the solvent include ethers such as dioxane, tetrahydrofuran, cyclopentyl methyl ether, and 1,2-dimethoxyethane.
- Step 1-3 introduction of alkyl chain
- the compound (a3) is reacted with an alkyl halide (preferably iodide) in the presence of a base to introduce an alkyl chain to obtain the compound (a4).
- an alkyl halide preferably iodide
- a base preferably iodide
- the base and the solvent those similar to those in the above step 1-2 can be used.
- Step 1-4 Deprotection
- the tert-butyl group of compound (a4) is deprotected under acid hydrolysis conditions to obtain compound (a5).
- the acid used for deprotection include trifluoroacetic acid (TFA) and hydrochloric acid.
- the solvent include methylene chloride.
- Step 1-5 Decarboxylation
- carboxylic acid (a6) is obtained by decarboxylation of compound (a5).
- the decarboxylation reaction can be performed, for example, by heating in a solvent.
- aromatic hydrocarbons such as benzene, toluene and xylene are used.
- Step 1-6 Reduction step
- Compound (a7) is obtained by reducing the carboxyl group of compound (a6) to a hydroxyl group in the presence of a reducing agent.
- the reducing agent include borane complexes such as borane (BH 3 ) -tetrahydrofuran complex and borane-dimethyl sulfide complex.
- solvent examples include ethers such as diethyl ether, tetrahydrofuran and dioxane, halogenated hydrocarbons such as chloroform, methylene chloride and dichloroethane, hydrocarbons such as hexane, benzene and toluene, and mixed solvents thereof.
- ethers such as diethyl ether, tetrahydrofuran and dioxane
- halogenated hydrocarbons such as chloroform, methylene chloride and dichloroethane
- hydrocarbons such as hexane, benzene and toluene
- mixed solvents thereof examples include ethers such as diethyl ether, tetrahydrofuran and dioxane, halogenated hydrocarbons such as chloroform, methylene chloride and dichloroethane, hydrocarbons such as hexane, benzene and toluene, and mixed solvents thereof.
- Step 1-7 Esterification
- a condensing agent and the base those similar to those in Step 1-1 can be used.
- Step 2-1 Esterification
- an ester halide (b1) is obtained by condensing a halogenated alkylcarboxylic acid to benzyl alcohol.
- the esterification conditions are the same as in step 1-1.
- Step 2-2 alkyl chain introduction
- the ester halide (b1) is reacted with di-tert-butyl malonate in the presence of a base to obtain the compound (b2).
- Step 2-3 introduction of alkyl chain
- the compound (b2) is reacted with an alkyl halide in the presence of a base to obtain the compound (b3).
- Step 2-4 Deprotection
- the tert-butoxycarbonyl group of compound (b3) is deprotected under acid hydrolysis conditions to give compound (b4).
- Step 2-5 Decarboxylation
- Step 2-6 Reduction step
- Step 2-7 Esterification
- the obtained compound (b6) is esterified with 1-methyl-piperidine-4-carboxylic acid or a derivative thereof (such as a hydrogen halide salt) in the presence of a condensing agent and a base to obtain a compound (b7).
- Step 2-8 Deprotection
- the benzyl protecting group is deprotected under reducing conditions to give compound (b8).
- Step 2-9 Esterification
- the compound of the above formula (1a) can also be synthesized according to the above scheme 1 or 2. Specifically, when ring P has the structure of formula (P-1), (P-4), or (P-5), for example, in step 1-7 or step 2-7, 1-methyl Instead of piperidine-4-carboxylic acid, an esterification reaction may be carried out using a carboxylic acid corresponding to the structure of formulas (P-1), (P-4) and (P-5).
- reaction reagent for example, chloroformate such as chloroformate 4-nitrophenyl
- N-alkylpiperazine or N-alkylhomopiperazine compounds having the structures of formulas (P-2) and (P-3)
- the present invention provides a lipid complex containing (I) the above-described cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol.
- the lipid complex includes (I) the above-described cationic lipid, and (II) at least one lipid selected from the group consisting of neutral lipid, polyethylene glycol-modified lipid, and sterol, III) a nucleic acid. Therefore, the lipid complex of the present invention may or may not contain a nucleic acid.
- the lipid complex of this embodiment enables nucleic acid to be efficiently released into the cytoplasm.
- the lipid complex of this embodiment when stored for a certain period (for example, 1 month or 3 months), an increase in particle diameter is suppressed, and excellent physical stability can be exhibited.
- Examples of the form of a complex formed of a lipid containing a cationic lipid and a nucleic acid include, for example, a complex of a nucleic acid and a membrane (reverse micelle) composed of a single lipid (single molecule) layer, and a complex of a nucleic acid and a liposome. Body, a complex of nucleic acid and micelle, and the like.
- the nucleic acid is encapsulated in microparticles composed of a lipid containing a cationic lipid.
- the lipid complex of this embodiment contains, for example, 10 to 100 mol%, such as 20 to 90 mol%, such as 40 to 80 mol%, of the above-described cationic lipid based on the total lipid contained in the lipid complex.
- Cationic lipids can be used singly or in combination of two or more.
- nucleic acid examples include siRNA, miRNA, shRNA expression vector, antisense oligonucleotide, mRNA, ribozyme and the like.
- the nucleic acid may be siRNA, miRNA, mRNA.
- the lipid complex of the present embodiment contains, for example, 0.01 to 50% by weight, for example 0.1 to 30% by weight, for example 1 to 10% by weight, of the nucleic acid with respect to the total weight of the lipid complex.
- the lipid complex of the present embodiment comprises, as a lipid component, (I) the above-described cationic lipid, and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol. contains.
- the lipid complex of the present embodiment contains, for example, 50 to 100% by weight, such as 70 to 99.99% by weight, for example 90 to 99% by weight, of the lipid component with respect to the total weight of the lipid complex.
- Neutral lipid means lipid present at physiological pH in either uncharged or neutral zwitterionic form.
- Neutral lipids include dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) , Diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), dilignocelloylphosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), sphingomyelin, ceramide, dioleoylphosphatidylglycerol (DOPG), dipalmitoyl Phosphatidylglycerol
- the lipid complex of the present embodiment may contain neutral lipid, for example, 0 to 50 mol%, for example 0 to 40 mol%, for example 0 to 30 mol%, based on the total lipid contained in the lipid complex. Good.
- polyethylene glycol-modified lipid examples include PEG2000-DMG (PEG2000-dimyristylglycerol), PEG2000-DPG (PEG2000-dipalmitoylglycerol), PEG2000-DSG (PEG2000-distearoylglycerol), PEG5000-DMG (PEG5000-dimyristylglycerol).
- PEG5000-DPG PEG5000-dipalmitoylglycerol
- PEG5000-DSG PEG5000-distearoylglycerol
- PEG-cDMA N-[(methoxypoly (ethylene glycol) 2000) carbamyl] -1,2-dimyristyloxylpropyl- 3-amine
- PEG-C-DOMG R-3-[( ⁇ -methoxy-poly (ethylene glycol) 2000) carbamoyl)]-1,2-dimyristyloxylpropyl-3-amine
- DAA PEG-dialkyloxypropyl
- PEG-phospholipid PEG-ceramide
- PEG-dialkyloxypropyl examples include PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, PEG-distearyloxypropyl, and the like.
- Polyethylene glycol-modified lipids can be used alone or in combination of two or more.
- the lipid complex of the present embodiment contains, for example, 0 to 30 mol%, such as 0 to 20 mol%, such as 0 to 10 mol%, of polyethylene glycol-modified lipid, based on the total lipid contained in the lipid complex. Also good.
- Sterol is an alcohol having a steroid skeleton.
- sterols include cholesterol, dihydrocholesterol, lanosterol, ⁇ -sitosterol, campesterol, stigmasterol, brassicasterol, ergocasterol, fucosterol, and 3 ⁇ - [N- (N ′, N′-dimethylaminoethyl) carbamoyl].
- examples include cholesterol (DC-Chol).
- a sterol can be used individually by 1 type or in mixture of 2 or more types.
- the lipid complex of this embodiment may contain, for example, 0 to 90 mol%, such as 10 to 80 mol%, such as 20 to 50 mol%, of sterol based on the total lipid contained in the lipid complex.
- the combination of lipid components in the lipid complex of the present embodiment is not particularly limited.
- the combination of the above-described cationic lipid, neutral lipid and sterol, the above-described cationic lipid, neutral lipid, polyethylene glycol-modified lipid, and Examples include sterol combinations.
- the present invention provides (I) the above-described cationic lipid, (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol, and (III) a nucleic acid.
- the composition containing this is provided.
- the composition of this embodiment makes it possible to efficiently release nucleic acids into the cytoplasm.
- the composition of this embodiment may contain the lipid complex described above, a pharmaceutically acceptable medium, and optionally other additives. Pharmaceutically acceptable media and other additives will be described later.
- composition of the present embodiment contains, for example, 10 to 100 mol%, such as 20 to 90 mol%, such as 40 to 70 mol%, of the above-described cationic lipid based on the total lipid contained in the composition.
- Cationic lipids can be used singly or in combination of two or more.
- Nucleic acids include those similar to those described above.
- the composition of the present embodiment contains, for example, 0.01 to 50% by weight, such as 0.1 to 30% by weight, for example 1 to 10% by weight, of nucleic acid, based on the total weight of the composition.
- composition of this embodiment contains, as a lipid component, (I) the cationic lipid described above, and (II) at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols. To do.
- composition of the present embodiment may contain, for example, 0 to 50 mol%, such as 0 to 40 mol%, such as 0 to 30 mol%, based on the total lipid contained in the composition.
- the composition of the present embodiment may contain, for example, 0 to 30 mol%, such as 0 to 20 mol%, such as 0 to 10 mol%, of polyethylene glycol-modified lipid, based on the total lipid contained in the composition. .
- composition of the present embodiment may contain, for example, 0 to 90 mol%, such as 10 to 80 mol%, such as 20 to 50 mol%, based on the total lipid contained in the composition.
- the combination of lipid components in the composition of the present embodiment is not particularly limited.
- composition of the present embodiment includes, as other additives, sugars such as sucrose, glucose, sorbitol, and lactose; amino acids such as glutamine, glutamic acid, sodium glutamate, and histidine; citric acid, phosphorus Acid salts such as acid, acetic acid, lactic acid, carbonic acid and tartaric acid may be contained.
- sugars such as sucrose, glucose, sorbitol, and lactose
- amino acids such as glutamine, glutamic acid, sodium glutamate, and histidine
- citric acid citric acid
- phosphorus Acid salts such as acid, acetic acid, lactic acid, carbonic acid and tartaric acid may be contained.
- composition of the present embodiment may be formulated as a pharmaceutical composition.
- dosage form of the pharmaceutical composition include injections.
- the composition of the present embodiment may be in a powder state from which the solvent has been removed by freeze drying or the like, or in a liquid state.
- the composition of one embodiment of the present invention is a powder composition comprising the lipid complex of the above-described embodiment.
- the powder composition may be prepared by removing the solvent from the liquid composition (dispersion) by, for example, filtration or centrifugation, or may be prepared by lyophilizing the dispersion.
- the composition When the composition is in a powder state, it can be suspended or dissolved in a pharmaceutically acceptable medium before use and used as an injection.
- the composition of one embodiment of the present invention is a liquid composition comprising the lipid complex of the above-described embodiment and a pharmaceutically acceptable medium. When the composition is in a liquid state, it can be used as an injection as it is or suspended or dissolved in a pharmaceutically acceptable medium.
- compositions of the present embodiment further comprises solubilizing agents such as alcohols such as ethanol, propylene glycol, and polyethylene glycol, stabilizers, antioxidants, preservatives, excipients, fillers, and weight increase commonly used in pharmaceutical production.
- solubilizing agents such as alcohols such as ethanol, propylene glycol, and polyethylene glycol, stabilizers, antioxidants, preservatives, excipients, fillers, and weight increase commonly used in pharmaceutical production.
- Additives such as agents, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, preservatives, flavoring agents, and soothing agents may be contained.
- Administration of the composition to a patient can be performed by parenteral methods such as intraarterial injection, intravenous injection, and subcutaneous injection.
- the dose of the composition varies depending on the administration subject, target organ, symptom, and administration method.
- the subject to which the composition is administered is not limited and can be applied to various animals, but in particular, it can be applied to mammals, preferably humans and laboratory animals in clinical tests, screenings, and experiments.
- the composition of the present embodiment forms a lipid complex in which a nucleic acid is encapsulated in fine particles composed of a lipid containing a cationic lipid.
- the “average particle diameter” of the lipid complex can be calculated by any one of volume average, number average, and Z-average.
- the average particle size (Z-average) of the lipid complex may be, for example, 10 to 1000 nm, such as 30 to 500 nm, such as 30 to 200 nm.
- the composition of the present embodiment is preferably one in which the particle size of the lipid complex does not increase as much as possible during storage, compared to before storage.
- the average particle size (Z-average) when stored at 4 ° C. for 3 months is preferably 1.3 times or less of the particle size before storage, preferably 1.2 times or less Are more preferable, and those of 1.1 times or less are particularly preferable.
- the composition of the present embodiment preferably has almost no surface charge in an environment of about pH 7.4, such as in blood, from the viewpoint of suppressing non-specific adsorption and immune reaction.
- an environment of about pH 7.4 such as in blood
- the present invention provides a polar organic solvent comprising (I) the above-described cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol.
- a composition comprising: a step (a) of obtaining a mixed solution by mixing an aqueous solution containing (III) an aqueous solution containing a nucleic acid; and a step (b) for reducing the content of a polar organic solvent in the mixed solution.
