JP6358661B2 - Surfactant-like compound - Google Patents
Surfactant-like compound Download PDFInfo
- Publication number
- JP6358661B2 JP6358661B2 JP2015506743A JP2015506743A JP6358661B2 JP 6358661 B2 JP6358661 B2 JP 6358661B2 JP 2015506743 A JP2015506743 A JP 2015506743A JP 2015506743 A JP2015506743 A JP 2015506743A JP 6358661 B2 JP6358661 B2 JP 6358661B2
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- JP
- Japan
- Prior art keywords
- peg
- dna
- surfactant
- compound
- complex
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 108020004414 DNA Proteins 0.000 description 163
- 229920001223 polyethylene glycol Polymers 0.000 description 96
- 238000012360 testing method Methods 0.000 description 35
- 125000001165 hydrophobic group Chemical group 0.000 description 25
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- 239000002245 particle Substances 0.000 description 18
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- 239000000243 solution Substances 0.000 description 17
- 125000002091 cationic group Chemical group 0.000 description 16
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- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 15
- 125000001424 substituent group Chemical group 0.000 description 15
- 238000012377 drug delivery Methods 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- RAXXELZNTBOGNW-UHFFFAOYSA-N 1H-imidazole Chemical group C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 11
- 238000001727 in vivo Methods 0.000 description 11
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 11
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol group Chemical group [C@@H]1(CC[C@H]2[C@@H]3CC=C4C[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)[C@H](C)CCCC(C)C HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 10
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- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- 238000004020 luminiscence type Methods 0.000 description 7
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- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 6
- 125000000217 alkyl group Chemical group 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
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- 108060001084 Luciferase Proteins 0.000 description 5
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- MPPPKRYCTPRNTB-UHFFFAOYSA-N 1-bromobutane Chemical compound CCCCBr MPPPKRYCTPRNTB-UHFFFAOYSA-N 0.000 description 4
- 101100297347 Caenorhabditis elegans pgl-3 gene Proteins 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- 239000005089 Luciferase Substances 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 125000003368 amide group Chemical group 0.000 description 4
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- 125000000524 functional group Chemical group 0.000 description 3
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- 238000000751 protein extraction Methods 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical group NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
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- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 125000003827 glycol group Chemical group 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 125000001183 hydrocarbyl group Chemical group 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 125000002883 imidazolyl group Chemical group 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- PBBAFLCORNAZCD-UHFFFAOYSA-N nonanoyl nonanoate Chemical compound CCCCCCCCC(=O)OC(=O)CCCCCCCC PBBAFLCORNAZCD-UHFFFAOYSA-N 0.000 description 2
- 231100000956 nontoxicity Toxicity 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 239000011882 ultra-fine particle Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- VMKOFRJSULQZRM-UHFFFAOYSA-N 1-bromooctane Chemical compound CCCCCCCCBr VMKOFRJSULQZRM-UHFFFAOYSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical group CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- LGCGXHIRLZORQA-UHFFFAOYSA-N 6-bromohexanamide Chemical compound NC(=O)CCCCCBr LGCGXHIRLZORQA-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
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- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical group NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101000934888 Homo sapiens Succinate dehydrogenase cytochrome b560 subunit, mitochondrial Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- RAXXELZNTBOGNW-UHFFFAOYSA-O Imidazolium Chemical group C1=C[NH+]=CN1 RAXXELZNTBOGNW-UHFFFAOYSA-O 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229920002873 Polyethylenimine Polymers 0.000 description 1
- 229920001218 Pullulan Polymers 0.000 description 1
- 239000004373 Pullulan Substances 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 102100025393 Succinate dehydrogenase cytochrome b560 subunit, mitochondrial Human genes 0.000 description 1
