WO2022158290A1 - Aminopolyester et nanoparticules lipidiques - Google Patents

Aminopolyester et nanoparticules lipidiques Download PDF

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WO2022158290A1
WO2022158290A1 PCT/JP2022/000164 JP2022000164W WO2022158290A1 WO 2022158290 A1 WO2022158290 A1 WO 2022158290A1 JP 2022000164 W JP2022000164 W JP 2022000164W WO 2022158290 A1 WO2022158290 A1 WO 2022158290A1
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group
integer
lipid nanoparticles
general formula
aminopolyester
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PCT/JP2022/000164
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Japanese (ja)
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敏文 佐藤
秀吉 原島
拓也 磯野
悠介 佐藤
マハモド マンソル アブドワキル
テンラク コウ
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国立大学法人北海道大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
    • C08G63/685—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/78—Preparation processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to lipid nanoparticles useful as gene delivery carriers that can be selectively delivered to lung tissue.
  • lipid nanoparticles have been used as carriers for encapsulating fat-soluble drugs and nucleic acids such as siRNA (short interfering RNA) or mRNA and delivering them to target cells.
  • lipid nanoparticles containing pH-sensitive cationic lipids as constituent lipids have been reported as lipid nanoparticles that serve as carriers for efficiently delivering nucleic acids such as siRNA into target cells (Patent Document 1).
  • Lipid nanoparticles have also been developed that can be incorporated into target cells via receptors by modifying the surfaces of the lipid nanoparticles with target ligands.
  • liver is easily accessible to systemically administered carriers due to its vascular structure and physiological properties. Therefore, there are many reports of liver-targeted gene delivery carriers. On the other hand, there are still few carriers that achieve gene expression specifically in organs other than the liver.
  • pulmonary diseases are diverse, including lung cancer, cystic fibrosis, pulmonary hypertension, and pulmonary fibrosis.
  • Lipid nanoparticles have been developed that can be incorporated into target cells via receptors by modifying the surface of the lipid nanoparticles with target ligands. Although there have been examples of the development of lung-specific delivery carriers so far, they are based on the existing idea of introducing artificial ligands. For example, the GALA peptide has been studied for its application as an endosomal escape element, but it has a high affinity for pulmonary vascular endothelial cells and is being studied as a ligand that targets pulmonary vessels. Lipid nanoparticles into which GALA peptide has been introduced serve as carriers that achieve gene expression specifically in lung tissue (Patent Document 2).
  • Non-Patent Document 1 Nanoparticles composed of aminopolyester (APE) are delivered to various organs such as the liver, lungs, and spleen.
  • APE aminopolyester
  • lipid nanoparticles modified with target ligands often migrate to the liver. Therefore, it is important to develop a carrier that selectively delivers to target tissues while suppressing translocation to the liver in order to realize systemic gene therapy.
  • the purpose of the present invention is to provide lipid nanoparticles that serve as gene delivery carriers that can be selectively delivered to the lung while suppressing translocation to the liver, and aminopolyesters that serve as constituent lipids of the lipid nanoparticles.
  • aminopolyester-containing lipid nanoparticles synthesized by ring-opening polymerization of ⁇ -decalactone are useful as gene delivery carriers that are highly expressed specifically in lung tissue, and have completed the present invention.
  • the present invention provides the following lipid nanoparticles and the like. [1] the following general formula (P-1)
  • R 11 to R 15 is an alkyl group having 1 to 12 carbon atoms and the rest are hydrogen atoms; n is an integer of 2 or more; is an integer; a black circle represents a bond with another group]
  • R 1 and R 2 are each independently a group represented by the following general formula (A- 3 ) or (A-4); p1 is an integer of 1 to 6; is a group represented by the following general formula (A-3) or (A-4); A is a 5- to 7-membered ring having two nitrogen atoms; p2 is an integer of 1-6 . ]
  • R 4 is a hydrogen atom, a hydroxy group, a piperidinyl group, a pyrrolidinyl group, or -NR 6 R 7 ;
  • p3 is an integer of 1 to 12;
  • A is a 5- to 7-membered ring having two nitrogen atoms;
  • R 8 is a hydrogen atom, a hydroxy group, a piperidinyl group, or a pyrrolidinyl group; p6 is an integer of 1 to 6; and an amino alcohol represented by the following general formula (L-1)
  • R 10 is a hydrogen atom or an alkyl group having 1 to 12 carbon atoms.
  • the amino alcohol represented by the general formula (A-1) or (A-2) is represented by the following general formulas (1) to (5)
  • R OH is — ( CH 2 )p10-R 20 ;
  • R 20 is a hydrogen atom or a hydroxy group;
  • p10 is an integer of 1 to 6; each independently an alkyl group having 1 to 12 carbon atoms or a dialkylaminoalkyl group having 3 to 12 carbon atoms;
  • R 23 is an alkyl group having 1 to 6 carbon atoms, a pyrrolidinylalkyl group, or piperidine a nylalkyl group;
  • p11 is an integer of 1 to 6;
  • R 24 and R 25 are each independently R OH or —(CH 2 )p12—N(R OH ) 2 ; is an integer of 1 to 6;
  • R 26 and R 27 are each independently R OH or -(CH 2 )p13-N(R OH ) 2 ;
  • p13 is an integer of 1 to 6
  • R 28 and R 29 are each independently R OH or —(CH 2 )
  • R OP is -(CH 2 )p10-R 30 ;
  • R 30 is a hydrogen atom, a hydroxy group, or -OR P ;
  • p10 is an integer of 1 to 6;
  • R 22 are each independently an alkyl group having 1 to 12 carbon atoms or a dialkylaminoalkyl group having 3 to 12 carbon atoms;
  • R 23 is an alkyl group having 1 to 6 carbon atoms, pyrrolidinylalkyl or a piperidinylalkyl group;
  • p11 is an integer of 1 to 6;
  • R 34 and R 35 are each independently R OP or —(CH 2 )p12-N(R OP ) p12 is an integer of 1 to 6 ;
  • R 36 and R 37 are each independently R OP or —(CH 2 )p13-N(R OP ) 2 ;
  • p13 is is an integer of 1 to 6;
  • R 38 and R 39 are each independently R
  • R 11 to R 15 is an alkyl group having 1 to 12 carbon atoms and the rest are hydrogen atoms; n is an integer of 2 or more; q is 1 to 6 is an integer; the black circle represents a bond with an oxygen atom) is a group represented by]
  • R 31 is a hydrogen atom or R P ;
  • one of R 11 to R 15 is an alkyl group having 1 to 12 carbon atoms and the rest are hydrogen atoms; n is an integer of 2 or more; q is 1 to 6 a black circle represents a bond with an oxygen atom)], the amino polyester according to any one of the above [1] to [7]. [9] the following general formula (A-1) or (A-2)
  • R 1 and R 2 are each independently a group represented by the following general formula (A- 3 ) or (A-4); p1 is an integer of 1 to 6; is a group represented by the following general formula (A-3) or (A-4); A is a 5- to 7-membered ring having two nitrogen atoms; p2 is an integer of 1-6 . ]
  • R 4 is a hydrogen atom, a hydroxy group, a piperidinyl group, a pyrrolidinyl group, or -NR 6 R 7 ;
  • p3 is an integer of 1 to 12;
  • A is a 5- to 7-membered ring having two nitrogen atoms;
  • R 8 is a hydrogen atom, a hydroxy group, a piperidinyl group, or a pyrrolidinyl group; p6 is an integer of 1 to 6; and an amino alcohol represented by the following general formula (L-1)
  • R 10 is a hydrogen atom or an alkyl group having 1 to 12 carbon atoms.