- a manufacturing method is provided. According to the production method of the present embodiment, a composition capable of efficiently releasing nucleic acid into the cytoplasm can be produced.
- a lipid complex in which nucleic acid is encapsulated in fine particles composed of lipid can be formed by electrostatic interaction between water-soluble nucleic acid and the above-mentioned cationic lipid and hydrophobic interaction between lipids.
- the polar organic solvent include alcohols such as ethanol.
- step (a) a polar organic in which (I) the above-described cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol are dissolved.
- a solvent-containing aqueous solution and (III) an aqueous solution containing a nucleic acid are mixed to obtain a mixed solution.
- the concentration of the polar organic solvent in the polar organic solvent-containing aqueous solution is not particularly limited as long as it satisfies the conditions for dissolving lipid molecules even after mixing with the nucleic acid aqueous solution.
- the concentration of the polar organic solvent in the aqueous solution containing the polar organic solvent in the step (a) may be 0 to 60% by weight.
- step (b) the content of the polar organic solvent is decreased by adding water or the like to the above mixed solution. Thereby, a lipid complex can be formed.
- the concentration of the polar organic solvent in the final aqueous solution containing the polar organic solvent in step (b) may be 0 to 5% by weight.
- the mixed liquid obtained in step (a) may be subjected to dialysis to remove the polar organic solvent and replace the solvent with a pharmaceutically acceptable medium. Since the content of the polar organic solvent in the solution decreases during the dialysis process, a lipid complex can thereby be formed.
- lipid complex in which nucleic acids are efficiently encapsulated inside fine particles can be obtained.
- Such lipid complexes can have excellent physical stability. For example, when stored for a certain period (for example, 1 month, for example, 3 months), an increase in particle diameter can be suppressed.
- the composition can be used as a pharmaceutical composition.
- the composition of the present invention can be used for treatment (for example, gene therapy) in which a desired nucleic acid is introduced into a target cytoplasm (for example, a cytoplasm that causes various diseases) in vivo or in vitro. Therefore, one embodiment of the present invention provides a method for treating various diseases (particularly a gene therapy method) using a pharmaceutical composition containing the lipid complex described above.
- the administration target, administration method and conditions are the same as described above.
- kits for nucleic acid drug delivery comprising the anionic lipid.
- the kit can be preferably used for treatment (for example, gene therapy) for various target cells.
- the storage state of the anionic lipid is not particularly limited, and may be set to an arbitrary state such as a solution or a powder in consideration of each stability (storage) and ease of use. it can.
- the kit of the present embodiment may include, for example, various nucleic acids, various media (pharmaceutically acceptable media, buffers), instructions for use (use manual), and the like.
- the kit of this embodiment is used to prepare a composition or lipid complex containing a desired nucleic acid to be introduced into a target cell and a lipid containing the anionic lipid.
- the prepared composition or lipid complex can be used effectively for nucleic acid delivery to target cells.
- an embodiment of the present invention may be a nucleic acid drug delivery kit including a pharmaceutical composition containing the anionic lipid.
- the kit of the present embodiment may include, for example, various media (pharmaceutically acceptable media), instructions for use (use manual), and the like.
- Root temperature in the following Examples and Production Examples usually indicates about 10 ° C. to about 35 ° C. % Indicates weight percent unless otherwise specified.
- YAMAZEN parallel prep ⁇ Column: YAMAZEN Hi-FlashTM Column (Silicagel), size; S (16 ⁇ 60 mm), M (20 ⁇ 75 mm), L (26 ⁇ 100 mm), 2 L (26 ⁇ 150 mm) ⁇ , or an automated flash purification system
- reaction mixture was cooled to room temperature, diethyl ether was added, washed with water and saturated brine in this order, and the organic phase was dried over anhydrous magnesium sulfate. After filtration, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (cyclohexane) to obtain the title compound (1.16 g).
- the reaction mixture was cooled in an ice-water bath, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with n-heptane.
- the organic phase was washed with saturated brine and dried over anhydrous magnesium sulfate. After filtration, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (n-heptane / ethyl acetate) to obtain the title compound (490 mg, 0.91 mmol).
- Example A-2- (1 (2) Synthesis of 1-benzyl 9,9-di-tert-butyl nonadecane-1,9,9-tricarboxylate According to the method of Example A-1- (2), Example A-2- (1 ), Obtained from 1-iododecane (0.54 mL, 2.55 mmol), 65% sodium hydride (71 mg, 1.91 mmol) and 1,4-dioxane (5 mL). The compound (470 mg, 0.78 mmol) was obtained.
- the reaction mixture was cooled with an ice-water bath, water was added, and the mixture was extracted with diethyl ether. The organic phase was washed with saturated brine and dried over anhydrous magnesium sulfate. After filtration, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (n-heptane / ethyl acetate) to obtain the title compound (456 mg, 1.02 mmol).
- Example A-4- According to the method of (1), Example A- The compound obtained in 4- (1) (456 mg, 1.02 mmol), the compound obtained in Production Example 3 (478 mg, 1.53 mmol), sodium iodide (46 mg, 0.31 mmol), 60% sodium hydride ( 49 mg, 1.22 mmol) and DMF (3.3 mL) gave the title compound (380 mg, 0.56 mmol).
- Example A-4- The title compound (234 mg, 0.45 mmol) was obtained from the compound obtained in (2) (380 mg, 0.56 mmol), methylene chloride (2 mL), TFA (1 mL), and xylene (2 mL).
- Example A-4- Compound (188 mg, 0.37 mmol), 1-methyl-piperidine-4-carboxylic acid hydrochloride (132 mg, 0.74 mmol), EDC (155 mg, 0.81 mmol), DIPEA (0.126 mL, .0. 74 mmol), DMAP (9.0 mg, 0.074 mmol) and methylene chloride (1.9 mL) gave the title compound (217 mg, 0.34 mmol).
- Example A-1- (5) According to the method of Example A-1- (5), the compound obtained in Example A-1- (4) (40 mg, 0.078 mmol) and the compound obtained in Production Example 10- (2) (28 mg, 0 .16 mmol), EDC (33 mg, 0.17 mmol), DIPEA (0.027 mL, 0.16 mmol), DMAP (1.9 mg, 0.016 mmol) and methylene chloride (1.0 mL) to give the title compound (40 mg,. 063 mmol).
- Example A-1- (5) According to the method of Example A-1- (5), the compound obtained in Example A-1- (4) (40 mg, 0.078 mmol) and the compound obtained in Production Example 11- (2) (27.8 mg) , 0.16 mmol), EDC (33 mg, 0.17 mmol), DIPEA (0.027 mL, 0.157 mmol), DMAP (1.9 mg, 0.016 mmol) and methylene chloride (1.0 mL) to give the title compound (38 mg, 0.060 mmol) was obtained.
- Example A-1- (4) The compound (11 mg, 0.022 mmol) obtained in Example A-1- (4) and pyridine (0.0043 mL, 0.054 mmol) were dissolved in methylene chloride (0.8 mL), and chloroformate 4 was added under ice cooling.
- -Nitrophenyl (12 mg, 0.060 mmol) was added, and the mixture was stirred at the same temperature for 10 minutes and at room temperature for 1.5 hours.
- 1-Methylpiperazine (0.010 mL, 0.090 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 3 hours.
- a saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate.
- reaction mixture was diluted with diethyl ether and washed with water, and then the aqueous layer was extracted with diethyl ether. The organic phases were combined, washed with saturated brine, and dried over anhydrous magnesium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the title compound (9.88 g, 25.2 mmol).
- Example A-14 2- (4-Oxo-4-((8-pentyltridecyl) oxy) butyl) dodecyl 1-methylpiperidine-4-carboxylate (cationic lipid 14)
- the compound obtained in Example A-13- (6) 120 mg, 0.30 mmol
- the compound obtained in Production Example 4- (4) (90 mg, 0 .33 mmol)
- EDC 64 mg, 0.33 mmol
- DMAP 4.0 mg, 0.033 mmol
- methylene chloride 1.5 mL
- Example A-15 2- ⁇ 4-[(4-nonyltridecyl) oxy] -4-oxobutyl ⁇ dodecyl 1-methylpiperidine-4-carboxylate (cationic lipid 15)
- the compound obtained in Example A-13- (6) 110 mg, 0.28 mmol
- the compound obtained in Production Example 5- (4) (99 mg, 0 .30 mmol)
- EDC 58 mg, 0.30 mmol
- DMAP 4.0 mg, 0.033 mmol
- methylene chloride 1.3 mL
- Example A-17 Bis (4-pentylnonyl) 9- ⁇ [(1-methylpiperidine-4-carbonyl) oxy] methyl ⁇ heptadecandioate (cationic lipid 17)
- the compound obtained in Example A-16- (1) (0.20 g, 0.44 mmol) and the compound obtained in Production Example 7- (2)
- the title compound (0.23 g, 0.27 mmol) was obtained from EDC (0.20 g, 1.05 mmol), DMAP (11 mg, 0.09 mmol) and methylene chloride (5 mL).
- Example A-16- (1) Synthesis of 5-[ ⁇ (1-methylpiperidine-4-carbonyl) oxy ⁇ methyl] dinonanoic acid According to the method of Example A-16- (1), the compound obtained in Example A-18- (4) (0.62 g, 1.19 mmol), 10% palladium-carbon (62 mg, 0.06 mmol, 50 The title compound (0.41 g, 1.19 mmol) was obtained from ethanol (2 mL) and ethanol (2 mL).
- the reaction mixture was diluted with diethyl ether and washed with water, and then the aqueous layer was extracted with diethyl ether. The organic phases were combined, washed with saturated brine, and dried over anhydrous magnesium sulfate. After filtration, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (cyclohexane / ethyl acetate) to obtain the title compound (6.01 g, 14.3 mmol).
- Example A-18- (1) Synthesis of 1,11-dibenzyl 6,6-di-tert-butyl undecane-1,6,6,11-tetracarboxylate According to the method of Example A-18- (1), the compound obtained in Example A-21- (1) (4.0 g, 9.51 mmol), 60% sodium hydride (0.42 g, 10.5 mmol) The title compound (5.18 g, 8.29 mmol) was obtained from the compound (3.25 g, 11.4 mmol) obtained in Preparation Example 9 and THF (30 mL).
- the synthesized cationic lipids 1 to 22 are shown in Table A below.
- composition (1) Preparation of composition (1)> [Example B-1]
- a composition was prepared using the cationic lipid 1 of Example A-1.
- cationic lipid 1 DSPC (Nippon Seika Co., Ltd.), Cholesterol (Nippon Seika Co., Ltd.), MPEG2000-DMG (Nippon Oil Co., Ltd.) is 60 / 8.5 / 30 / 1.5 (molar ratio).
- DSPC Nippon Seika Co., Ltd.
- Cholesterol Nippon Seika Co., Ltd.
- MPEG2000-DMG Nippon Oil Co., Ltd.
- the siRNA diluted solution and the lipid solution were added and mixed at a flow rate of 3 mL / min and 1 mL / min, respectively, to obtain a lipid complex aqueous solution.
- the obtained lipid complex aqueous solution was subjected to dialysis using a dialysis membrane (trade name “Float-A-Lyzer G2,” SPECTRUM, 50K MWCO), and the external solution was phosphate buffer (PBS, pH 7.4).
- concentration and filter sterilization were performed to obtain a liquid composition of Example B-1.
- Example B-2 A composition of Example A-7 was obtained in the same manner as Example B-1, except that the cationic lipid 2 of Example A-2 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-3 A composition of Example B-3 was obtained in the same manner as Example B-1, except that the cationic lipid 3 of Example A-3 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-4 A composition of Example B-4 was obtained in the same manner as Example B-1, except that the cationic lipid 4 of Example A-4 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-5 A composition of Example B-5 was obtained in the same manner as in Example B-1, except that the cationic lipid 5 of Example A-5 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-6 A composition of Example B-6 was obtained in the same manner as Example B-1, except that the cationic lipid 6 of Example A-6 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-7 A composition of Example B-7 was obtained in the same manner as Example B-1, except that the cationic lipid 7 of Example A-7 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-8 A composition of Example B-8 was obtained in the same manner as Example B-1, except that the cationic lipid 8 of Example A-8 was used instead of the cationic lipid 1 as the cationic lipid.
- the composition was diluted with RNase Free Water, and the siRNA concentration (A) measured using Quant-iT RiboGreen RNA Reagent (Invitrogen) was defined as siRNA present in the lipid complex external solution.
- the nucleic acid encapsulation rate was calculated by the following formula (F1).
- Encapsulation rate (%) 100 ⁇ (A / B) ⁇ 100 (F1)
- the average particle size of the lipid complex was measured with a particle size measuring device (trade name “Zetasizer Nano ZS”, manufactured by Malvern).
- Table 1 shows the siRNA encapsulation rate, the average particle diameter (Z-average), and the polydispersity index of the lipid complex.
- compositions of Examples B-1 to B-8 show a high siRNA encapsulation rate equivalent to that of Comparative Example B-1.
- composition (2) was prepared using the cationic lipid 1 of Example A-1.
- As the nucleic acid Firefly Luciferase (FLuc) mRNA (TriLink Biotechnology, hereinafter may be referred to as “FLuc mRNA”) was used.
- FLuc mRNA was dissolved in 27 ⁇ g / mL with 50 mM sodium acetate (pH 4.0) to prepare a diluted mRNA solution.
- cationic lipid 1, DSPC Nippon Seika Co., Ltd.
- Cholesterol Nippon Seika Co., Ltd.
- MPEG2000-DMG Nippon Oil Co., Ltd.
- aqueous solution of lipid complex was obtained by adding and mixing the diluted mRNA solution and the lipid solution at a flow rate of 3 mL / min and 1 mL / min, respectively.