- QNEPTKZEXBPDLF-JDTILAPWSA-N [(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-yl] carbonochloridate Chemical compound C1C=C2C[C@@H](OC(Cl)=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 QNEPTKZEXBPDLF-JDTILAPWSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 125000005037 alkyl phenyl group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000003816 antisense DNA Substances 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000001244 carboxylic acid anhydrides Chemical class 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
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- 238000004132 cross linking Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 125000002704 decyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
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- 238000005886 esterification reaction Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- ALBYIUDWACNRRB-UHFFFAOYSA-N hexanamide Chemical group CCCCCC(N)=O ALBYIUDWACNRRB-UHFFFAOYSA-N 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- QAFBDRSXXHEXGB-UHFFFAOYSA-N imidazol-1-ylacetic acid Chemical compound OC(=O)CN1C=CN=C1 QAFBDRSXXHEXGB-UHFFFAOYSA-N 0.000 description 1
- 125000005462 imide group Chemical group 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 125000001400 nonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 230000001293 nucleolytic effect Effects 0.000 description 1
- WVJVHUWVQNLPCR-UHFFFAOYSA-N octadecanoyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC(=O)CCCCCCCCCCCCCCCCC WVJVHUWVQNLPCR-UHFFFAOYSA-N 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000002165 photosensitisation Effects 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920002401 polyacrylamide Chemical group 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 235000019423 pullulan Nutrition 0.000 description 1
- 125000001725 pyrenyl group Chemical group 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 238000001226 reprecipitation Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N urea group Chemical group NC(=O)N XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
- C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
- C08G65/33303—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
- C08G65/33317—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group heterocyclic
-
- 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
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
- C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
- C08G65/3332—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing carboxamide group
- C08G65/33324—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing carboxamide group acyclic
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
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Description
本発明は、界面活性剤様化合物に関し、さらに詳しくは、生体適合性及び血液中での安定性が高く、カチオン過剰による毒性が低く、小粒子径で標的部位に移送され易く、標的部位での発現効率が高い核酸複合体を形成することができる界面活性剤様化合物に関するものである。 The present invention relates to a surfactant-like compound, and more specifically, has high biocompatibility and stability in blood, low toxicity due to excess cation, and is easily transported to a target site with a small particle size. The present invention relates to a surfactant-like compound capable of forming a nucleic acid complex with high expression efficiency.
近年、プラスミドＤＮＡやアンチセンスＤＮＡなどの核酸を用いる遺伝子治療が注目されている。遺伝子治療の重要な要素として、核酸を効率よく標的部位へ送る薬物伝達システム（ドラッグデリバリーシステム（ＤＤＳ））が挙げられる。
従来の核酸のドラッグデリバリーシステムでは、核酸デリバリーのために、核酸と核酸キャリアとの複合体が用いられてきた。
その複合体は、生体内の毒性が低く、核酸を核酸分解酵素から保護し、効率的に標的部位へ輸送することが求められており、複合体の形成には分子内に数多くのカチオン性基を有する核酸キャリアと核酸複合体とのポリイオンコンプレックス形成などの手法が用いられてきた。
たとえば、特許文献１には、ｓｉＲＮＡとも生理的条件下で安定な会合体を提供する共重合体として、親水性ポリマー鎖ブロックが該ポリアミノ酸鎖ブロックの主鎖のいずれか一方の末端に共有結合を介して結合しており、かつ、該ポリアミノ酸鎖ブロックにおけるアミノ酸反復単位１０パーセント以上から７０パーセント以下の側鎖に疎水性基が共有結合を介して結合している共重合体が提案されている。
特許文献２には、インビボでの肝臓への核酸のターゲティングの効率および取込の効率を高めるための製剤として、核酸とカチオン化プルラン誘導体から形成されるポリイオンコンプレックスを含み、前記カチオン化プルラン誘導体のカチオン化率が１０−２７％であることを特徴とする、肝臓デリバリー用製剤が提案されている。
特許文献３には、二本鎖リボ核酸と、多数の含窒素基を有するブロック共重合体とが静電結合されてなる非高分子ミセル形態のポリイオンコンプレックスであって、動的光散乱測定法で測定した場合に１００ｎｍ未満の平均粒径を有するポリイオンコンプレックスが提案されている。
特許文献４には、血清中において光増感性物質を十分に保持し構造安定性に優れたポリイオンコンプレックス、及びその構成成分である核酸ポリプレックスとして、複数の窒素含有カチオン性基を含むカチオン性ポリマーを含むポリイオンコンプレックスが提案されている。In recent years, gene therapy using nucleic acids such as plasmid DNA and antisense DNA has attracted attention. An important element of gene therapy is a drug delivery system (drug delivery system (DDS)) that efficiently sends nucleic acids to a target site.
In conventional nucleic acid drug delivery systems, a complex of a nucleic acid and a nucleic acid carrier has been used for nucleic acid delivery.
The complex has low toxicity in vivo, and is required to protect the nucleic acid from the nucleolytic enzyme and efficiently transport it to the target site. For the formation of the complex, many cationic groups are present in the molecule. Techniques such as formation of a polyion complex of a nucleic acid carrier having a nucleic acid and a nucleic acid complex have been used.
For example, Patent Document 1 discloses that a hydrophilic polymer chain block is covalently bonded to one end of the main chain of the polyamino acid chain block as a copolymer that provides a stable association with siRNA under physiological conditions. And a copolymer in which a hydrophobic group is bonded via a covalent bond to a side chain of 10% to 70% of amino acid repeating units in the polyamino acid chain block. Yes.