  • a method for producing an aminopolyester comprising ring-opening polymerization with a caprolactone derivative represented by to produce an aminopolyester.
  • the lipid nanoparticle of [12] wherein the nucleic acid is a gene to be expressed in lung tissue cells.
  • a pharmaceutical composition comprising the lipid nanoparticles of any one of [10] to [13] as an active ingredient.
  • the pharmaceutical composition of [14] above which is used for treatment of pulmonary diseases.
  • the lipid nanoparticles according to any one of the above [10] to [13], wherein the lipid nanoparticles encapsulating the exogenous gene to be expressed in the cells of lung tissue are used in a subject animal (excluding humans). ) to express the foreign gene in the lung tissue of the subject animal.
  • the lipid nanoparticles according to the present invention can highly express the enclosed gene in lung tissue. Therefore, the lipid nanoparticles are useful as lung tissue-specific gene delivery carriers for immunotherapy and gene therapy.
  • FI EGFP fluorescence intensity
  • FIG. 1 is a diagram showing measurement results of EGFP fluorescence intensity (FI) of HeLa cells introduced with each EGFP-expressing lipid nanoparticle in Example 1.
  • FIG. 2 lipid nanoparticles for EGFP expression containing AA03-DL-10, AA04-DL-10, AA05-DL-10, AA07-DL-10, AA09-DL-5, or AA11-DL-5 Pie chart showing expression ratios in lung, liver, spleen, kidney, and heart in treated mice.
  • Example 3 in mice to which lipid nanoparticles for EGFP expression containing AA03-DL-10 or AA03-VL-10 were administered, EGFP fluorescence intensity of the lung, liver, and spleen is a diagram showing the results of measuring the fluorescence intensity.
  • . 1 is a TEM image of lipid nanoparticles for EGFP expression containing AA03-DL-10 in Example 3.
  • FIG. 4 shows stained images of tissue sections of mice to which EGFP-expressing lipid nanoparticles containing AA03-DL-10 were administered and mice to which PBS was administered in Example 4.
  • mice administered EGFP-expressing lipid nanoparticles containing AA03-DL-10 and mice administered PBS Fig. 7(C)), ALP (Fig. 7(D)), BUN (Fig. 7(E)), and CRE (Fig. 7(F)).
  • X1 to X2 (X1 and X2 are real numbers satisfying X1 ⁇ X2)" means "X1 or more and X2 or less”.
  • the lipid nanoparticles according to the present invention are amino polyesters having a structure represented by the following general formula (P-1) (hereinafter sometimes referred to as “structure (P-1)”) (hereinafter referred to as “ It is characterized by containing the "amino polyester”.).
  • structure (P-1) hereinafter sometimes referred to as "structure (P-1)"
  • It is characterized by containing the "amino polyester”.
  • one of R 11 to R 15 is an alkyl group having 1 to 12 carbon atoms and the rest are hydrogen atoms.
  • n is an integer of 2 or more, and q is an integer of 1-6.
  • a black circle represents a bond with another group.
  • the lipid nanoparticles according to the present invention contain the aminopolyester according to the present invention as a lipid component that constitutes the lipid membrane.
  • the polyester portion in the aminopolyester according to the present invention is highly specific to lung tissue. Therefore, by containing the aminopolyester as a lipid component, the lipid nanoparticles according to the present invention are useful as a lung-specific delivery carrier without modifying the lipid nanoparticle surface with a target ligand or the like.
  • the aminopolyester according to the present invention since the aminopolyester according to the present invention has an amino group, it can efficiently entrap basic substances such as nucleic acids. Therefore, by containing the aminopolyester as a lipid component, the lipid nanoparticles according to the present invention are useful as a carrier that specifically delivers a basic substance to the lung tissue. It is an excellent lung-specific gene delivery carrier for exogenous genes.
  • the alkyl group represented by any one of R 11 to R 15 is not particularly limited as long as it is an alkyl group having 1 to 12 carbon atoms, and is a linear alkyl group. may be a branched alkyl group.
  • alkyl groups having 1 to 12 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group and isopentyl group.
  • the alkyl group which is any one of R 11 to R 15 in the structure (P-1) in the aminopolyester according to the present invention is preferably an alkyl group having 2 to 9 carbon atoms, and an alkyl group having 3 to 6 carbon atoms. is more preferable, a linear alkyl group having 3 to 6 carbon atoms is more preferable, and an n-butyl group is particularly preferable.
  • n is not particularly limited as long as it is an integer of 2 or more.
  • n is preferably an integer of 2 to 70, more preferably an integer of 10 to 70, and 15 to 60. More preferred.
  • q is not particularly limited as long as it is an integer of 1-6.
  • q is preferably an integer of 1 to 4, more preferably an integer of 1 to 3, and even more preferably 2. .
  • the structure (P-1) in the aminopolyester according to the present invention is preferably a structure in which R 11 to R 14 are hydrogen atoms and R 15 is an alkyl group having 1 to 12 carbon atoms; R 11 to R 14 is a hydrogen atom, R 15 is a linear alkyl group having 1 to 12 carbon atoms, and q is an integer of 1 to 4; is a linear alkyl group having 2 to 9 carbon atoms and q is an integer of 1 to 3; R 11 to R 14 are hydrogen atoms; A structure in which a chain alkyl group and q is an integer of 1 to 3 is more preferable; A structure in which R 11 to R 14 are hydrogen atoms, R 15 is an n-butyl group, and q is an integer of 2 is particularly preferred.
  • An aminopolyester having structure (P-1) can be synthesized, for example, by ring-opening polymerization of a seven-membered lactone using an aminoalcohol as an initiator.
  • This ring-opening polymerization reaction can be efficiently carried out at about room temperature by using a basic catalyst in an aprotic organic solvent with relatively low polarity such as toluene.
  • the basic catalyst may be an inorganic base catalyst such as a metal base, but an organic base catalyst is preferable.
  • phosphazene bases, organolithium compounds, and Bronsted strongly basic organic catalysts such as bicyclic guanidine type organic bases are preferred, and phosphazene bases are more preferred.
  • Preferred bicyclic guanidine-type organic bases are TBD (triazabicyclodecene, CAS No.: 5807-14-7) and MTBD (methyltriazabicyclodecene, CAS No.: 84030-20-6).