- the obtained lipid complex aqueous solution is subjected to dialysis using a dialysis membrane (trade name “Float-A-Lyzer G2,” SPECTRUM, 50K MWCO), and the external solution is phosphate buffer (PBS, pH 7.4).
- concentration and filter sterilization were performed to obtain the composition of Example B-9.
- Example B-10 A composition of Example B-10 was obtained in the same manner as Example B-9, except that the cationic lipid 2 of Example A-2 was used instead of the cationic lipid 1 as the cationic lipid.
- Comparative Example B-2 A composition of Comparative Example B-2 was obtained in the same manner as in Example B-9, except that ALN-319 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-9 to Example B-10 show the same high mRNA encapsulation rate as the composition of Comparative Example B-2.
- Example B-11 A composition was prepared using the cationic lipid 2 of Example A-2. Cationic lipid 2 is used instead of cationic lipid 1 as the cationic lipid, and human erythropoietin (hEPO) mRNA (TriLink Biotechnologies, hereinafter referred to as “EPO mRNA”) may be used as the nucleic acid instead of FLuc mRNA.
- EPO mRNA human erythropoietin
- the composition of Example B-11 was obtained in the same manner as in Example B-9 except that was used.
- Comparative Example B-3 A composition of Comparative Example B-3 was obtained in the same manner as in Example B-11 except that ALN-319 was used instead of the cationic lipid 2 as the cationic lipid.
- composition (3) ⁇ Analysis of composition (3)>
- the encapsulation rate of mRNA in the lipid complex and the average particle size of the lipid complex were measured for the compositions of Example B-11 and Comparative Example B-3.
- Table 3 shows the encapsulation rate of mRNA and the average particle size (Z-average) of the lipid complex.
- Example B-11 exhibits a high encapsulation rate of mRNA equivalent to the composition of Comparative Example B-3.
- Example B-12 ⁇ Preparation of composition (4)> [Example B-12]
- the composition of Example B-12 containing Factor VII siRNA was obtained using the cationic lipid 1 of Example A-1.
- Example B-13 A composition of Example B-13 was obtained in the same manner as in Example B-12 except that the cationic lipid 2 of Example A-2 was used instead of the cationic lipid 1 as the cationic lipid.
- Comparative Example B-4 A composition of Comparative Example B-4 was obtained in the same manner as in Example B-12, except that ALN-319 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-12 and Example B-13 showed little change in average particle size even after storage for 3 months, indicating that they were physically more stable than the composition of Comparative Example B-4. It was done.
- Example B-14 ⁇ Preparation of composition (5)> [Example B-14] In the same manner as in the preparation of the composition (1), the composition of Example B-14 containing Factor VII siRNA was obtained using the cationic lipid 9 of Example A-9.
- Example B-15 A composition of Example B-15 was obtained in the same manner as in Example B-14 except that the cationic lipid 10 of Example A-10 was used in place of the cationic lipid 9 as the cationic lipid.
- Example B-16 A composition of Example B-16 was obtained in the same manner as in Example B-14 except that the cationic lipid 11 of Example A-11 was used in place of the cationic lipid 9 as the cationic lipid.
- Example B-17 A composition of Example B-17 was obtained in the same manner as in Example B-14 except that the cationic lipid 12 of Example A-12 was used in place of the cationic lipid 9 as the cationic lipid.
- Example B-18 A composition of Example B-18 was obtained in the same manner as in Example B-14 except that the cationic lipid 13 of Example A-13 was used instead of the cationic lipid 9 as the cationic lipid.
- Example B-19 A composition of Example B-19 was obtained in the same manner as in Example B-14 except that the cationic lipid 14 of Example A-14 was used instead of the cationic lipid 9 as the cationic lipid.
- Example B-20 A composition of Example B-20 was obtained in the same manner as in Example B-14 except that the cationic lipid 15 of Example A-15 was used in place of the cationic lipid 9 as the cationic lipid.
- Example B-21 A composition of Example B-21 was obtained in the same manner as Example B-14, except that the cationic lipid 2 of Example A-2 was used instead of the cationic lipid 9 as the cationic lipid.
- Example B-22 ⁇ Preparation of composition (6)> [Example B-22] In the same manner as in the preparation of the composition (1), the composition of Example B-22 containing Factor VII siRNA was obtained using the cationic lipid 16 of Example A-16.
- Example B-23 A composition of Example B-23 was obtained in the same manner as in Example B-22 except that the cationic lipid 17 of Example A-17 was used instead of the cationic lipid 16 as the cationic lipid.
- Example B-24 A composition of Example B-24 was obtained in the same manner as in Example B-22 except that the cationic lipid 18 of Example A-18 was used instead of the cationic lipid 16 as the cationic lipid.
- Example B-25 A composition of Example B-25 was obtained in the same manner as in Example B-22 except that the cationic lipid 19 of Example A-19 was used instead of the cationic lipid 16 as the cationic lipid.
- Example B-26 A composition of Example B-26 was obtained in the same manner as in Example B-22 except that the cationic lipid 20 of Example A-20 was used instead of the cationic lipid 16 as the cationic lipid.
- Example B-27 A composition of Example B-27 was obtained in the same manner as in Example B-22 except that the cationic lipid 21 of Example A-21 was used in place of the cationic lipid 16 as the cationic lipid.
- Example B-28 A composition of Example B-28 was obtained in the same manner as in Example B-22, except that the cationic lipid 22 of Example A-22 was used instead of the cationic lipid 16 as the cationic lipid.
- Example B-29 A composition of Example B-29 was obtained in the same manner as in Example B-22 except that the cationic lipid 2 of Example A-2 was used instead of the cationic lipid 16 as the cationic lipid.
- ⁇ Analysis of composition (6)> In the same manner as in the analysis (1) of the composition, the encapsulation rate of siRNA into the lipid complex and the average particle size of the lipid complex were measured for the compositions of Example B-22 to Example B-29. Table 6 shows the encapsulation rate of siRNA, the average particle size (Z-average) and polydispersity index of lipid complexes.
- Example B-30 ⁇ Preparation of composition (7)> [Example B-30] In the same manner as in the preparation of the composition (1), the composition of Example B-30 containing Factor VII siRNA was obtained using the cationic lipid 1 of Example A-1.
- Example B-31 A composition of Example B-30 was obtained in the same manner as in Example B-30 except that the cationic lipid 2 of Example A-2 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-32 A composition of Example B-32 was obtained in the same manner as in Example B-30 except that the cationic lipid 3 of Example A-3 was used instead of the cationic lipid 1 as the cationic lipid.
- Example B-33 A composition of Example B-33 was obtained in the same manner as in Example B-30 except that the cationic lipid 4 of Example A-4 was used instead of the cationic lipid 1 as the cationic lipid.
- Comparative Example B-5 A composition of Comparative Example B-5 was obtained in the same manner as in Example B-30 except that ALN-319 described above was used in place of the cationic lipid 1 as the cationic lipid.
- compositions of Examples B-30 to B-33 showed little change in average particle size even after storage for 6 months, indicating that they were physically more stable than the composition of Comparative Example B-5. It was done.
- composition (8) was prepared using the cationic lipid 2 of Example A-2. EPO mRNA was used as the nucleic acid.
- EPO mRNA was dissolved in 80 ⁇ g / mL with 10 mM sodium citrate (pH 4.0) to prepare a diluted mRNA solution.
- cationic lipid 2 DOPE (Nippon Oil Co., Ltd.), Cholesterol (Nippon Seika Co., Ltd.), and MPEG2000-DMG (Nippon Oil Co., Ltd.) were mixed at 60 / 5.0 / 33.5 / 1.5 (molar ratio ) And dissolved in ethanol to a total lipid concentration of 2.25 mM to obtain a lipid solution.
- aqueous solution of lipid complex was obtained by adding and mixing the diluted mRNA solution and the lipid solution at a flow rate of 3 mL / min and 1 mL / min, respectively.
- the obtained lipid complex aqueous solution is subjected to dialysis using a dialysis membrane (trade name “Float-A-Lyzer G2,” SPECTRUM, 50K MWCO), and the external solution is phosphate buffer (PBS, pH 7.4).
- concentration and filter sterilization were performed to obtain the composition of Example B-34.
- Example B-35 The same as the preparation of the composition (1) except that Luciferase siRNA was used instead of Factor VII siRNA as the nucleic acid, and Cationic lipid 2 of Example A-2 was used instead of Cationic lipid 1 as the cationic lipid. Thus, a composition of Example B-35 was obtained.
- compositions of Example B-34 and Example B-35 exhibit high nucleic acid encapsulation rates. This result shows that the compositions of the examples can be used for nucleic acid delivery regardless of the type of nucleic acid.
- the Factor VII protein concentration in the PBS administration group was taken as 100%, and the Factor VII protein concentration in the composition administration group was calculated as a relative value. The results are shown in FIG.
- compositions of Example B-1 to Example B-3 are more effective in inhibiting the expression of Factor VII protein than the composition of Comparative Example B-1. This result indicates that the composition of the example efficiently releases the nucleic acid into the cytoplasm.
- Example B-4 has a higher inhibitory effect on the expression of Factor VII protein than the composition of Comparative Example B-1. This result indicates that the composition of the example efficiently releases the nucleic acid into the cytoplasm.
- Example B-5 is highly effective in inhibiting the expression of Factor VII protein. This result indicates that the composition of the example efficiently releases the nucleic acid into the cytoplasm.
- Example B-11 has a higher hEPO protein expression effect than the composition of Comparative Example B-3. This result indicates that the composition of the example efficiently releases the nucleic acid into the cytoplasm.
- the Factor VII protein concentration in the PBS administration group was taken as 100%, and the Factor VII protein concentration in the composition administration group was calculated as a relative value. The results are shown in Table 13.
- HEPO was not detected in the untreated group and Luc siRNA administration group, and hEPO was detected 1 day after administration in the EPO mRNA administration group. It was also confirmed that the reticulocyte count increased 4 days after administration due to the action of the produced EPO. This result indicates that the composition of the example efficiently releases the nucleic acid into the cytoplasm.
- nucleic acid can be efficiently released into the cytoplasm. Moreover, according to the cationic lipid of the present invention, it is possible to suppress an increase in the particle size of the lipid complex when stored for a certain period.
- a cationic lipid capable of efficiently releasing nucleic acid into the cytoplasm can be provided.