Patent Document 2 includes a polyion complex formed from a nucleic acid and a cationized pullulan derivative as a preparation for increasing the efficiency of targeting and uptake of the nucleic acid to the liver in vivo. A preparation for liver delivery characterized by a cationization rate of 10-27% has been proposed.
Patent Document 3 discloses a polyion complex in the form of a non-polymeric micelle in which double-stranded ribonucleic acid and a block copolymer having a large number of nitrogen-containing groups are electrostatically bonded, and a dynamic light scattering measurement method. A polyion complex has been proposed having an average particle size of less than 100 nm when measured by.
Patent Document 4 discloses a cationic polymer containing a plurality of nitrogen-containing cationic groups as a polyion complex that sufficiently retains a photosensitizing substance in serum and excellent in structural stability, and a nucleic acid polyplex that is a constituent component thereof. There have been proposed polyion complexes containing.
しかしながら、特許文献１〜４にかかる提案では、生体への毒性があるという問題や、イオンコンプレックスの粒子径が大きいため血管から標的部位への移送効率が悪く、標的部位での発現効率が悪いという問題があった。
このため、生体への毒性が低く、小粒子径で標的部位に移送され易く、標的部位での発現効率が高い核酸複合体を形成できる化合物の開発が要望されている。However, in the proposals according to Patent Documents 1 to 4, there is a problem that there is toxicity to the living body, and because the particle size of the ion complex is large, the transfer efficiency from the blood vessel to the target site is poor, and the expression efficiency at the target site is poor. There was a problem.
For this reason, there is a demand for the development of a compound that can form a nucleic acid complex that has low toxicity to a living body, is easily transported to a target site with a small particle size, and has high expression efficiency at the target site.
したがって、本発明の目的は、生体への毒性が低く、小粒子径で標的部位に移送され易く、標的部位での発現効率が高い核酸複合体を形成することができる界面活性剤様化合物を提供することにある。 Accordingly, an object of the present invention is to provide a surfactant-like compound that can form a nucleic acid complex that has low toxicity to a living body, is easily transported to a target site with a small particle size, and has high expression efficiency at the target site. There is to do.
本発明者らは、上記課題を解消すべく鋭意検討した結果、核酸と複合体を形成させる化合物の構造においてカチオン性基や窒素含有基の数をコントロールすることにより、当該化合物と核酸との複合体の物理的性質及び生物学的性質が変わることを知見し、さらに検討した結果、本発明を完成するに至った。
すなわち、本発明は以下の各発明を提供するものである。
１．親水性基と疎水性基とそれらの間に位置する１〜５個の含窒素基とを具備することを特徴とする界面活性剤様化合物。
２．上記親水性基が
非イオン性の親水性基である
１に記載の界面活性剤様化合物。
３． 上記親水性基が
下記式（１）で表されるエチレングリコール基である
１または２に記載の界面活性剤様化合物。
Ｒ−（ＣＨ２−ＣＨ２−Ｏ）ｎ− ・・・・・（１）
（式中、ｎは４０〜１２０の整数を表す。Ｒは炭素数１〜１８の炭化水素基を示す。）
４．上記疎水性基が
炭素数１〜１８の直鎖又は分岐鎖のアルキル基又は、コレステロール基である
１〜３のいずれかに記載の界面活性剤様化合物。
５．上記疎水性基が、その一部に疎水性ではない置換基を有する、
１〜４記載のいずれかに記載の界面活性剤様化合物。
６．上記置換基が、水素結合性官能基含有置換基である
５に記載の界面活性剤様化合物。
７．上記置換基が、上記疎水性基の末端に位置する
６に記載の界面活性剤様化合物。
８．上記疎水性基が、下記式１に示す１−アミド−アルキル基、１−ヒドロキシ−アルキル基、１−チミン−アルキル基である
７に記載の界面活性剤様化合物。
９．上記含窒素基が
アミド基又はイミダゾリウム基である
１〜８のいずれかに記載の界面活性剤様化合物。
１０．上記疎水性基と上記含窒素基との間にカチオン性基を有する
１〜９のいずれかに記載の界面活性剤様化合物。
１１．上記カチオン性基がイミダゾリウム基である
１０に記載の界面活性剤様化合物。
１２．核酸複合体形成用である
１〜１１のいずれかに記載の界面活性剤様化合物。As a result of diligent studies to solve the above-mentioned problems, the present inventors have controlled the number of cationic groups and nitrogen-containing groups in the structure of a compound that forms a complex with a nucleic acid, thereby combining the compound with the nucleic acid. As a result of finding out and further examining the physical and biological properties of the body, the present invention has been completed.