  • TBD triazabicyclodecene
  • MTBD methyltriazabicyclodecene
  • phosphazene base t-Bu-P 4 base (CAS No.: 111324-04-0) is particularly preferred.
  • the size of the portion of structure (P-1) in the aminopolyester according to the present invention is not particularly limited depending on the number of repeating structural units in parentheses.
  • the aminopolyester according to the present invention preferably has a number average molecular weight of 3,000 to 10,000 (3 kDa to 10 kDa) in the portion of structure (P-1).
  • the number average molecular weight of portions of structure (P-1) can be determined by size exclusion chromatography (SEC) analysis.
  • a caprolactone derivative represented by the following general formula (L-1) is preferable as the 7-membered ring lactone used in the synthesis of the aminopolyester according to the present invention.
  • R 10 is a hydrogen atom or an alkyl group having 1 to 12 carbon atoms.
  • Examples of the alkyl group having 1 to 12 carbon atoms for R 10 include the same alkyl groups as the alkyl groups for any one of R 11 to R 15 described above, preferably alkyl groups having 2 to 9 carbon atoms, and 3 to 3 carbon atoms. 6 alkyl groups are more preferred.
  • the ⁇ -caprolactone derivative is preferably a compound in which R 10 is bonded to the ⁇ carbon (the carbon atom bonded to the oxygen atom in the 7-membered ring) in the following general formula (L-1).
  • the aminoalcohol used for synthesizing the aminopolyester of the present invention is preferably, for example, an aminoalcohol represented by the following general formula (A-1) or (A-2).
  • p1 is an integer of 1-6.
  • p1 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and even more preferably 2 or 3.
  • p2 is an integer of 1-6.
  • p2 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and even more preferably 2 or 3.
  • A is a 5- to 7-membered ring having 2 nitrogen atoms.
  • A include a piperazinyl group, a diazepanyl group, an imidazolidinyl group and the like.
  • the amino alcohol represented by the general formula (A-2) compounds in which A is a piperazinyl group or a diazepanyl group are preferred, and compounds in which A is a piperazinyl group are more preferred.
  • R 1 and R 2 are each independently a group represented by general formula (A-3) or (A-4) below.
  • R 3 is a group represented by general formula (A-3) or (A-4) below.
  • R 1 and R 2 may be the same group or different groups.
  • A is the same as A in general formula (A-2).
  • R 4 is a hydrogen atom, hydroxy group, piperidinyl group, pyrrolidinyl group, or -NR 6 R 7 .
  • R 6 and R 7 are each independently a group represented by general formula (A-5) below.
  • R 8 is a hydrogen atom, a hydroxy group, a piperidinyl group, or a pyrrolidinyl group.
  • R 8 is preferably a hydrogen atom or a hydroxy group, more preferably a hydroxy group.
  • p6 is an integer of 1-6. p6 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and even more preferably 2 or 3.
  • a black circle represents a bond with another group. That is, the black circled hands in general formulas (A-3) and (A-4) are the nitrogen atoms to which R 1 and R 2 in general formula (A-1) are bonded, or the general formula It is a bond with the nitrogen atom to which R 3 in (A-2) is bonded.
  • the black circled hand in general formula (A-5) is a bond with the nitrogen atom to which R 6 and R 7 in —NR 6 R 7 are bonded.
  • p3 is an integer of 1-12.
  • p3 is preferably an integer of 1 to 10, more preferably an integer of 1 to 6, even more preferably an integer of 1 to 4, and even more preferably an integer of 1 to 3. is preferred, and 2 is particularly preferred.
  • p4 and p5 in one molecule may be the same integer or different integers.
  • p4 and p5 are each independently preferably an integer of 1 to 4, and more preferably both p4 and p5 are 2 or 3.
  • aminoalcohol represented by general formula (A-1) or general formula (A-2) compounds represented by any of the following general formulas (1) to (5) are preferable.
  • R OH is -(CH 2 )p10-R 20 .
  • R20 is a hydrogen atom or a hydroxy group.
  • p10 is an integer of 1-6.
  • p10 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and even more preferably 2 or 3.
  • a plurality of R OH in one molecule may be the same group or different groups.
  • R 21 and R 22 are each independently an alkyl group having 1 to 12 carbon atoms or a dialkylaminoalkyl group having 3 to 12 carbon atoms. In general formula (1), R 21 and R 22 may be the same group or different groups.
  • the alkyl group is not particularly limited as long as it is an alkyl group having 1 to 12 carbon atoms, and may be a branched alkyl group. A linear alkyl group is preferred. Further, when R 21 and R 22 are dialkylaminoalkyl groups, the dialkylaminoalkyl group is not particularly limited as long as it has 3 to 12 carbon atoms, and the alkyl group portion in the group is linear. It may be an alkyl group or a branched alkyl group.
  • dialkylaminoalkyl group examples include dimethylaminoethyl group, dimethylaminopropyl group, dimethylaminobutyl group, diethylaminoethyl group, diethylaminopropyl group, diethylaminobutyl group and the like.
  • the aminoalcohol represented by the general formula (1) may be a compound in which both R 21 and R 22 are alkyl groups having 1 to 12 carbon atoms, and both R 21 and R 22 have 3 carbon atoms.
  • at least one of R 21 and R 22 is preferably a compound having a dialkylaminoalkyl group having 3 to 12 carbon atoms, and R 21 has 1 to 6 carbon atoms.
  • R 22 is a dialkylaminoalkyl group having 3 to 8 carbon atoms, wherein R 21 is an alkyl group having 1 to 6 carbon atoms and R 22 is dimethyl having 3 to 8 carbon atoms. More preferred are compounds that are aminoalkyl groups.
  • R 23 is an alkyl group having 1 to 6 carbon atoms, a pyrrolidinylalkyl group, or a piperidinylalkyl group.
  • the alkyl group portion in the pyrrolidinylalkyl group and piperidinylalkyl group is not particularly limited, and may be, for example, a linear or branched alkyl group having 1 to 6 carbon atoms.
  • As the aminoalcohol represented by the general formula (2) compounds in which R 23 is a C 1-6 alkyl group are preferred, and compounds in which R 23 is a C 1-3 alkyl group are more preferred.
  • p11 is an integer of 1-6. p11 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and even more preferably 2 or 3.
  • R 24 and R 25 are each independently R OH or -(CH 2 )p12-N(R OH ) 2 .
  • p12 is an integer of 1-6.
  • p12 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and even more preferably 2 or 3.
  • R 24 and R 25 may be the same group or different groups.
  • Examples of the amino alcohol represented by the general formula (3) include compounds in which p11 is 2 or 3 and both R 24 and R 25 are R OH , and compounds in which p11 is 2 or 3 and R 24 and Compounds in which R 25 are both —(CH 2 )p12—N(R OH ) 2 are preferred, and compounds in which p11 is 2 or 3 and R 24 and R 25 are both R OH are particularly preferred.
  • R 26 and R 27 are each independently R OH or -(CH 2 )p13-N(R OH ) 2 .
  • p13 is an integer of 1-6.
  • p13 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and even more preferably 2 or 3.