Abstract
Description
[2]下記式(1)で表される、[1]に記載の化合物又はその薬学的に許容される塩。
[3b]上記式(A1)、(A2)、(A3)、(A4)、(A5)、(A9)、(A12)、(A15)、(A20)で表される化合物からなる群より選択される、[3]に記載の化合物又はその薬学的に許容される塩。
[3c]上記式(A1)~(A5)で表される化合物からなる群より選択される、[3]に記載の化合物又はその薬学的に許容される塩。
[3d]上記式(A6)~(A8)で表される化合物からなる群より選択される、[3]に記載の化合物又はその薬学的に許容される塩。
(1)本発明のカチオン性脂質によれば、核酸を細胞質に効率よく放出することを可能とする。
(2)本発明のカチオン性脂質によれば、一定期間保管した場合に脂質複合体の粒子径の増大を抑制することを可能とする。
したがって、本発明のカチオン性脂質は、細胞質への核酸送達用の脂質としての利用可能性を有している。
一実施形態において、本発明は、下記式(1a)で表される化合物又はその薬学的に許容される塩であり、カチオン性脂質として用いることができる。カチオン性脂質は、前記塩の水和物又は前記塩の溶媒和物であってもよい。
一実施形態において、環Pは下記式(P-1)を表す。
本明細書において、「アルケニル」とは、指定された数の炭素原子および少なくとも1つの炭素-炭素二重結合を有する直鎖または分岐の炭化水素基を意味する。例えば、モノエン、ジエン、トリエン及びテトラエンなどが挙げられるが、これらに限定されるものではない。
本明細書において、「アルキレン」とは、指定された数の炭素原子を有する直鎖状、環状または分岐状の2価の飽和脂肪族炭化水素基を意味する。
本明細書において、「ハロゲン」は、F、Cl、Br、Iを意味する。
したがって、本発明の一実施形態は、下記式(1b)で表される化合物又はその薬学的に許容される塩である。
したがって、本発明の一実施形態は、下記式(1c)で表される化合物又はその薬学的に許容される塩である。
本発明の一実施形態は、上記式(A1)~(A5)、(A9)~(A22)のいずれかで表される化合物又はその薬学的に許容される塩であり、カチオン性脂質として用いることができる。カチオン性脂質は、前記塩の水和物又は前記塩の溶媒和物であってもよい。
本発明の一実施形態は、上記式(A1)、(A2)、(A3)、(A4)、(A5)、(A9)、(A12)、(A15)、(A16)、(A17)、(A19)、(A20)、(A22)のいずれかで表される化合物又はその薬学的に許容される塩であり、カチオン性脂質として用いることができる。カチオン性脂質は、前記塩の水和物又は前記塩の溶媒和物であってもよい。
本発明の一実施形態は、上記式(A1)、(A2)、(A3)、(A4)、(A5)、(A9)、(A12)、(A15)、(A20)のいずれかで表される化合物又はその薬学的に許容される塩であり、カチオン性脂質として用いることができる。カチオン性脂質は、前記塩の水和物又は前記塩の溶媒和物であってもよい。
本発明の一実施形態は、上記式(A1)、(A2)、(A3)、(A4)、(A5)のいずれかで表される化合物又はその薬学的に許容される塩であり、カチオン性脂質として用いることができる。カチオン性脂質は、前記塩の水和物又は前記塩の溶媒和物であってもよい。
本発明の一実施形態は、上記式(A6)、(A7)、(A8)のいずれかで表される化合物又はその薬学的に許容される塩であり、カチオン性脂質として用いることができる。カチオン性脂質は、前記塩の水和物又は前記塩の溶媒和物であってもよい。
本発明のカチオン性脂質は、幾何異性体、光学異性体等の立体異性体、互変異性体等が存在し得るが、本発明のカチオン性脂質は、これらを含め、全ての可能な異性体およびそれらの混合物を包含する。
本発明のカチオン性脂質の製造方法について説明する。下記式(10)および(11)に、カチオン性脂質の合成スキームの一実施形態を示す。本明細書に記載されている化合物はすべて、化合物として本発明に含まれる。本発明の化合物は、以下のスキームにおいて記載されている方法の少なくとも1つに従って合成することができる。
(工程1-1:エステル化)
まず、アルコール(a1)とハロゲン化アルキルカルボン酸 X-L1-COOH(Xはハロゲン原子であり、L1は前記と同義)(好ましくは臭化アルキルカルボン酸)とを縮合剤の存在下で反応させてエステルのハロゲン化物(a2)を得る。縮合剤としては、例えば、1-[3-(ジメチルアミノ)プロピル]-3-エチルカルボジイミド(EDC)塩酸塩、N,N’-ジシクロヘキシルカルボジイミド(DCC)等が挙げられる。必要に応じて、塩基を加えても良い。塩基としては、例えば、NMM、TEA、DIPEA、DMAP、ピリジン、ピコリン、ルチジン等が挙げられる。溶媒としては、例えば、テトラヒドロフラン(THF)、塩化メチレン、クロロホルム、ベンゼン、ヘキサン、酢酸エチル等が挙げられる。
(工程1-2:アルキル鎖導入)
次いで、エステルハロゲン化物(a2)とジ-tert-ブチル マロネートとを塩基の存在下で反応させる。これによりマロン酸ジエステルの活性メチレンの水素原子が引き抜かれて、アルキルエステル鎖が導入され、化合物(a3)を得る。塩基としては例えばNaHが挙げられる。溶媒としては、例えば、ジオキサン、テトラヒドロフラン、シクロペンチルメチルエーテル、1,2-ジメトキシエタン等のエーテル類等が挙げられる。
(工程1-3:アルキル鎖導入)
次いで、化合物(a3)とハロゲン化アルキル(好ましくはヨウ化物)を塩基の存在下で反応させ、アルキル鎖を導入し、化合物(a4)を得る。塩基および溶媒は上記工程1-2と同様のものを使用できる。
(工程1-4:脱保護)
次いで、化合物(a4)のtert-ブチル基を酸加水分解条件下で脱保護し、化合物(a5)を得る。脱保護に使用する酸としては、例えば、トリフルオロ酢酸(TFA)、塩酸等が挙げられる。溶媒としては、例えば、塩化メチレン等が挙げられる。
(工程1-5:脱炭酸)
次いで、化合物(a5)の脱炭酸によりカルボン酸(a6)を得る。脱炭酸反応は、例えば、溶媒中、加熱することにより行うことができる。溶媒としては、例えば、ベンゼン、トルエン、キシレン、などの芳香族炭化水素類等が用いられる。
(工程1-6:還元工程)
化合物(a6)のカルボキシル基を還元剤の存在下でヒドロキシル基へと還元して化合物(a7)を得る。還元剤としては、例えばボラン(BH3)-テトラヒドロフラン錯体、ボラン-ジメチルスルフィド錯体等のボラン錯体などが挙げられる。溶媒としては、例えばジエチルエーテル、テトラヒドロフラン、ジオキサンなどのエーテル類、クロロホルム、塩化メチレン、ジクロロエタンなどのハロゲン化炭化水素類、ヘキサン、ベンゼン、トルエンなどの炭化水素類、およびこれらの混合溶媒等が用いられる。
(工程1-7:エステル化)
得られたアルコール(a7)と1-メチル-ピペリジン-4-カルボン酸またはその誘導体(ハロゲン化水素塩など)とを縮合剤、塩基の存在下で反応させて最終物である化合物(a8)(R=L2-X1-R2)(式(1)のカチオン性脂質に相当する化合物)が得られる。縮合剤および塩基としては工程1-1と同様のものを使用できる。
上記スキーム2は、当業者が、異なる方法によって式(1)のカチオン性脂質(X1が単結合である化合物)を合成する方法を示したものである。
(工程2-1:エステル化)
まず、ベンジルアルコールに対してハロゲン化アルキルカルボン酸を縮合させることでエステルのハロゲン化物(b1)を得る。エステル化条件は工程1-1と同様である。
(工程2-2:アルキル鎖導入)
次いで、工程1-2と同様に、エステルのハロゲン化物(b1)とジ-tert-ブチル マロネートとを塩基の存在下で反応させ、化合物(b2)を得る。
(工程2-3:アルキル鎖導入)
次いで、工程1-3と同様に、化合物(b2)とハロゲン化アルキルを塩基の存在下で反応させ、化合物(b3)を得る。
(工程2-4:脱保護)
次いで、工程1-4と同様に、化合物(b3)のtert-ブトキシカルボニル基を酸加水分解条件下で脱保護し、化合物(b4)を得る。
(工程2-5:脱炭酸)
次いで、工程1-5と同様に、カルボン酸(b5)を得る。
(工程2-6:還元工程)
さらにこれを、工程1-6と同様に還元剤の存在下で還元して化合物(b6)を得る。
(工程2-7:エステル化)
得られた化合物(b6)と1-メチル-ピペリジン-4-カルボン酸またはその誘導体(ハロゲン化水素塩など)とを縮合剤、塩基の存在下でエステル化反応させて、化合物(b7)を得る。
(工程2-8:脱保護)
続いて、還元条件下で、ベンジル保護基を脱保護して化合物(b8)を得る。脱保護は例えば、パラジウム/炭素等の金属触媒存在下、接触水素添加反応することにより行えばよい。
(工程2-9:エステル化)
最後に、化合物(b8)をアルコール(R1OH)と反応させることにより化合物(b9)(R=L2-X1-R2)(式(1)のカチオン性脂質に相当する化合物)を得ることができる。
本発明は、(I)上述したカチオン性脂質と、(II)中性脂質、ポリエチレングリコール修飾脂質及びステロールからなる群より選択される少なくとも一種の脂質と、を含有する脂質複合体を提供する。本発明の一実施形態として、脂質複合体は、(I)上述したカチオン性脂質と、(II)中性脂質、ポリエチレングリコール修飾脂質及びステロールからなる群より選択される少なくとも一種の脂質と、(III)核酸と、を含有する。したがって、本発明の脂質複合体は、核酸を含んでいても含んでいなくてもよい。本実施形態の脂質複合体は、核酸を細胞質に効率よく放出することを可能とする。また、本実施形態の脂質複合体は、一定期間(例えば1か月又は3か月)保管した場合に粒子径の増大が抑制され、優れた物理的安定性を示し得る。
カチオン性脂質を含有する脂質と核酸とで形成される複合体の形態としては、例えば、核酸と脂質一重(一分子)層からなる膜(逆ミセル)との複合体、核酸とリポソームとの複合体、核酸とミセルとの複合体等が挙げられる。本発明の一実施形態の脂質複合体において、核酸はカチオン性脂質を含有する脂質で構成された微粒子内に封入されている。
一実施形態において、本発明は、(I)上述したカチオン性脂質と、(II)中性脂質、ポリエチレングリコール修飾脂質及びステロールからなる群より選択される少なくとも一種の脂質と、(III)核酸と、を含有する組成物を提供する。本実施形態の組成物は、核酸を細胞質に効率よく放出することを可能とする。本実施形態の組成物は、上述した脂質複合体、薬学的に許容される媒体、及び場合によりその他の添加剤を含有するものであってもよい。薬学的に許容される媒体、その他の添加剤については後述する。
本実施形態の組成物は、保管期間中、脂質複合体の粒子径が保管前と比べて極力増大しないものが好ましい。例えば、4℃で3ヶ月保管した場合の平均粒子径(Z-平均)の大きさが保管前の粒子径の大きさに対して1.3倍以下であるものが好ましく、1.2倍以下であるものがより好ましく、1.1倍以下であるものが特に好ましい。
一実施形態において、本発明は、(I)上述したカチオン性脂質と、(II)中性脂質、ポリエチレングリコール修飾脂質及びステロールからなる群より選択される少なくとも一種の脂質とを含有する極性有機溶媒含有水溶液と、(III)核酸を含有する水溶液とを混合して混合液を得る工程(a)と、混合液中の極性有機溶媒の含有率を減少させる工程(b)とを備える、組成物の製造方法を提供する。本実施形態の製造方法によれば、核酸を細胞質に効率よく放出することが可能な組成物を製造することができる。
本発明の化合物を合成するために利用される出発原料、試薬、酸、塩基、脱水剤、溶媒、および触媒はすべて、市販されているか、または当業者に公知の有機合成法によって生成することができる。さらに、本発明の化合物は、以下の実施例で示されている通り、当業者に公知の有機合成法により製造することができる。
DIPEA:N,N-ジイソプロピルエチルアミン
DMAP:4-(ジメチルアミノ)ピリジン
DMF:N,N-ジメチルホルムアミド
EDC:1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩
n-:ノルマル
tert-:ターシャリー
EtOAc:酢酸エチル
TFA:トリフルオロ酢酸
THF:テトラヒドロフラン
1H-NMR:プロトン核磁気共鳴スペクトルメトリー
s:シングレット、d:ダブレット、t:トリプレット、q:カルテット、quin:クインテット、m:マルチプレット、br:ブロード。
クロマトグラフィーに関しては、YAMAZEN社製パラレルプレップ{カラム:YAMAZEN社製 Hi-FlashTM Column(Silicagel)、サイズ;S(16×60mm)、M(20×75mm)、L(26×100mm)、2L(26×150mm)}、あるいはBiotage社製フラッシュ自動精製システムIsoleraTM{カラム:SNAP Cartridge KP-Sil(10g、25g、50g、100g、340g)}を用いた。
1H-NMR(400MHz,CDCl3)δ(ppm):0.82-0.95(m,6H),1.17-1.49(m,24H),1.52-1.70(m,3H),1.75-1.93(m,2H),2.23-2.36(m,2H),3.34-3.47(m,2H),3.91-4.02(m,2H).
1H-NMR(400MHz,CDCl3)δ(ppm):1.22-1.48(m,8H),1.58-1.71(m,2H),1.78-1.90(m,2H),2.30-2.40(m,2H),3.35-3.44(m,2H),5.12(s,2H),7.26-7.45(m,5H).
1H-NMR(400MHz,CDCl3)δ(ppm):1.22-1.48(m,6H),1.58-1.71(m,2H),1.78-1.90(m,2H),2.30-2.38(m,2H),3.33-3.45(m,2H),5.12(s,2H),7.28-7.44(m,5H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):1.22-1.59(m,8H),2.12-2.25(m,2H),3.47-3.62(m,2H),7.19-7.28(m,2H),7.34-7.44(m,2H),7.71-7.85(m,10H),7.85-7.94(m,3H),11.97(br s,1H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):0.80-0.89(m,6H),1.16-1.38(m,18H),1.43-1.53(m,2H),1.89-1.99(m,4H),2.17(t,J=7.43Hz,2H),5.07(t,J=6.90Hz,1H),11.83(br s,1H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):0.85(t,J=7.1Hz,6H),1.14-1.32(m,25H),1.42-1.52(m,2H),2.18(t,J=7.43Hz,2H).
1H-NMR(600MHz,CDCl3)δ(ppm):0.88(t,J=7.24Hz,6H),1.13-1.41(m,28H),1.54-1.60(m,2H),3.59-3.68(m,2H).
1H-NMR(600MHz,CDCl3)δ(ppm):0.08(s,6H),0.91(s,9H),2.05(quin,J=6.05Hz,2H),3.53(t,J=6.42Hz,2H),3.75(t,J=5.69Hz,2H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):0.01(s,6H),0.84(s,9H),1.65-1.75(m,2H),3.48-3.57(m,2H),3.69(t,J=6.14Hz,2H),7.73-7.84(m,12H),7.87-7.93(m,3H).
1H-NMR(600MHz,CDCl3)δ(ppm):0.07(s,6H),0.90(m,15H),1.21-1.41(m,28H),1.92-2.04(m,4H),2.24(q,J=7.34Hz,2H),3.58(t,J=7.24 Hz,2H),5.09(t,J=7.15Hz,1H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):0.80-0.90(m,6H),1.13-1.30(m,35H),1.32-1.39(m,2H),3.32-3.38(m,2H),4.30(t,J=5.23Hz,1H).
1H-NMR(600Hz,CDCl3)δ(ppm):0.07(s,6H),0.84-0.95(m,15H),0.89(s,1H),1.21-1.41(m,20H),1.92-2.05(m,4H),2.24(q,J=7.34Hz,2H),3.58(t,J=7.24Hz,2H),5.09(t,J=7.24Hz,1H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):0.82-0.89(m,6H),1.14-1.31(m,27H),1.33-1.39(m,2H),3.32-3.38(m,2H),4.30(t,J=5.14 Hz,1H).
1H-NMR(600Hz,CDCl3)δ(ppm):0.07(s,6H),0.90(m,15H),1.19-1.43(m,13H),1.92-2.05(m,4H),2.21-2.29(m,2H),3.58(t,J=7.24Hz,2H),5.10(t,J=7.15Hz,1H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):0.87-0.93(m,6H),1.18-1.36(m,19H),1.37-1.45(m,2H),3.36-3.42(m,2H),4.35(t,J=5.23Hz,1H).