That is, the present invention provides the following inventions.
1. A surfactant-like compound comprising a hydrophilic group, a hydrophobic group, and 1 to 5 nitrogen-containing groups located therebetween.
2. 2. The surfactant-like compound according to 1, wherein the hydrophilic group is a nonionic hydrophilic group.
3. The surfactant-like compound according to 1 or 2, wherein the hydrophilic group is an ethylene glycol group represented by the following formula (1).
R— (CH 2 —CH 2 —O) n − (1)
(In the formula, n represents an integer of 40 to 120. R represents a hydrocarbon group having 1 to 18 carbon atoms.)
4). The surfactant-like compound according to any one of 1 to 3, wherein the hydrophobic group is a linear or branched alkyl group having 1 to 18 carbon atoms or a cholesterol group.
5). The hydrophobic group has a non-hydrophobic substituent in a part thereof,
The surfactant-like compound according to any one of 1 to 4.
6). 6. The surfactant-like compound according to 5, wherein the substituent is a hydrogen-bonding functional group-containing substituent.
7). 7. The surfactant-like compound according to 6, wherein the substituent is located at the end of the hydrophobic group.
8). 8. The surfactant-like compound according to 7, wherein the hydrophobic group is a 1-amido-alkyl group, 1-hydroxy-alkyl group, or 1-thymine-alkyl group represented by the following formula 1.
9. The surfactant-like compound according to any one of 1 to 8, wherein the nitrogen-containing group is an amide group or an imidazolium group.
10. The surfactant-like compound according to any one of 1 to 9, which has a cationic group between the hydrophobic group and the nitrogen-containing group.
11. 11. The surfactant-like compound according to 10, wherein the cationic group is an imidazolium group.
12 The surfactant-like compound according to any one of 1 to 11, which is used for forming a nucleic acid complex.
本発明の界面活性剤様化合物は、生体適合性及び血液中での安定性が高く、生体への毒性が低く、小粒子径で標的部位に移送され易く、標的部位での発現効率が高い核酸複合体を形成することができる界面活性剤様化合物で、核酸複合体形成用に好ましく用いることができる。 The surfactant-like compound of the present invention is a nucleic acid having high biocompatibility and stability in blood, low toxicity to the living body, small particle size, easy transport to the target site, and high expression efficiency at the target site. A surfactant-like compound capable of forming a complex and can be preferably used for forming a nucleic acid complex.
以下、本発明をさらに詳細に説明する。
本発明の界面活性剤様化合物は、親水性基と疎水性基とそれらの間に位置する特定個数の含窒素基とを具備することを特徴とする。Hereinafter, the present invention will be described in more detail.
The surfactant-like compound of the present invention is characterized by comprising a hydrophilic group, a hydrophobic group, and a specific number of nitrogen-containing groups located therebetween.
＜親水性基＞
上記親水性基としては、特に非イオン性の親水性基が好ましく挙げられる。
上記非イオン性の親水性基としては、エチレングリコール基、デキストラン基、ポリアクリルアミド基などが挙げられ、下記式（１）で表されるエチレングリコール基が好ましく挙げられる。
Ｒ−（ＣＨ２−ＣＨ２−Ｏ）ｎ− ・・・・・（１）
（式中、ｎは４０〜１２０の整数を表す。Ｒは炭素数１〜１８の直鎖又は分岐鎖のアルキル基を示す。）
これにより、生体適合性が付与され、核酸と複合体を形成した場合における複合体の水に対する溶解性を高め、且つ、血中などの生理条件下での血清タンパク質や細胞表面などとの非特異的な相互作用を抑制し血中での安定性が高くなる。
具体的には以下に示すエチレングリコール基が好ましく挙げられる。
ＣＨ３−（ＣＨ２−ＣＨ２−Ｏ）ｎ−、
Ｃ２Ｈ５−（ＣＨ２−ＣＨ２−Ｏ）ｎ−、
Ｃ３Ｈ７−（ＣＨ２−ＣＨ２−Ｏ）ｎ−、
Ｃ４Ｈ９−（ＣＨ２−ＣＨ２−Ｏ）ｎ−、
Ｃ５Ｈ１１−（ＣＨ２−ＣＨ２−Ｏ）ｎ−、
なお、これらの例示の基において重合度を示すｎは４０〜１２０となる範囲である。<Hydrophilic group>
As the hydrophilic group, a nonionic hydrophilic group is particularly preferable.