  • R 26 and R 27 may be the same group or different groups.
  • Examples of the aminoalcohol represented by general formula (4) include compounds in which both R 26 and R 27 are R OH , and compounds in which both R 26 and R 27 are —(CH 2 )p13—N(R OH ) 2 are preferred, and compounds in which both R 26 and R 27 are R 2 OH are particularly preferred.
  • R 28 and R 29 are each independently R OH or -(CH 2 )p14-N(R OH ) 2 .
  • p14 is an integer of 1-6.
  • p14 is preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and even more preferably 2 or 3.
  • R 28 and R 29 may be the same group or different groups.
  • Examples of the aminoalcohol represented by the general formula (5) include compounds in which both R 28 and R 29 are R OH , and compounds in which both R 28 and R 29 are -(CH 2 )p14 - N(R OH ) are preferred, and compounds in which both R 28 and R 29 are R 2 OH are particularly preferred.
  • An amino polyester having a structure (P-1) obtained by ring-opening polymerization of a caprolactone derivative represented by the general formula (L-1) and an amino alcohol represented by the general formulas (1) to (5) includes: Examples thereof include compounds represented by the following general formulas (1P) to (5P).
  • R 21 and R 22 are the same as R 21 and R 22 in general formula (1)
  • R 23 in general formula (2P) is R in general formula (2)
  • 23 , and p11 in general formula (3P) is the same as p11 in general formula (3).
  • R OP is - ( CH2 )p10-R30.
  • p10 is an integer of 1 to 6, preferably an integer of 1 to 5, more preferably an integer of 2 to 4, and even more preferably 2 or 3.
  • R 30 is a hydrogen atom, a hydroxy group, or -OR P.
  • R P is a group represented by the following general formula (P-2).
  • R 11 to R 15 are the same as R 11 to R 15 in general formula (P-1), and n is the same as n in general formula (P-1) is.
  • a black circle represents a bond with an oxygen atom. That is, the hands marked with black circles are bonding hands with oxygen atoms.
  • R 34 and R 35 are each independently R OP or -(CH 2 )p12-N(R OP ) 2 .
  • p12 is an integer of 1-6.
  • R 36 and R 37 are each independently R OP or -(CH 2 )p13-N(R OP ) 2 .
  • p13 is an integer of 1-6.
  • R 38 and R 39 are each independently R OP or -(CH 2 )p14-N(R OP ) 2 .
  • p14 is an integer of 1-6.
  • a plurality of R 1 P in one molecule of the aminopolyesters represented by general formulas (2P) to (5P) may be the same groups or different groups.
  • aminopolyesters represented by any of general formulas (1P) to (5P) include aminopolyesters represented by the following general formulas (AP-01) to (AP-11).
  • R 31 is a hydrogen atom or R 3 P.
  • R P is the same as R P in R 30 above.
  • a plurality of R 2 P 's in one molecule of the aminopolyesters represented by general formulas (AP-01) to (AP-11) may be the same groups or different groups.
  • aminopolyester in particular, general formula (AP-03), general formula (AP-04), general formula (AP-05), general formula (AP-07), general formula (AP-09) , Or an amino polyester represented by any of the general formula (AP-11) is preferable, and from the viewpoint of higher selectivity to lung tissue, general formula (AP-03), general formula (AP-04), general More preferred are aminopolyesters represented by formula (AP-05) or general formula (AP-09).
  • lipid nanoparticles according to the present invention all the lipids constituting the lipid nanoparticles may be the aminopolyester according to the present invention, or the aminopolyester and other lipids may be contained. Since the selectivity to lung tissue can be sufficiently high, the content of the aminopolyester according to the present invention with respect to the total amount of lipids constituting the lipid nanoparticles according to the present invention is preferably 30 to 100% (mol), and 50 to 100% (mol) is more preferred, 70-100% (mol) is more preferred, and 85-100% (mol) is even more preferred.
  • lipids other than the aminopolyester according to the present invention can be lipids that are generally used for forming liposomes.
  • lipids include, for example, phospholipids, sterols, saturated or unsaturated fatty acids, and the like. These can be used singly or in combination of two or more.
  • Phospholipids include phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, ceramide phosphorylglycerol phosphate, and phosphatidic acid.
  • sterols include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol and brassicasterol; Examples thereof include microorganism-derived sterols such as zymosterol and ergosterol.
  • the lipid nanoparticles according to the present invention preferably contain sterols, more preferably cholesterol.
  • the lipid nanoparticles according to the present invention preferably contain polyalkylene glycol-modified lipids as lipid components.
  • Polyalkylene glycol is a hydrophilic polymer, and the surface of lipid nanoparticles can be modified with polyalkylene glycol by constructing lipid nanoparticles using polyalkylene glycol-modified lipids as lipid membrane-constituting lipids. Stability such as blood retention of lipid nanoparticles can sometimes be enhanced by surface modification with polyalkylene glycol.
  • polyalkylene glycol for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, etc. can be used.
  • the molecular weight of polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, more preferably about 1,000 to 5,000.
  • stearylated polyethylene glycol eg, PEG45 stearate (STR-PEG45), etc.
  • PEG45 stearate STR-PEG45
  • Others N-[carbonyl-methoxypolyethyleneglycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[methoxypolyethyleneglycol-2000]-1,2-dimyristoyl-sn -glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-750 ]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-
  • the ratio of the polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles of the present invention is particularly limited as long as it is an amount that does not impair the lung tissue-specific gene expression activity of the aminopolyester of the present invention. is not.
  • the ratio of the polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles is preferably 1 to 3 mol%.
  • the lipid nanoparticles according to the present invention can be subjected to appropriate surface modification or the like as necessary.
  • the lipid nanoparticles according to the present invention can be enhanced in blood retention by modifying the surface with a hydrophilic polymer or the like.
  • surface modification can be performed by using lipids modified with these modifying groups as constituent lipids of lipid nanoparticles.
  • examples of lipid derivatives for enhancing blood retention include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, and the like. can also be used.
  • examples of lipid derivatives for enhancing blood retention include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, and the like.
  • polyalkylene glycol, dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, and divinyl ether-maleic anhydride alternating copolymer are also used as hydrophilic polymers for increasing blood retention.
  • the lipid nanoparticles can be surface-modified with an oligosaccharide compound having three or more sugars.
  • the type of tri- or more oligosaccharide compound is not particularly limited, but for example, an oligosaccharide compound in which about 3 to 10 sugar units are bonded can be used, and preferably about 3 to 6 sugar units are bonded.
  • Oligosaccharide compounds can be used. Among them, oligosaccharide compounds that are trimers or hexamers of glucose are preferably used, and oligosaccharide compounds that are trimers or tetramers of glucose are more preferably used.
  • isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, maltohexaose and the like can be preferably used.
  • Triose, maltotetraose, maltopentaose or maltohexaose are more preferred.
  • Particularly preferred is maltotriose or maltotetraose, most preferred is maltotriose.