ベンジル 4-ブロモブタノエートの合成
1H-NMR(600MHz,CDCl3)δ(ppm):2.20(quin,J=6.83Hz,2H),2.56(t,J=7.15 Hz,2H),3.46(t,J=6.51Hz,2H),5.13(s,2H),7.28-7.42(m,5H).
ベンジル 6-ブロモヘキサノエートの合成
1H-NMR(600MHz,CDCl3)δ(ppm):1.44-1.51(m,2H),1.64-1.72(m,2H),1.83-1.90(m,2H),2.38(t,J=7.43 Hz,2H),3.39(t,J=6.69 Hz,2H),5.12(s,2H),7.30-7.40(m,5H).
(1R,5S,6r)-エチル 3-アザビシクロ[3.1.0]ヘキサン-6-カルボキシレート(CAS 174456-77-0)(0.89g,5.74mmol)をTHF(20mL)に溶解し、酢酸(0.49mL,8.6mmol)、ホルムアルデヒド溶液(11.7mL,161.5mol)を室温で順に加え、30分間撹拌した。ナトリウムトリ(アセトキシ)ボロヒドリド(2.43g,11.5mmol)を加え、室温で3時間撹拌した。反応混合物を、減圧下、濃縮したのち、飽和炭酸水素ナトリウム水溶液を加え、塩化メチレンで抽出した。無水硫酸ナトリウムで乾燥した。濾過後、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(塩化メチレン/メタノール)にて精製し、標記化合物(0.70g,4.14mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):1.25(t,J=7.2Hz,3H),1.94(br s,2H),2.01(br s,1H),2.29(s,3H),2.35(d,J=9.2Hz,2H),3.05(d,J=9.2Hz,2H),4.09(q,J=7.2Hz,2H).
製造例10-(1)で得た化合物(0.60g,3.55mmol)に濃塩酸(10mL)を加え、70℃で12時間撹拌した。反応混合物を、減圧下、濃縮し、標記化合物(0.52g,3.05mmol)を得た。
1H-NMR(400MHz,DMSO-d6)δ(ppm):2.14(br s,2H),2.25(br s,1H),2.74(br s,3H),3.25-3.37(m,2H),3.56-3.67(m,2H)、10.91(br s,1H),12.49(br s,1H).
製造例10-(1)の方法に準じ、(1R,5S,6s)-エチル 3-アザビシクロ[3.1.0]ヘキサン-6-カルボキシレート(CAS 1144099-54-6)(2.5g,16.1mmol)、酢酸(1.4mL,24.2mmol)、ホルムアルデヒド溶液(10.0mL,134.3mmol)、ナトリウムトリ(アセトキシ)ボロヒドリド(6.8g,32.2mmol)、およびTHF(50mL)から、標記化合物(2.5g,14.8mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):1.23(t,J=7.1Hz,3H),1.48-1.68(m,3H),2.22(s,3H),2.33(br d,J=8.8Hz,2H),3.02(d,J=8.8Hz,2H),4.10(q,J=7.1Hz,2H).
製造例10-(2)の方法に準じ、製造例10-(2)で得た化合物(500mg,2.96mmol)と濃塩酸(10mL)とから、標記化合物(350mg,1.97mmol)を得た。
1H-NMR(400MHz,DMSO-d6)δ(ppm):1.88-1.94(m,0.45H),1.94-2.00(m,0.55H),2.35-2.45(m,2H),2.68(br s,1.65H),2.67(br s,1.35H),3.08-3.18(m,1.1H),3.45-3.57(m,0.9H)、3.73-3.88(m,2H),9.16(br s,1H),11.52(br s,1H).
60%水素化ナトリウム(59mg,1.48mmol)を1,4-ジオキサン(6.5mL)に懸濁させ、室温でジ-tert-ブチル マロネート(0.30mL,1.35mmol)をゆっくり加え、20分間撹拌した。製造例1で得た化合物(573mg,1.41mmol)を1,4-ジオキサン(1mL)を使って加え、70℃で3時間、95℃で15時間撹拌した。反応混合物を氷水バスで冷却し、飽和塩化アンモニウム水溶液を加え、n-ヘプタンで抽出した。有機相を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した。濾過後、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(n-ヘプタン/酢酸エチル)にて精製し、標記化合物(490mg,0.91mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.80-0.98(m,6H),1.18-1.38(m,24H),1.39-1.54(m,2H),1.46(s,18H),1.55-1.69(m,3H),1.71-1.87(m,2H),2.24-2.35(m,2H),3.06-3.15(m,1H),3.91-4.02(m,2H).
実施例A-1-(1)で得られた化合物(490mg,0.91mmol)を1,4-ジオキサン(4mL)に溶解し、水冷下、60%水素化ナトリウム(47mg,1.18mmol)を加え、室温で5分間撹拌した。1-ヨードデカン(0.39mL,1.81mmol)を加え、80℃で15時間撹拌した。反応混合物を氷水バスで冷却し、飽和塩化アンモニウム水溶液を加え、n-ヘプタンで抽出した。有機相を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した。濾過後、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(n-ヘプタン/酢酸エチル)にて精製し、標記化合物(450mg,0.66mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.80-0.96(m,9H),1.05-1.36(m,40H),1.39-1.49(m,2H),1.44(s,18H),1.53-1.68(m,3H),1.70-1.82(m,4H),2.23-2.34(m,2H),3.92-4.01(m,2H).
実施例A-1-(2)で得られた化合物(450mg,0.66mmol)を塩化メチレン(2mL)に溶解し、氷冷下、TFA(1mL)を加え、室温で1.5時間撹拌した。反応混合物にトルエンを加え、減圧下、溶媒を留去した。トルエンの添加および留去を2回繰り返すことにより乾燥し、2-{9-[(2-ブチルオクチル)オキシ]-9-オキソノニル}-2-デシルマロン酸の粗生成物を得た。得られた粗生成物をキシレン(5mL)に溶解し、150℃で8時間撹拌した。反応混合物を室温に戻し、減圧下濃縮した。得られた残渣をカラムクロマトグラフィー(n-ヘプタン/酢酸エチル)にて精製し、標記化合物(240mg,0.46mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.80-0.97(m,9H),1.16-1.70(m,49H),2.22-2.41(m,3H),3.92-4.02(m,2H).
実施例A-1-(3)で得た化合物(240mg,0.46mmol)をTHF(4mL)に溶解し、-15℃で0.92Mボラン-THF錯体(0.75mL,0.69mmol)を滴下し、0℃で2時間撹拌した。飽和炭酸水素ナトリウム水溶液を加え、室温で5分間撹拌し、酢酸エチルで抽出した。有機相を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した。濾過後、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(n-ヘプタン/酢酸エチル)にて精製し、標記化合物(197mg,0.39mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.80-0.98(m,9H),1.10-1.39(m,46H),1.39-1.50(m,1H),1.54-1.69(m,4H),2.23-2.36(m,2H),3.48-3.59(m,2H),3.92-4.02(m,2H).
実施例A-1-(4)で得た化合物(38mg,0.074mmol)、DIPEA(0.054mL,0.31mmol)、1-メチル-ピペリジン-4-カルボン酸 塩酸塩(27mg,0.15mmol)およびDMAP(1.8mg,0.015mmol)を塩化メチレン(0.8mL)に溶解し、氷冷下、EDC(31mg,0.16mmol)を加え、室温で15時間撹拌した。反応混合物に飽和炭酸水素ナトリウム水溶液を加え、クロロホルムで抽出した。有機相を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した。濾過後、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(n-ヘプタン/酢酸エチル/メタノール)にて精製し、標記化合物(38mg,0.060mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.77-0.97(m,9H),1.13-1.41(m,46H),1.50-1.69(m,4H),1.69-1.85(m,2H),1.85-2.09(m,4H),2.18-2.35(m,3H),2.27(s,3H),2.72-2.88(m,2H),3.90-4.03(m,4H).
60%水素化ナトリウム(83mg,2.07mmol)を1,4-ジオキサン(9.4mL)に懸濁させ、室温でジ-tert-ブチル マロネート(0.42mL,1.88mmol)をゆっくり加え、10分間撹拌した。製造例2で得た化合物(650mg,1.99mmol)を加え、95℃で13時間撹拌した。反応混合物を氷水バスで冷却し、飽和塩化アンモニウム水溶液を加え、n-ヘプタンで抽出した。有機相を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した。濾過後、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(n-ヘプタン/酢酸エチル)にて精製し、標記化合物(536mg,1.16mmol)を得た。
実施例A-1-(2)の方法に準じ、実施例A-2-(1)で得られた化合物(590mg,1.28mmol)、1-ヨードデカン(0.54mL,2.55mmol)、65%水素化ナトリウム(71mg,1.91mmol)および1,4-ジオキサン(5mL)から標記化合物(470mg,0.78mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.83-0.93(m,3H),1.05-1.36(m,26H),1.44(s,18H),1.57-1.68(m,2H),1.71-1.82(m,4H),2.29-2.39(m,2H),5.11(s,2H),7.28-7.42(m,5H).
実施例A-1-(3)の方法に準じ、実施例A-2-(2)で得られた化合物(470mg,0.78mmol)、塩化メチレン(2mL)、TFA(1mL)、およびキシレン(2mL)から標記化合物(286mg,0.64mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.81-0.95(m,3H),1.18-1.37(m,26H),1.39-1.52(m,2H),1.54-1.69(m,4H),2.28-2.41(m,3H),5.11(s,2H),7.28-7.40(m,5H).
実施例A-1-(4)の方法に準じ、実施例A-2-(3)で得た化合物(285mg,0.64mmol)、0.92Mボラン-THF錯体(1.0mL,0.96mmol)、およびTHF(3.2mL)から標記化合物(223mg,0.52mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.81-0.95(m,3H),1.12-1.38(m,31H),1.39-1.50(m,1H),1.58-1.72(m,2H),2.23-2.36(m,2H),3.48-3.59(m,2H),5.11(s,2H),7.28-7.41(m,5H).
実施例A-1-(5)の方法に準じ、実施例A-2-(4)で得た化合物(114mg,0.26mmol)、1-メチル-ピペリジン-4-カルボン酸 塩酸塩(95mg,0.53mmol)、EDC(111mg,0.58mmol)、DIPEA(0.090mL,0.53mmol)、DMAP(6.4mg,0.053mmol)および塩化メチレン(1.3mL)から標記化合物(128mg,0.23mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.81-0.96(m,3H),1.15-1.41(m,31H),1.52-1.70(m,2H),1.70-1.84(m,2H),1.85-2.06(m,4H),2.18-2.40(m,3H),2.27(s,3H),2.73-2.89(m,2H),3.92-4.03(m,2H),5.12(s,2H),7.28-7.44(m,5H).
実施例A-2-(5)で得た化合物(127mg,0.23mmol)を酢酸エチル(2mL)に溶解し、室温で10%パラジウム-炭素(24mg,50%含水)を加え、水素雰囲気下、常圧で3時間撹拌した。反応液を濾過し、酢酸エチルで洗浄した。濾液を減圧下濃縮し、得られた残渣をカラムクロマトグラフィー(クロロホルム/メタノール)にて精製し、標記化合物(94mg,0.20mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.81-0.95(m,3H),1.12-1.44(m,31H),1.53-1.71(m,2H),1.74-1.95(m,2H),1.97-2.10(m,2H),2.11-2.37(m,5H),2.40(s,3H),3.11-3.28(m,2H),3.79-3.92(m,1H),4.14-4.25(m,1H).
製造例2の方法に準じ、実施例A-2-(6)で得た化合物(93mg,0.20mmol)、3-ペンチルオクタン-1-オール(CAS 1443519-63-8)(60mg,0.30mmol)、EDC(42mg,0.22mmol)、DMAP(4.9mg,0.040mmol)および塩化メチレン(1.5mL)から標記化合物(103mg,0.16mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.82-0.95(m,9H),1.14-1.46(m,46H),1.50-1.68(m,6H),1.70-1.84(m,2H),1.85-2.06(m,4H),2.19-2.33(m,3H),2.27(s,3H),2.74-2.87(m,2H),3.93-4.02(m,2H),4.03-4.14(m,2H).
実施例A-2-(1)で得られた化合物(536mg,1.16mmol)をTHF(6mL)に溶解し、水冷下、60%水素化ナトリウム(58mg,1.45mmol)を加え、室温で5分間撹拌した。1-ヨードノナン(0.23mL,1.16mmol)を加え、80℃で20時間撹拌した。反応混合物を氷水バスで冷却し、飽和塩化アンモニウム水溶液を加え、n-ヘプタンで抽出した。有機相を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した。濾過後、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(n-ヘプタン/酢酸エチル)にて精製し、標記化合物(340mg,0.577mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.82-0.92(m,3H),1.03-1.36(m,24H),1.44(s,18H),1.57-1.68(m,2H),1.71-1.81(m,4H),2.28-2.40(m,2H),5.11(s,2H),7.28-7.41(m,5H).
実施例A-1-(3)の方法に準じ、実施例A-3-(1)で得られた化合物(340mg,0.58mmol)、塩化メチレン(2mL)、TFA(1mL)、およびキシレン(2mL)から標記化合物(96mg,0.22mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.80-0.95(m,3H),1.12-1.74(m,30H),2.28-2.41(m,3H),5.11(s,2H),7.28-7.40(m,5H).
実施例A-1-(4)の方法に準じ、実施例A-3-(2)で得た化合物(96mg,0.22mmol)、0.92Mボラン-THF錯体(0.36mL,0.33mmol)およびTHF(1.1mL)から標記化合物(80mg,0.19mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.81-0.94(m,3H),1.09-1.38(m,29H),1.38-1.50(m,1H),1.58-1.71(m,2H),2.30-2.40(m,2H),3.48-3.58(m,2H),5.11(s,2H),7.29-7.42(m,5H).