  • the amount of surface modification of the lipid nanoparticles with the oligosaccharide compound is not particularly limited. is.
  • the method of surface-modifying lipid nanoparticles with an oligosaccharide compound is not particularly limited, for example, liposomes in which the surface of lipid nanoparticles is modified with monosaccharides such as galactose and mannose (International Publication No. 2007/102481) are known. Therefore, the surface modification method described in this publication can be employed. The entire disclosure of the above publications is incorporated herein by reference.
  • the lipid nanoparticles according to the present invention can be endowed with one or more functions such as temperature change sensitive function, membrane permeation function, gene expression function, and pH sensitive function. Appropriate addition of these functions improves the retention of lipid nanoparticles in blood, efficiently escapes lipid nanoparticles from endosomes after endocytosis in target cells, and releases encapsulated nucleic acids. It can be expressed more efficiently in lung tissue cells.
  • functions such as temperature change sensitive function, membrane permeation function, gene expression function, and pH sensitive function.
  • the lipid nanoparticles according to the present invention are one or more selected from the group consisting of antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charged substances, and membrane polypeptides.
  • Membrane polypeptides include, for example, surface-to-membrane polypeptides and integral-to-membrane polypeptides. The blending amount of these substances is not particularly limited, and can be appropriately selected according to the purpose.
  • the average particle size of the lipid nanoparticles is preferably 500 nm or less, more preferably 100 to 400 nm, because high delivery efficiency to lung tissue cells present in vivo is likely to be obtained. is more preferably 100 to 350 nm, even more preferably 200 to 350 nm.
  • the average particle size of lipid nanoparticles means the number average particle size measured by dynamic light scattering (DLS). Measurement by the dynamic light scattering method can be performed by a conventional method using a commercially available DLS device or the like.
  • the polydispersity index (PDI) of the lipid nanoparticles according to the present invention is about 0.05 to 0.7, preferably about 0.1 to 0.6, more preferably about 0.15 to 0.4.
  • the zeta potential can be in the range -50 mV to -10 mV, preferably in the range -45 mV to -15 mV, more preferably in the range -20 mV to -10 mV.
  • the form of the lipid nanoparticles according to the present invention is not particularly limited, it is preferably in the form of being dispersed in an aqueous solvent.
  • examples of such forms include nanoparticles in which a monomolecular layer of a hydrophilic substance is formed on the surface of a lipid-containing core.
  • unilamellar liposomes, multilamellar liposomes, spherical micelles, etc. can be mentioned, and amorphous layered structures composed of amphipathic lipid molecules can also be used.
  • lipid nanoparticles composed of an aminopolyester, a polyalkylene glycol-modified lipid, and a nucleic acid are nanoparticles having a core composed of an aminopolyester and a nucleic acid, and a polyalkylene glycol layer on the nanoparticle surface.
  • the lipid nanoparticles according to the present invention contain the target components to be delivered into the target cells inside the particles covered with a lipid membrane.
  • the components that the lipid nanoparticles according to the present invention enclose inside the particles are not particularly limited as long as they have sizes that can be encapsulated.
  • the lipid nanoparticles according to the present invention include nucleic acids, sugars, peptides, Any substance such as a low-molecular-weight compound or a metal compound can be enclosed.
  • a nucleic acid is preferable as a component to be encapsulated in the lipid nanoparticles according to the present invention.
  • the nucleic acid may be DNA, RNA, analogues or derivatives thereof (eg, peptide nucleic acid (PNA), phosphorothioate DNA, etc.).
  • the nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention may be a single-stranded nucleic acid, a double-stranded nucleic acid, a linear nucleic acid, or a circular nucleic acid.
  • the nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention preferably contains a foreign gene to be expressed in the target cell, and is a nucleic acid that functions to express the foreign gene in the cell by being taken up into the cell. It is more preferable to have The foreign gene may be a gene originally contained in the genomic DNA of target cells (preferably lung tissue cells) or a gene not contained in the genomic DNA. Examples of such nucleic acids include gene expression vectors containing a nucleic acid consisting of a base sequence encoding a gene to be expressed. The gene expression vector may exist as an extrachromosomal gene in the introduced cell, or may be incorporated into genomic DNA by homologous recombination.
  • the gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is not particularly limited, and vectors generally used in gene therapy and the like can be used.
  • the gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is preferably a nucleic acid vector such as a plasmid vector.
  • the plasmid vector may remain circular, or may be preliminarily cut into a linear shape and encapsulated in the lipid nanoparticles of the present invention.
  • Gene expression vectors can be designed by conventional methods using commonly used molecular biological tools based on the base sequence information of the gene to be expressed, and can be produced by various known methods. .
  • the nucleic acid to be encapsulated in the lipid nanoparticles of the present invention is also preferably a functional nucleic acid that controls the expression of target genes present in target cells.
  • Such functional nucleic acids include antisense oligonucleotides, antisense DNA, antisense RNA, siRNA, microRNA and the like. It may also be an siRNA expression vector that expresses siRNA in cells.
  • the siRNA expression vector can be prepared from a commercially available siRNA expression vector, which may be modified as appropriate.
  • the method for producing lipid nanoparticles according to the present invention is not particularly limited, and any method available to those skilled in the art can be adopted.
  • all the lipid components are dissolved in an organic solvent such as chloroform, dried under reduced pressure by an evaporator or spray-dried by a spray dryer to form a lipid film, and then components to be encapsulated in the lipid nanoparticles.
  • it can be produced by adding an aqueous solvent containing nucleic acid or the like to the above dried mixture and further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure jet emulsifier.
  • extrusion extrusion filtration
  • membrane filter with a uniform pore size
  • the composition of the aqueous solvent (dispersion medium) is not particularly limited. can be done.
  • These aqueous solvents (dispersion media) can stably disperse lipid nanoparticles, and furthermore, monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, xylose sugar, lactose, sucrose, cellobiose, trehalose, Disaccharides such as maltose, trisaccharides such as raffinose and melezinose, polysaccharides such as cyclodextrin, sugars (aqueous solutions) such as sugar alcohols such as erythritol, xylitol, sorbitol, mannitol and maltitol, glycerin, diglycerin and polyglycerin Polyhydric alcohol (aqueous solution) such as propylene glycol, polypropylene glycol, ethylene glyco
  • the pH of the aqueous solvent should be set from weakly acidic to near neutral (about pH 3.0 to 8.0), and/or the dissolved oxygen should be removed by nitrogen bubbling or the like. is desirable.
  • lipid nanoparticles for example, glucose, galactose, mannose, fructose, inositol, ribose, xylose monosaccharides, lactose, sucrose, cellobiose, trehalose
  • sugars aqueous solutions
  • disaccharides such as maltose, trisaccharides such as raffinose and melezinose
  • polysaccharides such as cyclodextrin
  • sugar alcohols such as erythritol, xylitol, sorbitol, mannitol and maltitol.
  • aqueous dispersion for example, the above sugars, glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether , diethylene glycol monoalkyl ether, 1,3-butylene glycol, and other polyhydric alcohols (aqueous solutions) may improve stability.