実施例A-1-(5)の方法に準じ、実施例A-3-(3)で得た化合物(80mg,0.19mmol)、1-メチル-ピペリジン-4-カルボン酸 塩酸塩(69mg,0.38mmol)、EDC(81mg,0.42mmol)、DIPEA(0.066mL,0.38mmol)、DMAP(4.7mg,0.038mmol)および塩化メチレン(1.0mL)から標記化合物(97mg,0.18mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.83-0.94(m,3H),1.15-1.39(m,29H),1.52-1.70(m,2H),1.70-1.85(m,2H),1.85-2.07(m,4H),2.19-2.40(m,3H),2.27(s,3H),2.73-2.88(m,2H),3.94-4.02(m,2H),5.12(s,2H),7.28-7.43(m,5H).
実施例A-2-(6)の方法に準じ、実施例A-3-(4)で得た化合物(96mg,0.18mmol)を酢酸エチル(2mL)に溶解し、室温で10%パラジウム-炭素(19mg,50%含水)を加え、水素雰囲気下、常圧で3時間撹拌した。反応液を濾過し、酢酸エチルで洗浄した。濾液を減圧下濃縮し、得られた残渣をカラムクロマトグラフィー(クロロホルム/メタノール)にて精製し、標記化合物(74mg,0.163mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.81-0.95(m,3H),1.12-1.44(m,29H),1.53-1.71(m,2H),1.74-1.94(m,2H),1.98-2.08(m,2H),2.08-2.38(m,5H),2.39(s,3H),3.10-3.30(m,2H),3.80-3.91(m,1H),4.14-4.25(m,1H).
製造例2の方法に準じ、実施例A-3-(5)で得た化合物(74mg,0.16mmol)、3-ペンチルオクタン-1-オール(CAS 1443519-63-8)(49mg,0.25mmol)、EDC(38mg,0.20mmol)、DMAP(4.0mg,0.033mmol)および塩化メチレン(1.2mL)から標記化合物(81mg,0.13mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.81-0.95(m,9H),1.15-1.47(m,44H),1.50-1.68(m,6H),1.70-1.84(m,2H),1.84-2.06(m,4H),2.18-2.33(m,3H),2.27(s,3H),2.73-2.87(m,2H),3.94-4.01(m,2H),4.04-4.12(m,2H).
60%水素化ナトリウム(59mg,1.48mmol)をDMF(5.4mL)に懸濁させ、氷冷下、ジ-tert-ブチル マロネート(0.30mL,1.35mmol)をゆっくり加え、0℃で5分間撹拌したのちバスを取り外して20分間撹拌した。氷冷下、ナトリウムヨージド(61mg,0.40mmol)、製造例3で得た化合物(443mg,1.41mmol)を加え、室温で15時間撹拌した。反応混合物を氷水バスで冷却し、水を加え、ジエチルエーテルで抽出した。有機相を飽和食塩水で洗浄し、無水硫酸マグネシウムで乾燥した。濾過後、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(n-ヘプタン/酢酸エチル)にて精製し、標記化合物(456mg,1.02mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):1.23-1.36(m,8H),1.45(s,18H),1.58-1.69(m,2H),1.72-1.84(m,2H),2.30-2.40(m,2H),3.05-3.15(m,1H),5.11(s,2H),7.28-7.41(m,5H).
実施例A-4-(1)の方法に準じ、実施例A-4-(1)で得られた化合物(456mg,1.02mmol)、製造例3で得た化合物(478mg,1.53mmol)、ナトリウムヨージド(46mg,0.31mmol)、60%水素化ナトリウム(49mg,1.22mmol)およびDMF(3.3mL)から標記化合物(380mg,0.56mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):1.03-1.17(m,4H),1.23-1.36(m,12H),1.43(s,18H),1.56-1.68(m,4H),1.70-1.80(m,4H),2.29-2.38(m,4H),5.11(s,4H),7.27-7.41(m,10H).
実施例A-1-(3)の方法に準じ、実施例A-4-(2)で得られた化合物(380mg,0.56mmol)、塩化メチレン(2mL)、TFA(1mL)、およびキシレン(2mL)から標記化合物(234mg,0.45mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):1.18-1.37(m,16H),1.38-1.71(m,8H),2.26-2.41(m,5H),5.11(s,4H),7.28-7.43(m,10H).
実施例A-1-(4)の方法に準じ、実施例A-4-(3)で得た化合物(234mg,0.45mmol)、0.92Mボラン-THF錯体(0.73mL,0.67mmol)とTHF(1.8mL)とから標記化合物(188mg,0.37mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):1.12-1.51(m,22H),1.56-1.72(m,4H),2.29-2.41(m,4H),3.47-3.58(m,2H),5.11(s,4H),7.28-7.44(m,10H).
実施例A-1-(5)の方法に準じ、実施例A-4-(4)で得た化合物(188mg,0.37mmol)、1-メチル-ピペリジン-4-カルボン酸 塩酸塩(132mg,0.74mmol)、EDC(155mg,0.81mmol)、DIPEA(0.126mL,0.74mmol)、DMAP(9.0mg,0.074mmol)および塩化メチレン(1.9mL)から標記化合物(217mg,0.34mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):1.16-1.38(m,21H),1.53-1.69(m,4H),1.70-1.84(m,2H),1.85-2.04(m,4H),2.19-2.40(m,5H),2.26(s,3H),2.74-2.86(m,2H),3.92-4.01(m,2H),5.11(s,4H),7.28-7.41(m,10H).
実施例A-4-(5)で得た化合物(217mg,0.34mmol)をTHF(4mL)およびメタノール(2mL)に溶解し、室温で10%パラジウム-炭素(19mg,50%含水)を加え、水素雰囲気下、常圧で2時間撹拌した。反応系を窒素で置換したのち反応液を濾過し、メタノールで洗浄した。濾液を減圧下濃縮し、9-{[(1-メチルピペリジン-4-カルボニル)オキシ]メチル}ヘプタデカン二酸の粗生成物(160mg)を得た。
製造例2の方法に準じ、得られた粗生成物(40mg)、3-ペンチルオクタン-1-オール(CAS 1443519-63-8)(44mg,0.22mmol)、EDC(37mg,0.19mmol)、DMAP(2.1mg,0.018mmol)およびTHF(1mL)から標記化合物(34mg,0.041mmol)を得た。
1H-NMR(400MHz,CDCl3)δ(ppm):0.83-0.94(m,12H),1.14-1.48(m,53H),1.51-1.67(m,10H),1.70-1.84(m,2H),1.85-2.07(m,4H),2.18-2.34(m,5H),2.27(s,3H),2.74-2.88(m,2H),3.93-4.01(m,2H),4.03-4.12(m,4H).
1H-NMR(400MHz,CDCl3)δ(ppm):0.81-0.96(m,6H),1.15-1.44(m,35H),1.52-1.68(m,6H),1.70-1.84(m,2H),1.85-2.05(m,4H),2.05-2.15(m,4H),2.21-2.35(m,5H),2.27(s,3H),2.74-2.88(m,2H),3.93-4.01(m,2H),4.57-4.67(m,4H),5.47-5.58(m,2H),5.59-5.70(m,2H).
[実施例A-6]
(1R,5S,6r)-2-{9-[(2-ブチルオクチル)オキシ]-9-オキソノニル}ドデシル 3-メチル-3-アザビシクロ[3.1.0]ヘキサン-6-カルボキシレート(カチオン性脂質6)
1H-NMR(400MHz,CDCl3)δ(ppm):0.80-1.00(m,9H),1.16-1.40(m,46H),1.52-1.70(m,4H),1.89-1.97(m,2H),1.98-2.06(m,1H),2.23-2.42(m,4H),2.30(s,3H),3.00-3.12(m,2H),3.87-4.03(m,4H).
[実施例A-7]
(1R,5S,6s)-2-{9-[(2-ブチルオクチル)オキシ]-9-オキソノニル}ドデシル 3-メチル-3-アザビシクロ[3.1.0]ヘキサン-6-カルボキシレート(カチオン性脂質7)
1H-NMR(400MHz,CDCl3)δ(ppm):0.81-0.97(m,9H),1.15-1.40(m,46H),1.46-1.70(m,7H),2.21(s,3H),2.24-2.38(m,4H),2.96-3.07(m,2H),3.85-4.02(m,4H).
1H-NMR(400MHz,CDCl3)δ(ppm):0.80-0.98(m,9H),1.16-1.42(m,46H),1.52-1.77(m,4H),2.23-2.44(m,6H),2.30(s,3H),3.42-3.57(m,4H),3.90-4.04(m,4H).
[実施例A-9]
2-[9-(ヘキシルオキシ)-9-オキソノニル]ドデシル 1-メチルピペリジン-4-カルボキシレート(カチオン性脂質9)
1H-NMR(600MHz,CDCl3)δ(ppm):0.85-0.92(m,6H),1.19-1.39(m,37H),1.57-1.66(m,5H),1.73-1.84(m,2H),1.87-1.94(m,2H),1.96-2.07(m,2H),2.22-2.32(m,6H),2.73-2.87(m,2H),3.98(d,J=5.50Hz,2H),4.06(t,J=6.79Hz,2H).
[実施例A-10]
2-[9-(オクチルオキシ)-9-オキソノニル]ドデシル 1-メチルピペリジン-4-カルボキシレート(カチオン性脂質10)
1H-NMR(600MHz,CDCl3)δ(ppm):0.91(t,J=6.97Hz,6H),1.23-1.40(m,40H),1.60-1.68(m,5H),1.76-1.85(m,2H),1.89-1.95(m,2H),1.97-2.06(m,2H),2.25-2.33(m,6H),2.78-2.86(m,2H),4.00(d,J=5.69 Hz,2H),4.08(t,J=6.79 Hz,2H).
[実施例A-11]
2-[9-(デシルオキシ) -9-オキソノニル]ドデシル 1-メチルピペリジン-4-カルボキシレート(カチオン性脂質11)
1H-NMR(600MHz,CDCl3)δ(ppm):0.91(t,J=7.06Hz,6H),1.17-1.42(m,44H),1.60-1.68(m,5H),1.75-1.86(m,2H),1.88-1.96(m,2H),1.97-2.07(m,2H),2.24-2.35(m,6H),2.78-2.87(m,2H),4.00(d,J=5.50Hz,2H),4.08(t,J=6.79 Hz,2H).
[実施例A-12]
2-{9-オキソ-9-[(4-ペンチルノニル)オキシ]ノニル}ドデシル 1-メチルピペリジン-4-カルボキシレート(カチオン性脂質12)
1H-NMR(600MHz,CDCl3)δ(ppm):0.85-0.92(m,9H),1.16-1.34(m,49H),1.51-1.66(m,5H),1.73-1.84(m,2H),1.86-1.94(m,2H),1.95-2.05(m,2H),2.23-2.31(m,6H),2.75-2.85(m,2H),3.98(d,J=5.50Hz,2H),4.04(t,J=6.79 Hz,2H).
1H-NMR(600MHz,CDCl3)δ(ppm):1.42-1.49(m,18H),1.65-1.72(m,2H),1.80-1.87(m,2H),2.39(t,J=7.52 Hz,2H),3.13(t,J=7.52Hz,1H),5.11(s,2H),7.29-7.40(m,5H).
1H-NMR(600MHz,CDCl3)δ(ppm):0.85-0.91(m,3H),1.07-1.17(m,2H),1.21-1.33(m,16H),1.44(s,18H),1.49-1.54(m,2H),1.74-1.84(m,4H),2.36(t,J=7.34 Hz,2H),5.11(s,2H),7.28-7.38(m,5H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):0.82-0.89(m,3H),1.17-1.31(m,16H),1.32-1.42(m,3H),1.43-1.57(m,4H),2.16-2.22(m,1H),2.32-2.38(m,2H),5.08(s,2H),7.29-7.40(m,5H),12.06(br s,1H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):0.85(t,J=6.88Hz,3H),1.08-1.37(m,18H),1.49-1.58(m,2H),2.27-2.36(m,2H),3.21-3.29(m,2H),4.28(t,J=5.14Hz,1H),5.01-5.12(m,2H),7.30-7.40(m,5H).
1H-NMR(600MHz,CDCl3)δ(ppm):0.88(t,J=7.06 Hz,3H),1.17-1.38(m,21H),1.60-1.70(m,3H),1.77-1.87(m,2H),1.89-1.99(m,2H),2.23-2.38(m,6H),2.74-2.91(m,2H),3.94-4.02(m,2H),5.09-5.13(m,2H),7.30-7.41(m,5H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):0.85(t,J=6.88 Hz,3H),1.21-1.32(m,20H),1.44-1.66(m,5H),1.75-1.83(m,2H),1.95-1.98(m,1H),1.99-2.06(m,1H),2.13-2.23(m,5H),2.23-2.30(m,1H),2.64-2.77(m,2H),3.88-3.97(m,2H).
1H-NMR(600MHz,CDCl3)δ(ppm):0.88(t,J=6.7Hz,6H),1.26(br s,41H),1.57-1.69(m,6H),1.73-1.82(m,2H),1.90(br dd,J=13.5,3.03Hz,2H),1.94-2.04(m,2H),2.22-2.34(m,6H),2.67-2.90(br d,J=9.8Hz,2H),3.95-4.02(m,2H),4.03-4.08(t,J=6.8Hz,2H).
[実施例A-14]
2-(4-オキソ-4-((8-ペンチルトリデシル)オキシ)ブチル)ドデシル 1-メチルピペリジン-4-カルボキシレート(カチオン性脂質14)
1H-NMR(600MHz,CD3OD)δ(ppm):0.87-0.97(m,9H),1.18-1.45(m,47H),1.60-1.73(m,5H),1.73-1.83 (m,2H),1.88-1.99(m,2H),2.06-2.19(m,2H),2.23-2.43(m,6H),2.77-2.89(m,2H),3.99-4.15(m,4H).