  • the gene expression vectors encapsulated in the lipid nanoparticles are selectively expressed in lung tissue rather than other organs.
  • the siRNA expression vector encapsulated in the lipid nanoparticles is selectively expressed in lung tissue over other organs, The expression of the gene targeted by the expression vector is suppressed.
  • the lipid nanoparticles of the present invention encapsulating a foreign gene to be expressed in lung tissue cells are administered to a subject animal, the foreign gene can be expressed in the lung tissue of the subject animal.
  • the lipid nanoparticles according to the present invention function as a delivery carrier targeting lung tissue. Therefore, the lipid nanoparticles according to the present invention are useful as a carrier for delivering basic medicinal ingredients to lung tissue for the treatment of pulmonary diseases. It is useful as an active ingredient of pharmaceutical compositions used for gene therapy.
  • Pulmonary diseases include, for example, pneumonia, viral infections such as SARS, acute respiratory distress syndrome, lung cancer, pulmonary hypertension, pulmonary fibrosis, cystic fibrosis, and the like.
  • the animals to which the lipid nanoparticles according to the present invention are administered are not particularly limited, and may be humans or animals other than humans.
  • Non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters and guinea pigs, and birds such as chickens, quails and ducks.
  • the administration route for administering the lipid nanoparticles according to the present invention to animals is not particularly limited, and includes intravenous administration, enteral administration, intramuscular administration, subcutaneous administration, transdermal administration, and intranasal administration. Parenteral administration such as administration and pulmonary administration is preferred.
  • Methyl acrylate manufactured by TCI, >99.0%
  • lithium aluminum hydride LiAlH4 ; manufactured by TCI, >95.0%
  • didecylamine A02; manufactured by TCI, >97.0%
  • ethylenediamine anhydride A05; manufactured by TCI, > 98.0%
  • 3,3′-diaminodipropylamine A06; manufactured by TCI, >98.0%
  • tris(2-aminoethyl)amine A07; manufactured by TCI, >98.0%
  • 1-(2-aminoethyl ) pyrrolidine A08; Sigma-Aldrich, 98%)
  • 1,4-bis(3-aminopropyl)piperazine A10; TCI, >98.0%
  • homopiperazine A11, TCI, >98.0%
  • t-Bu-P4 ( ⁇ 0.8 mol/L solution in hexane; manufactured by Sigma-Aldrich Chemicals), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD; manufactured by TCI, >98.0%) and 1,4-bis(2-hydroxyethyl)piperazine (AA09; manufactured by TCI, >98.0%) were used after being stored in a glove box.
  • EGFP Enhanced Green Fluorescent Protein
  • mRNA encoding luciferase were purchased from TriLink Biotechnologies.
  • DMG-PEG2k was purchased from NOF Corporation.
  • Cholesterol and DiD were purchased from Sigma-Aldrich.
  • 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Avanti polar lipids.
  • the number average molecular weight of the aminopolyester was measured by the method of size exclusion chromatography (SEC). SEC was performed on a Shodex 3GPC-101 system equipped with a Shodex KG guard column and a set of two Shodex KF-804L columns (linear 8 mm x 300 mm; bead size 5 ⁇ m; exclusion limit 4 x 10 g/mol) in tetrahydrofuran (THF). 1.0 mL/min) and run at 40°C.
  • SEC size exclusion chromatography
  • M n,SEC Number average molecular weight
  • D dispersity of aminopolyesters determined by SEC were generated from standard polystyrene (PS) samples ranging from 1,200 to 1,320,000 g/mol. calculated by molecular weight calibration curve.
  • lipid nanoparticles encapsulating nucleic acids were prepared as follows, unless otherwise specified.
  • the constituent lipids of the lipid nanoparticles are aminopolyester and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (ammonium salt) (DMG-PEG2k , Avanti), and EGFP mRNA was used as the encapsulated nucleic acid.
  • a lipid film was created at the bottom of the tube by evaporating the ethanol by incubating in the bottom overnight.
  • mRNA was added to the test tube in which the lipid membrane had been prepared, incubated at room temperature for 30 minutes, and then sonicated for 1 minute with a bath sonicator.
  • the liposome solution in the test tube was transferred to a centrifugal ultrafiltration device (product name: "Amicon Ultra (100kDa)", manufactured by Merck) and centrifuged at 1500 x g for 30 minutes for ultrafiltration. Then, it was collected with 600 ⁇ L of phosphate buffer (PBS, pH 7.4). A PBS suspension of the collected liposomes (lipid nanoparticles) was used as a lipid nanoparticle solution.
  • phosphate buffer PBS, pH 7.4
  • nucleic acid recovery rate and encapsulation rate in lipid nanoparticles were evaluated using a nucleic acid quantification reagent (product name: "Quant-iT RiboGreen assay", manufactured by Thermo Fisher Scientific).
  • the lipid nanoparticle solution was diluted 40-fold with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8), and equal amounts of the RiboGreen dilution containing dextran sulfate and the dilution containing dextran sulfate and Triron X-100 were mixed and analyzed.
  • a sample was prepared for The fluorescence of this analytical sample was measured with a plate reader. Calculate the amount of nucleic acid after preparation of lipid nanoparticles and the amount of nucleic acid not encapsulated in lipid nanoparticles based on a calibration curve created using nucleic acid solutions with known concentrations, and calculate the recovery rate and encapsulation rate using the following formula. did.
  • [encapsulation rate (%)] 100 - [amount of nucleic acid outside ( ⁇ g) (fluorescence when only dextran sulfate is added)] / [amount of total nucleic acid after preparation ( ⁇ g) (amount of dextran sulfate and Triton X-100 Nucleic acid amount when added)] ⁇ 100
  • HeLa cells (ATCC Number: CRM-CCL-2) were used as cultured cells. HeLa cells were cultured in Dulbecco's modified Eagle's medium (containing 4500 mg/L glucose) supplemented with 10% heat-inactivated fetal bovine serum (hiFBS, manufactured by Gibco) and penicillin/streptomycin at 37°C under 5% CO 2 environment. did.
  • Dulbecco's modified Eagle's medium containing 4500 mg/L glucose
  • hiFBS heat-inactivated fetal bovine serum
  • penicillin/streptomycin at 37°C under 5% CO 2 environment. did.
  • EGFP fluorescence intensity was quantified using a Tecan Infinite M200 Pro plate reader (manufactured by Tecan).
  • Lipid nanoparticles for EGFP expression were administered to mice, and gene expression activity in each organ was evaluated using EGFP fluorescence as an indicator.
  • lipid nanoparticles for EGFP expression were administered to ICR mice via the tail vein so that the EGFP mRNA dose per mouse was 0.25 mg / kg, and 8 hours after administration, lungs, liver, spleen, kidneys , the heart was harvested and its fluorescence intensity was measured. The higher the fluorescence intensity, the more EGFP-expressing lipid nanoparticles were taken up by the organ, indicating that EGFP was expressed in cells.
  • Example 1 Lipid nanoparticles containing aminopolyesters synthesized from various aminoalcohols and ⁇ -decanolactone (DL) or caprolactone (CL) were prepared.
  • the resulting ester (12.0 g, 29.7 mmol, 1 eq) was then suspended in anhydrous THF (30 mL) and the reaction flask was cooled to 0°C. After stirring the resulting suspension at 0° C. for 30 minutes under an argon atmosphere, LiAlH 4 (6.3 g, 166.1 mmol, 5.6 eq) was added very slowly to the flask and the resulting mixture was stirred at room temperature for 48 hours. The reaction was then quenched by adding water (6.3 mL) to the flask at 0° C. very slowly. Further NaOH solution (15 wt%, 6.14 mL) was added to the mixture in the flask and stirred until all the gray solid turned white.
  • organic base catalysts four types of diphenyl phosphate (DPP), triazabicyclodecene (TBD), and t-Bu-P1 and t-Bu-P4 catalysts, which are phosphazene bases, were used. Synthesis of the desired aminopolyester was confirmed in all reactions using organic base catalysts. When the reactions using each organic base catalyst were compared, the t-Bu-P4 catalyst was able to achieve a monodisperse, stable synthesis in a short period of time.
  • DPP diphenyl phosphate
  • TBD triazabicyclodecene
  • t-Bu-P1 and t-Bu-P4 catalysts which are phosphazene bases
  • t-Bu-P4 catalyst specifically, an initiator amino alcohol, ⁇ -decalactone or caprolactone, and t-Bu-P4 (8.67 mg, 13.68 ⁇ mol) are added in a glove box. , was mixed with toluene in a 15 mL vial.
  • the amount of ⁇ -decalactone or caprolactone and amino alcohol in the reaction mixture is such that the ratio of ⁇ -decalactone or caprolactone and the hydroxyl group of the amino alcohol is a structural unit derived from ⁇ -decalactone or caprolactone (structure (P-1) ) was adjusted to have a degree of polymerization of 3 kDa, 5 kDa, or 10 kDa.
  • the polymerization reaction was carried out while stirring the resulting mixture at room temperature. When monomer conversion reached about 80-90%, benzoic acid was added to quench the reaction.
  • the synthesized aminopolyester was purified three times by precipitation in cold methanol and then dried overnight. The synthesized aminopolyester was prepared in N,N-dimethylformamide (DMF) and stored at -20°C until further use.
  • DMF N,N-dimethylformamide
  • the synthesized aminopolyester was named as "(aminoalcohol used)-(lactone used)-(structure (P-1) moiety size [kDa])". Properties of each aminopolyester synthesized using the t-Bu-P4 catalyst are shown in Tables 1-2. In the table, “Conv (%)” is the monomer conversion rate (%) calculated from the 1 H NMR spectrum, and “ Mn,th " is calculated from the molar ratio of the monomer and the aminoalcohol and the monomer conversion rate.
  • M n,SEC is the number average molecular weight calculated based on the SEC measurement results
  • D is the dispersity.
  • lipid and nucleic acid amounts > AA03-DL-5 and DMG-PEG2k are used as constituent lipids, EGFP mRNA is prepared so that the total lipid amount to RNA ratio (molar ratio) is 5: 1 to 100: 1, and lipid nanoparticles for EGFP expression are prepared. was prepared. The obtained lipid nanoparticles were introduced into HeLa cells, and EGFP expression efficiency was examined by measuring EGFP fluorescence. The results are shown in FIG. Lipid nanoparticles for EGFP expression prepared at a total lipid to RNA ratio (molar ratio) of 30:1 exhibited the highest EGFP expression efficiency.
  • lipid nanoparticles for EGFP expression were prepared at a total lipid to EGFP mRNA ratio (molar ratio) of 30:1.
  • the average particle size, PDI, and zeta potential of these EGFP-expressing lipid nanoparticles were measured, and the nucleic acid recovery rate and nucleic acid encapsulation rate in the lipid nanoparticles were also measured.
  • Tables 3 and 4 show the measurement results of each lipid nanoparticle. All lipid nanoparticles had a sufficiently small average particle size of 200 to 350 nm. The lipid nanoparticles had a zeta potential of -20 mV to -10 mV and had a negative surface charge. Furthermore, all lipid nanoparticles had a sufficiently large nucleic acid encapsulation rate of 80% or more. From these results, the lipid nanoparticles having aminopolyesters as constituent lipids according to the present invention are sufficiently small to reach the lung tissue when administered to the living body, and can efficiently encapsulate nucleic acids such as mRNA. was found to be useful as a gene delivery carrier targeting lung tissue.
  • lipid nanoparticles containing aminopolyester prepared using caprolactone as a monomer have a larger average particle size (>800 nm) and a PdI value (>0 .6) also tended to be large. That is, the aminopolyester prepared using caprolactone as a monomer hardly formed lipid nanoparticles (theocLogP ⁇ 0.94), while the aminopolyester prepared using ⁇ -decalactone as a monomer easily formed lipid nanoparticles (theo cLogP ⁇ 2.42). These results indicated that the hydrophobicity of the monomers used in aminopolyester synthesis is an important factor in the formation of lipid nanoparticles containing the aminopolyester as a constituent lipid.
  • EGFP-expressing lipid nanoparticles comprising aminopolyesters synthesized from linear aminoalcohols
  • EGFP-expressing lipid nanoparticles comprising aminopolyesters synthesized from aminoalcohols of moderate molecular weight (5000 g/mol) were used.
  • EGFP delivery efficiency to HeLa cells tended to be higher.
  • EGFP-expressing lipid nanoparticles containing an aminopolyester synthesized from a linear aminoalcohol having a large number of nitrogen atoms and a large number of hydroxyalkyl groups hereinafter sometimes referred to as "hydroxy tails" had high EGFP delivery efficiency.
  • lipid nanoparticles for EGFP expression containing aminopolyesters synthesized from AA03-DL-5, AA05-DL-5, and AA07-DL-5 with high hydroxy tails in the aminoalcohol showed very high EGFP delivery efficiency. achieved.
  • lipid nanoparticles for EGFP expression comprising aminopolyesters synthesized from aminoalcohols containing rings, more hydroxy tails or more nitrogen atoms in the aminoalcohol tended to lead to more efficient mRNA delivery.
  • Lipid nanoparticles for EGFP expression containing DL-10 were the highest performing gene expression carriers.
  • the cell viability of HeLa cells transfected with any of the EGFP-expressing lipid nanoparticles was 75% or more.
  • twice as many EGFP-expressing lipid nanoparticles were introduced in the MTT assay as in the previous EGFP-expressing experiments, cell viability was sufficiently high that this dose was well tolerated.
  • EGFP-expressing lipid nanoparticles comprising aminopolyesters synthesized from linear aminoalcohols except AA06
  • EGFP-expressing lipid nanoparticles comprising aminopolyesters synthesized from ring-containing aminoalcohols. showed better biocompatibility.