[実施例A-15]
2-{4-[(4-ノニルトリデシル)オキシ]-4-オキソブチル}ドデシル 1-メチルピペリジン-4-カルボキシレート(カチオン性脂質15)
1H-NMR(600MHz,CD3OD)δ(ppm):0.85-0.99(m,9H),1.15-1.44(m,55H),1.56-1.84(m,7H),1.94(m,J=13.2Hz,2H),2.06-2.19(m,2H),2.23-2.44(m,6H),2.76-2.90(m,2H),3.96-4.14(m,4H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):1.17-1.30(m,20H),1.43-1.52(m,4H),1.52-1.62(m,3H),1.77-1.82(m,2H),1.92-2.03(m,2H),2.12-2.22(m,7H),2.23-2.33(m,1H),2.66-2.75(m,2H),3.93(d,J=5.50Hz,2H).
1H-NMR(600MHz,CDCl3)δ(ppm):0.83-0.92(m,6H),1.21-1.39(m,40H),1.55-1.66(m,9H),1.72-1.83(m,2H),1.85-1.95(m,2H),1.95-2.06(m,2H),2.22-2.33(m,8H),2.75-2.87(m,2H),3.97(d,J=5.69Hz,2H),4.05(t,J=6.69Hz,4H).
[実施例A-17]
ビス(4-ペンチルノニル) 9-{[(1-メチルピペリジン-4-カルボニル)オキシ]メチル}ヘプタデカンジオエート(カチオン性脂質17)
1H-NMR(600MHz,CDCl3)δ(ppm):0.88(t,J=7.24Hz,12H),1.17-1.35(m,58H),1.58-1.65(m,9H),1.73-1.83(m,2H),1.87-1.93(m,2H),1.95-2.04(m,2H),2.23-2.32(m,8H),2.75-2.86(m,2H),3.97(d,J=5.50Hz,2H),4.04(t,J=6.79 Hz,4H).
1H-NMR(600MHz,CDCl3)δ(ppm):1.43(s,18H),1.47-1.54(m,4H),1.78-1.85(m,4H),2.36(t,J=7.34Hz,4H),5.10(s,4H),7.27-7.40(m,10H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):1.31-1.58(m,8H),2.19-2.25(m,1H),2.33-2.37(m,4H),5.05-5.11(m,4H),7.27-7.40(m,10H),12.14(br s,1H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):1.09-1.37(m,6H),1.48-1.56(m,4H),2.32(t,J=7.34Hz,4H),3.26(t,J=5.23Hz,2H),4.32(t,J=5.14Hz,1H),5.08(s,4H),7.28-7.41(m,10H).
1H-NMR(600MHz,CDCl3)δ(ppm):1.27-1.38(m,4H),1.59-1.69(m,5H),1.70-1.81(m,2H),1.85-1.92(m,2H),1.92-2.02(m,2H),2.26(s,4H),2.33(t,J=7.34Hz,4H),2.74-2.83(m,2H),3.97(d,J=5.69Hz,2H),5.11(s,4H),7.28-7.39(m,10H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):1.20-1.31(m,4H),1.44-1.66(m,7H),1.72-1.81(m,4H),1.88-1.97(m,2H),2.13(s,3H),2.16-2.22(m,4 H),2.22-2.30(m,1H),2.64-2.71(m,2H),3.93(d,J=5.32Hz,2H).
1H-NMR(600MHz,CDCl3)δ(ppm):0.88(t,J=7.06Hz,6H),1.20-1.41(m、45H),1.58-1.71(m,9H),1.71-1.82(m,2H),1.86-1.94(m,2H),1.95-2.04(m,2H),2.23-2.30(m,8H),2.77-2.83(m,2H),4.00(d,J=5.5Hz,2H),4.05(t,J=6.88Hz,4H).
[実施例A-19]
ビス(8-ペンチルトリデシル) 5-{[(1-メチルピペリジン-4-カルボニル)オキシ]メチル}ノナンジオエート(カチオン性脂質19)
1H-NMR(600MHz,CDCl3)δ(ppm):0.88(t,J=7.24Hz,12H),1.15-1.40(m,60H),1.59-1.71(m,10H),1.72-1.82(m,2H),1.86-1.94(m,2H),1.94-2.04(m,2H),2.23-2.32(m,8H),2.77-2.85(m,2H),4.00(d,J=5.5Hz,2H),4.05(t,J=6.88Hz,4H).
[実施例A-20]
ビス(4-ノニルトリデシル) 5-{[(1-メチルピペリジン-4-カルボニル)オキシ]メチル}ノナンジオエート(カチオン性脂質20)
1H-NMR(600MHz,CDCl3)δ(ppm):0.88(t,J=6.97 Hz,12H),1.17-1.41(m,77H),1.57-1.72(m,9H),1.72-1.81(m,2H),1.85-1.94(m,2H),1.94-2.03(m,2H),2.23-2.31(m,8H),2.80(m,J=10.50Hz,2H),4.00(d,J=5.50Hz,2H),4.03(t,J=6.88 Hz,4H).
1H-NMR(600MHz,CDCl3)δ(ppm):1.27-1.38(m,4H),1.45(s,18H),1.62-1.68(m,2H),1.73-1.82(m,2H),2.35(t,J=7.52Hz,2H),3.09(t,J=7.61Hz,1H),5.11(s,2H),7.29-7.40(m,5H).
1H-NMR(600MHz,CDCl3)δ(ppm):1.09-1.19(m,4H),1.28-1.36(m,5H),1.43(s,18H),1.60-1.69(m,4H),1.72-1.80(m,4H),2.34(t,J=7.52Hz,4H),5.05-5.15(m,4H),7.28-7.41(m,9H).
1H-NMR(600MHz,DMSO-d6)δ(ppm):1.15-1.57(m,16H),2.11-2.20(m,1H),2.33(t,J=7.34Hz,4H),5.08(s,4H),7.23-7.43(m,10H),12.01(br s,1H).
1H-NMR(600MHz,CDCl3)δ(ppm):1.11-1.36(m,13H),1.39-1.46(m,1H),1.60-1.70(m,4H),2.35(t,J=7.52 Hz,4H),3.51(d,J=5.32Hz,2H),5.11(s,4H),7.29-7.40(m,10H).
1H-NMR(600MHz,CDCl3)δ(ppm):1.22-1.35(m,12H),1.61-1.68(m,4H),1.70-1.84(m,3H),1.85-1.93(m,2H),1.95-2.06(m,2H),2.21-2.30(m,4H),2.35(t,J=7.52Hz,4H),2.74-2.85 (m,2H),3.96(d,J=5.50Hz,2H),5.11(s,4H),7.29-7.39 (m,10H).
1H-NMR (600MHz,DMSO-d6)δ(ppm):1.19-1.30(m,12H),1.44-1.53(m,4H),1.53-1.63(m,3H),1.72-1.81(m,2H),1.90-1.98(m,2H),2.14(s,3H),2.18(t,J=7.43 Hz,4H),2.23-2.31(m,1H),2.64-2.73(m,2H),3.93(d,J=5.50Hz,2H).
1H-NMR(600MHz,CDCl3)δ(ppm):0.88(t,J=7.06Hz,6H),1.19-1.39(m,44H),1.57-1.68(m,9H)1.71-1.86(m,2H),1.84-1.95(m,2H),1.95-2.06(m,2H),2.21-2.34(m,8H),2.75-2.87(m,2H),3.97(d,J=5.69Hz,2H),4.05(t,J=6.79Hz,4H).
[実施例A-22]
ビス(4-ヘプチルウンデシル) 7-{[(1-メチルピペリジン-4-カルボニル)オキシ]メチル}トリデカンジオエート(カチオン性脂質22)
1H-NMR(600MHz,CDCl3)δ(ppm):0.88(t,J=6.97Hz,12H),1.11-1.36(m,66H),1.49-1.67(m,9H),1.71-1.84(m,2H),1.84-1.95(m,2H),1.95-2.05(m,2H),2.21-2.33(m,8H),2.74-2.88(m,2H),3.97(d,J=5.50Hz,2H),4.04(t,J=6.88Hz,4H).
<組成物の調製(1)>
[実施例B-1]
実施例A-1のカチオン性脂質1を用いて組成物を調製した。核酸としては、センス鎖5’-GGAfUfCAfUfCfUfCAAGfUfCfUfUAfCT*T-3’(T:DNA、fU,fC=2’-Fluoro RNA、*=Phosphorothioate linkage)(配列番号1)、アンチセンス鎖5’-GfUAAGAfCfUfUGAGAfUGAfUfCfCT*T-3’(T:DNA、fU,fC=2’-Fluoro RNA、*=Phosphorothioate linkage)(配列番号2)の塩基配列からなる、Factor VII(血液凝固第VII因子)遺伝子の発現を抑制するsiRNAであり、アニーリング済みのもの(株式会社ジーンデザイン、以下「Factor VII siRNA」という場合がある。)を使用した。
カチオン性脂質として、カチオン性脂質1の代わりに実施例A-2のカチオン性脂質2を用いた以外は実施例B-1と同様にして、実施例A-7の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに実施例A-3のカチオン性脂質3を用いた以外は実施例B-1と同様にして、実施例B-3の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに実施例A-4のカチオン性脂質4を用いた以外は実施例B-1と同様にして、実施例B-4の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに実施例A-5のカチオン性脂質5を用いた以外は実施例B-1と同様にして、実施例B-5の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに実施例A-6のカチオン性脂質6を用いた以外は実施例B-1と同様にして、実施例B-6の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに実施例A-7のカチオン性脂質7を用いた以外は実施例B-1と同様にして、実施例B-7の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに実施例A-8のカチオン性脂質8を用いた以外は実施例B-1と同様にして、実施例B-8の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに、特許文献2に記載された下記式(12)で表されるジ((Z)-ノン-2-エン-1-イル)9-((4-(ジメチルアミノ)ブタノイル)オキシ)ヘプタデカンジオエート(以下、「ALN-319」という場合がある。)を、特許文献2に記載された方法にしたがって合成して用いた以外は実施例B-1と同様にして、比較例B-1の組成物を得た。
実施例B-1~実施例B-8及び比較例B-1の組成物について、脂質複合体へのsiRNAの封入率を測定した。
封入率(%)=100-(A/B)×100 (F1)
[実施例B-9]
実施例A-1のカチオン性脂質1を用いて組成物を調製した。核酸としては、Firefly Luciferase (FLuc)のmRNA(TriLink Biotechnologies、以下「FLuc mRNA」という場合がある。)を使用した。
カチオン性脂質として、カチオン性脂質1の代わりに実施例A-2のカチオン性脂質2を用いた以外は実施例B-9と同様にして、実施例B-10の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに、上述したALN-319を用いた以外は実施例B-9と同様にして、比較例B-2の組成物を得た。
組成物の解析(1)と同様にして、実施例B-9、実施例B-10及び比較例B-2の組成物について、脂質複合体へのmRNAの封入率、及び脂質複合体の平均粒子径を測定した。表2に、mRNAの封入率及び脂質複合体の平均粒子径(Z-平均)を示す。
[実施例B-11]
実施例A-2のカチオン性脂質2を用いて組成物を調製した。カチオン性脂質として、カチオン性脂質1の代わりに、カチオン性脂質2を用い、核酸として、FLuc mRNAの代わりに、Human Erythropoietin (hEPO)のmRNA(TriLink Biotechnologies、以下「EPO mRNA」という場合がある。)を使用した以外は実施例B-9と同様にして、実施例B-11の組成物を得た。
カチオン性脂質として、カチオン性脂質2の代わりに、上述したALN-319を用いた以外は実施例B-11と同様にして、比較例B-3の組成物を得た。
組成物の解析(1)と同様にして、実施例B-11および比較例B-3の組成物について、脂質複合体へのmRNAの封入率、及び脂質複合体の平均粒子径を測定した。表3に、mRNAの封入率及び脂質複合体の平均粒子径(Z-平均)を示す。
[実施例B-12]
組成物の調製(1)と同様にして、実施例A-1のカチオン性脂質1を用いて、Factor VII siRNAを含む、実施例B-12の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに、実施例A-2のカチオン性脂質2を用いた以外は実施例B-12と同様にして、実施例B-13の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに、上述したALN-319を用いた以外は実施例B-12と同様にして、比較例B-4の組成物を得た。
組成物の解析(1)と同様にして、実施例B-12、実施例B-13および比較例B-4の組成物について、脂質複合体の平均粒子径(保管前平均粒子径)を測定した。さらに、各組成物を密閉したバイアルにて4℃で3ヶ月間保管し、脂質複合体の平均粒子径(保管後平均粒子径)を測定した。表4に、脂質複合体の平均粒子径(Z-平均)の変化を示す。表中、平均粒子径の変化(%)は、保管後平均粒子径/保管前平均粒子径×100で算出した。
[実施例B-14]
組成物の調製(1)と同様にして、実施例A-9のカチオン性脂質9を用いて、Factor VII siRNAを含む、実施例B-14の組成物を得た。
カチオン性脂質として、カチオン性脂質9の代わりに、実施例A-10のカチオン性脂質10を用いた以外は実施例B-14と同様にして、実施例B-15の組成物を得た。
カチオン性脂質として、カチオン性脂質9の代わりに、実施例A-11のカチオン性脂質11を用いた以外は実施例B-14と同様にして、実施例B-16の組成物を得た。
カチオン性脂質として、カチオン性脂質9の代わりに、実施例A-12のカチオン性脂質12を用いた以外は実施例B-14と同様にして、実施例B-17の組成物を得た。
カチオン性脂質として、カチオン性脂質9の代わりに、実施例A-13のカチオン性脂質13を用いた以外は実施例B-14と同様にして、実施例B-18の組成物を得た。
カチオン性脂質として、カチオン性脂質9の代わりに、実施例A-14のカチオン性脂質14を用いた以外は実施例B-14と同様にして、実施例B-19の組成物を得た。
カチオン性脂質として、カチオン性脂質9の代わりに、実施例A-15のカチオン性脂質15を用いた以外は実施例B-14と同様にして、実施例B-20の組成物を得た。
カチオン性脂質として、カチオン性脂質9の代わりに、実施例A-2のカチオン性脂質2を用いた以外は実施例B-14と同様にして、実施例B-21の組成物を得た。
組成物の解析(1)と同様にして、実施例B-14~実施例B-21の組成物について、脂質複合体へのsiRNAの封入率、及び脂質複合体の平均粒子径を測定した。表5に、siRNAの封入率ならびに脂質複合体の平均粒子径(Z-平均)および多分散指数を示す。
[実施例B-22]
組成物の調製(1)と同様にして、実施例A-16のカチオン性脂質16を用いて、Factor VII siRNAを含む、実施例B-22の組成物を得た。
カチオン性脂質として、カチオン性脂質16の代わりに、実施例A-17のカチオン性脂質17を用いた以外は実施例B-22と同様にして、実施例B-23の組成物を得た。
カチオン性脂質として、カチオン性脂質16の代わりに、実施例A-18のカチオン性脂質18を用いた以外は実施例B-22と同様にして、実施例B-24の組成物を得た。
カチオン性脂質として、カチオン性脂質16の代わりに、実施例A-19のカチオン性脂質19を用いた以外は実施例B-22と同様にして、実施例B-25の組成物を得た。
カチオン性脂質として、カチオン性脂質16の代わりに、実施例A-20のカチオン性脂質20を用いた以外は実施例B-22と同様にして、実施例B-26の組成物を得た。
カチオン性脂質として、カチオン性脂質16の代わりに、実施例A-21のカチオン性脂質21を用いた以外は実施例B-22と同様にして、実施例B-27の組成物を得た。
カチオン性脂質として、カチオン性脂質16の代わりに、実施例A-22のカチオン性脂質22を用いた以外は実施例B-22と同様にして、実施例B-28の組成物を得た。
カチオン性脂質として、カチオン性脂質16の代わりに、実施例A-2のカチオン性脂質2を用いた以外は実施例B-22と同様にして、実施例B-29の組成物を得た。
<組成物の解析(6)>
組成物の解析(1)と同様にして、実施例B-22~実施例B-29の組成物について、脂質複合体へのsiRNAの封入率、及び脂質複合体の平均粒子径を測定した。表6に、siRNAの封入率ならびに脂質複合体の平均粒子径(Z-平均)および多分散指数を示す。
[実施例B-30]
組成物の調製(1)と同様にして、実施例A-1のカチオン性脂質1を用いて、Factor VII siRNAを含む、実施例B-30の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに、実施例A-2のカチオン性脂質2を用いた以外は実施例B-30と同様にして、実施例B-30の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに、実施例A-3のカチオン性脂質3を用いた以外は実施例B-30と同様にして、実施例B-32の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに、実施例A-4のカチオン性脂質4を用いた以外は実施例B-30と同様にして、実施例B-33の組成物を得た。
カチオン性脂質として、カチオン性脂質1の代わりに、上述したALN-319を用いた以外は実施例B-30と同様にして、比較例B-5の組成物を得た。
組成物の解析(1)と同様にして、実施例B-30~実施例B-33及び比較例B-5の組成物について、脂質複合体の平均粒子径(保管前平均粒子径)を測定した。さらに、各組成物を密閉したバイアルに4℃で保管し、3ヶ月後および6ヶ月後の脂質複合体の平均粒子径(保管後粒子径)を測定した。表7に、脂質複合体の平均粒子径(Z-平均)の変化を示す。表中、平均粒子径の変化(%)は、保管後平均粒子径(6ヶ月後)/保管前平均粒子径×100で算出した。
[実施例B-34]
実施例A-2のカチオン性脂質2を用いて組成物を調製した。核酸としては、EPO mRNAを使用した。
核酸として、Factor VII siRNAの代わりにLuciferase siRNAを、カチオン性脂質として、カチオン性脂質1の代わりに、実施例A-2のカチオン性脂質2を用いた以外は組成物の調製(1)と同様にして、実施例B-35の組成物を得た。なお、Luciferase siRNAは、センス鎖5’-CUUACGCUGAGUACUUCGAT*T-3’(T:DNA、*=Phosphorothioate linkage)(配列番号3)、アンチセンス鎖5’-UCGAAGUACUCAGCGUAAGT*T-3’(T:DNA、*=Phosphorothioate linkage)(配列番号4)の塩基配列からなり、アニーリング済みのもの(株式会社ジーンデザイン)を使用した。
組成物の解析(1)と同様にして、実施例B-34および実施例B-35の組成物について、脂質複合体へのmRNAまたはsiRNAの封入率、及び脂質複合体の平均粒子径を測定した。表8に、mRNAまたはsiRNAの封入率及び脂質複合体の平均粒子径(Z-平均)を示す。
[試験例1]
実施例B-1~実施例B-3及び比較例B-1の組成物を、脂質複合体に封入されたFactor VII siRNA濃度が1μg/mL又は5μg/mLとなるようにPBSで希釈した。各組成物を、ICRマウス(5週齢、メス、平均体重25g、n=3)に10mL/kgの投与容量で尾静脈内投与し、投与から24時間後に麻酔下で採血及び肝臓の採取を実施した。遠心により血液から血漿を分離し、血漿中のFactor VIIタンパク質濃度を市販のキット(商品名「BIOPHEN FVII」、HYPHEN BioMed社)により定量した。陰性対照として、PBSを投与した群に同様の処理を行った。
実施例B-4及び比較例B-1の組成物を試験例1と同様にしてICRマウス(5週齢、メス、平均体重25g、n=3)に投与し、投与から24時間後の血漿中のFactor VIIタンパク質濃度の相対値を算出した。結果を図2および表10に示す。
実施例B-5の組成物を試験例1と同様にしてICRマウス(5週齢、メス、平均体重25g、n=3)に投与し、投与から24時間後の血漿中のFactor VIIタンパク質濃度の相対値を算出した。結果を表11に示す。
実施例B-11と比較例B-3の組成物を、脂質複合体に封入されたhEPO mRNA濃度が1μg/mL又は3μg/mLとなるようにPBSで希釈した。各組成物を、ICRマウス(5週齢、メス、平均体重25g、n=3)に10mL/kgの投与容量で尾静脈内投与し、投与から24時間後に麻酔下で採血を実施した。遠心により血液から血漿を分離し、血漿中のhEPOタンパク質濃度を市販のキット(商品名「Human Erythropoietin Quantikine IVD ELISA Kit」、R&D Systems)により定量した。陰性対照として、何も投与していない(無処理)群とPBSを投与した群に同様の処理を行った。結果を表12に示す。
実施例B-14~実施例B-21の組成物を、脂質複合体に封入されたFactor VII siRNA濃度が3μg/mL又は30μg/mLとなるようにPBSで希釈した。各組成物を、ICRマウス(5週齢、メス、平均体重25g、n=3)に10mL/kgの投与容量で尾静脈内投与し、投与から24時間後に麻酔下で採血及び肝臓の採取を実施した。遠心により血液から血漿を分離し、血漿中のFactor VIIタンパク質濃度を市販のキット(商品名「BIOPHEN FVII」、HYPHEN BioMed社)により定量した。陰性対照として、PBSを投与した群に同様の処理を行った。
実施例B-22~実施例B-29の組成物を試験例5と同様にしてICRマウス(5週齢、メス、平均体重25g、n=3)に投与し、投与から24時間後の血漿中のFactor VIIタンパク質濃度の相対値を算出した。結果を表14に示す。
EPO mRNAを封入した実施例B-34の組成物を、脂質複合体に封入されたRNA濃度が30μg/mLとなるようにPBSで希釈した。各組成物を、BALB/cマウス(メス、n=4)に、10mL/kgの投与容量で尾静脈内投与し、投与から1日及び4日後に麻酔下で採血を実施した。遠心により血液から血漿を分離し、血漿中のhEPOタンパク濃度を市販のキット(商品名「Human Erythropoietin Quantikine IVD ELISA Kit」、R&D Systems)により定量した。また、網状赤血球数を測定した。陰性対照として、何も投与していない(無処理)群とLuciferase siRNAを封入した実施例B-35の組成物を投与した群に同様の処理を行った。結果を表15に示す
Claims (15)
- (I)請求項1~12のいずれか一項に記載の化合物又はその薬学的に許容される塩と、(II)中性脂質、ポリエチレングリコール修飾脂質、及びステロールからなる群から選ばれる少なくとも一種の脂質と、を含有する脂質複合体。
- (I)請求項1~12のいずれか一項に記載の化合物又はその薬学的に許容される塩と、(II)中性脂質、ポリエチレングリコール修飾脂質、及びステロールからなる群から選ばれる少なくとも一種の脂質と、(III)核酸と、を含有する組成物。
- (I)請求項1~12のいずれか一項に記載の化合物又はその薬学的に許容される塩と、(II)中性脂質、ポリエチレングリコール修飾脂質、及びステロールからなる群から選ばれる少なくとも一種の脂質とを含有する極性有機溶媒含有水溶液と、(III)核酸を含有する水溶液とを混合して混合液を得る工程と、混合液中の極性有機溶媒の含有率を減少させる工程と、を含む組成物の製造方法。
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AU2017282459A AU2017282459B2 (en) | 2016-06-24 | 2017-06-22 | Cationic lipid |
BR112018075123A BR112018075123A2 (pt) | 2016-06-24 | 2017-06-22 | lipídio catiônico |
SG11201808928YA SG11201808928YA (en) | 2016-06-24 | 2017-06-22 | Cationic lipid |
ES17815482T ES2948971T3 (es) | 2016-06-24 | 2017-06-22 | Lípido catiónico |
MX2018015236A MX2018015236A (es) | 2016-06-24 | 2017-06-22 | Lipido cationico. |
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JP2021104994A (ja) * | 2019-12-26 | 2021-07-26 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | 脂質複合体を含む医薬組成物及び脂質ナノ粒子を含む医薬組成物 |
WO2022168884A1 (ja) | 2021-02-04 | 2022-08-11 | 塩野義製薬株式会社 | カチオン性脂質 |
RU2781954C1 (ru) * | 2018-10-26 | 2022-10-21 | Эйсай Ар Энд Ди Менеджмент Ко., Лтд. | Двухцепочечная рибонуклеиновая кислота, ингибирующая экспрессию компонента комплемента c5 |
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WO2023053016A1 (en) * | 2021-09-28 | 2023-04-06 | Seqirus Inc. | Ionizable cationic compound |
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- 2017-06-22 BR BR112018075123A patent/BR112018075123A2/pt active Search and Examination
- 2017-06-22 RU RU2018143237A patent/RU2740921C2/ru active
- 2017-06-22 CN CN201780035314.7A patent/CN109311809B/zh active Active
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WO2020085456A1 (ja) | 2018-10-26 | 2020-04-30 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | 補体c5の発現を抑制する二本鎖リボ核酸 |
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RU2781954C1 (ru) * | 2018-10-26 | 2022-10-21 | Эйсай Ар Энд Ди Менеджмент Ко., Лтд. | Двухцепочечная рибонуклеиновая кислота, ингибирующая экспрессию компонента комплемента c5 |
JP2021104994A (ja) * | 2019-12-26 | 2021-07-26 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | 脂質複合体を含む医薬組成物及び脂質ナノ粒子を含む医薬組成物 |
JP6961860B1 (ja) * | 2019-12-26 | 2021-11-05 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | 補体c5の発現を抑制する二本鎖リボ核酸を含む医薬組成物 |
WO2021132462A1 (ja) | 2019-12-26 | 2021-07-01 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | 補体c5の発現を抑制する二本鎖リボ核酸を含む医薬組成物 |
WO2022168884A1 (ja) | 2021-02-04 | 2022-08-11 | 塩野義製薬株式会社 | カチオン性脂質 |
Also Published As
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JPWO2017222016A1 (ja) | 2019-04-18 |
CA3020271A1 (en) | 2017-12-28 |
KR20190021218A (ko) | 2019-03-05 |
TW201803849A (zh) | 2018-02-01 |
BR112018075123A2 (pt) | 2019-06-18 |
US10501416B2 (en) | 2019-12-10 |
RU2740921C2 (ru) | 2021-01-21 |
RU2018143237A3 (ja) | 2020-07-24 |
ES2948971T3 (es) | 2023-09-22 |
RU2018143237A (ru) | 2020-07-24 |
US20190218180A1 (en) | 2019-07-18 |
KR102358341B1 (ko) | 2022-02-07 |
CN109311809B (zh) | 2022-04-08 |
MX2018015236A (es) | 2019-04-11 |
TWI745386B (zh) | 2021-11-11 |
EP3476832A4 (en) | 2020-02-19 |
SG11201808928YA (en) | 2018-11-29 |
EP3476832A1 (en) | 2019-05-01 |
AU2017282459B2 (en) | 2021-05-06 |
IL262574B (en) | 2021-10-31 |
AU2017282459A1 (en) | 2018-11-01 |
IL262574A (en) | 2018-12-31 |
CN109311809A (zh) | 2019-02-05 |
JP6883034B2 (ja) | 2021-06-02 |
EP3476832B1 (en) | 2023-04-19 |
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