  • Example 2 The lipid nanoparticles for EGFP expression prepared in Example 1 were administered to mice, and 8 hours after administration, the major organs, lung, liver, spleen, kidney, and heart were collected, and mRNA delivery to each organ. Efficiency was measured. Tables 6 and 7 show the fluorescence intensity measurements of lung, liver, and spleen.
  • the gene expression activity in the lung, liver, and spleen differed greatly among the lipid nanoparticles, but almost all lipid nanoparticles for EGFP expression differed in molecular weight of the aminopolyester used and synthesis.
  • Preferential expression of EGFP in lung tissue was confirmed regardless of the type of amino alcohol used.
  • EGFP-expressing lipid nanoparticles, including AA11-DL-3, AA11-DL-5, and AA11-DL-10 showed comparable mRNA delivery in lung and liver.
  • lipid nanoparticles for EGFP expression containing aminopolyesters synthesized from linear aminoalcohols had higher molecular weight aminopolyesters (10kDa) than those with lower molecular weight aminopolyesters (3kDa) and Expression was higher in the lung than with a medium molecular weight aminopolyester (5 kDa).
  • aminopolyesters with large molecular weights and 2 or 4 hydroxy tails (AA03-DL-5, AA03-DL-10, AA05-DL-5, AA05-DL-10, AA09-DL-5, AA09- Lipid nanoparticles for EGFP expression containing DL-10) had high lung tissue-specific gene expression activity.
  • Lipid nanoparticles for EGFP expression including AA03-DL-10, AA04-DL-10, AA05-DL-10, and AA09-DL-5, which had high lung tissue-specific gene expression activity, and relatively lung tissue expression activity
  • a pie chart shows the expression ratios in the lung, liver, spleen, kidney, and heart of EGFP-expressing lipid nanoparticles containing AA07-DL-10 and AA11-DL-5, for which the expression activity of EGFP was high (FIG. 3).
  • EGFP-expressing lipid nanoparticles containing AA03-DL-10 or AA05-DL-10 were administered, more than 90% of the EGFP fluorescence was from the lung.
  • administration of EGFP-expressing lipid nanoparticles containing AA07-DL-10 or AA09-DL-5 resulted in lung tissue selectivity of 66% and 85%, respectively.
  • fluorescence intensities of the BL6 mouse lung, liver and spleen were almost the same as those of the ICR mouse lung, liver and spleen. That is, BL6 mice were also confirmed to have high lung tissue-selective gene expression activity, as in ICR mice.
  • Example 3 Gene expression activity in each tissue was examined for lipid nanoparticles containing an aminopolyester synthesized using a seven-membered ring lactone and lipid nanoparticles containing an aminopolyester synthesized using a six-membered ring lactone.
  • lipid nanoparticles containing an aminopolyester synthesized using a seven-membered ring lactone lipid nanoparticles for EGFP expression containing AA03-DL-10, which had the highest lung tissue-selective gene expression activity in Example 2, were used. Using.
  • lipid nanoparticles containing an aminopolyester synthesized using a six-membered ring lactone those prepared by the following method were used.
  • ⁇ -decalactone also called valerolactone (VL)
  • VL valerolactone
  • AA03 aminopolyester having a structure (P-1) portion of 10 kDa. -VL-10) was obtained (D ⁇ 1.2).
  • lipid nanoparticles for EGFP expression were prepared in the same manner as described above except that AA03-VL-10 was used. This lipid nanoparticle for EGFP expression was used as a lipid nanoparticle containing an aminopolyester synthesized using a six-membered ring lactone.
  • Lipid nanoparticles for EGFP expression containing AA03-DL-10 and lipid nanoparticles for EGFP expression containing AA03-VL-10 were administered to mice in the same manner as in Example 2, and 8 hours after administration, major organs Certain lungs, livers, spleens, kidneys, and hearts were harvested to measure the efficiency of mRNA delivery to each organ.
  • FIG. 4 shows the measurement results of the fluorescence intensity of the lung, liver and spleen. Unlike the EGFP-expressing lipid nanoparticles containing AA03-DL-10, the EGFP-expressing lipid nanoparticles containing AA03-VL-10 showed almost no gene expression in the lung tissue, and gene expression was mainly in the liver and spleen.
  • TEM Transmission electron microscopy
  • the TEM image was taken at an acceleration voltage of 120 kW using a field emission transmission electron microscope "FEI Tecnai (registered trademark) G2 SpiritBiotwin" (manufactured by Thermo Fisher Scientific).
  • FEI Tecnai registered trademark
  • G2 SpiritBiotwin manufactured by Thermo Fisher Scientific.
  • the formed lipid nanoparticles were first placed on a TEM grid covered with a carbon membrane and excess liquid was sucked up with filter paper. The copper grid was then dried under vacuum for 1 hour before being imaged by TEM.
  • Fig. 5 shows a TEM image of lipid nanoparticles for EGFP expression containing AA03-DL-10.
  • the lipid nanoparticles for EGFP expression containing AA03-DL-10 were spherical nanoparticles with an average particle size of about 150 nm.
  • Example 4 the safety of EGFP-expressing lipid nanoparticles containing AA03-DL-10, which had the highest lung tissue-selective gene expression activity, was examined. Specifically, lipid nanoparticles for EGFP expression containing AA03-DL-10 were administered to ICR mice through the tail vein so that the dose of EGFP mRNA per animal was 0.5 mg/kg. As a control, PBS was similarly administered to ICR mice. Eight hours after dosing, major organs were collected for histological analysis, and serum was collected for determination of lung, liver, kidney, and heart toxicity markers.
  • Fig. 6 shows the stained images of the collected tissue sections. Histological analysis of each tissue section revealed no signs of toxicity in mice administered EGFP-expressing lipid nanoparticles containing AA03-DL-10 compared to PBS-treated mice.
  • lipid nanoparticles containing AA03-DL-10 can be safely administered even at twice the dose (0.5 mg/kg) of the lung tissue-selective gene expression dose.
  • lipid nanoparticles containing aminopolyesters of the present invention, including AA03-DL-10 can efficiently encapsulate nucleic acids for gene expression, and can be efficiently carried into lung tissue. In addition to being taken up, it is also relatively safe. Therefore, the lipid nanoparticles are useful as carriers for delivering basic medicinal ingredients to lung tissue for treatment of pulmonary diseases. It can be an active ingredient of a pharmaceutical composition for pulmonary diseases for which there are few effective pharmaceuticals.

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  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Pulmonology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Polyesters Or Polycarbonates (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un aminopolyester ayant une structure représentée par la formule générale (P-1) [dans laquelle la fraction R11 ou R15 est un groupe alkyle en C1-C12 et le reste est un atome d'hydrogène; n est un nombre entier supérieur ou égal à 2; q est un nombre entier de 1 à 6; et les points noirs indiquent chacun une liaison avec d'autres groupes] et des nanoparticules lipidiques comprenant l'aminopolyester.
PCT/JP2022/000164 2021-01-20 2022-01-06 Aminopolyester et nanoparticules lipidiques WO2022158290A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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