Examples of the nonionic hydrophilic group include an ethylene glycol group, a dextran group, and a polyacrylamide group, and an ethylene glycol group represented by the following formula (1) is preferable.
R— (CH 2 —CH 2 —O) n − (1)
(In the formula, n represents an integer of 40 to 120. R represents a linear or branched alkyl group having 1 to 18 carbon atoms.)
This provides biocompatibility, enhances the solubility of the complex in water when it forms a complex with nucleic acid, and is non-specific to serum proteins and cell surfaces under physiological conditions such as blood. Suppresses natural interactions and increases stability in blood.
Specifically, the following ethylene glycol groups are preferred.
CH 3 - (CH 2 -CH 2 -O) n -,
C 2 H 5 - (CH 2 -CH 2 -O) n -,
C 3 H 7 - (CH 2 -CH 2 -O) n -,
C 4 H 9 - (CH 2 -CH 2 -O) n -,
C 5 H 11 - (CH 2 -CH 2 -O) n -,
In these exemplary groups, n indicating the degree of polymerization is in the range of 40 to 120.
＜疎水性基＞
上記疎水性基としては、疎水性の炭化水素基、直鎖又は分岐鎖のアルキル基、アリール基、コレステロール基、ポルフィリン基、脱プロトン化したイミダゾール基などが挙げられ、炭素数１〜１８の直鎖又は分岐鎖のアルキル基が好ましく挙げられる。
具体的には以下の疎水性基などが挙げられる。
直鎖又は分岐鎖のアルキル基：メチル基、エチル基、ブチル基、プロピル基、ペンチル基、ヘキシル基、オクチル基、ノニル基、デキル基、ラウリル基、ステアリル基、イソプロピル基、ｔｅｒｔ−ブチル基
アリール基：フェニル基、ベンジル基、ナフチル基、アルキルフェニル基、ピレニル基
コレステロール基：下記化式に示すコレステロール基
ポルフィリン基：下記化式に示すポルフィリン基
脱プロトン化したイミダゾール基：下記化式に示すイミダゾール基
これにより、ＤＮＡと疎水性の相互作用をすることができ、核酸と安定した複合体を形成することができる。
Examples of the hydrophobic group include a hydrophobic hydrocarbon group, a linear or branched alkyl group, an aryl group, a cholesterol group, a porphyrin group, a deprotonated imidazole group, and the like. A chain or branched alkyl group is preferred.
Specific examples include the following hydrophobic groups.
Linear or branched alkyl group: methyl group, ethyl group, butyl group, propyl group, pentyl group, hexyl group, octyl group, nonyl group, decyl group, lauryl group, stearyl group, isopropyl group, tert-butyl group aryl Group: phenyl group, benzyl group, naphthyl group, alkylphenyl group, pyrenyl group cholesterol group: cholesterol group shown in the following formula: porphyrin group: porphyrin group shown in the following formula: deprotonated imidazole group: imidazole shown in the following formula By this, hydrophobic interaction with DNA can be performed, and a stable complex with nucleic acid can be formed.
また、上記疎水性基は、その一部に疎水性ではない置換基を有していてもよい。
上記疎水性ではない置換基としては、水素結合性官能基含有置換基などが好ましく挙げられ、中でも、アミド基、ヒドロキシル基、核酸塩基（チミン、シトシン、アデニン、グアニン、ウラシル）、修飾核酸塩基（５−メチルシトシン、５−ヒドロキシメチルシトシン）等の水素結合性官能基含有置換基であるのが好ましい。
上記疎水性基が、その一部に疎水性ではない置換基を有する場合、該置換基の位置は、上記疎水性基の末端に位置するのが好ましい。
このような一部に疎水性ではない置換基を有する疎水性基としては、上述のアルキル基等の基に上記疎水性ではない置換基が導入されてなる疎水性基であれば特に制限なく用いることができるが、下記化式に示す１−アミド−アルキル基、１−ヒドロキシ−アルキル基、１−チミン−アルキル基等を特に好ましく挙げることができる。
これにより、核酸と、より安定した複合体を形成することができる。
具体的には以下の疎水性基を挙げることができる。
Preferred examples of the non-hydrophobic substituent include hydrogen-bonding functional group-containing substituents. Among them, amide groups, hydroxyl groups, nucleobases (thymine, cytosine, adenine, guanine, uracil), modified nucleobases ( A hydrogen-containing functional group-containing substituent such as 5-methylcytosine and 5-hydroxymethylcytosine) is preferable.
When the hydrophobic group has a non-hydrophobic substituent in a part thereof, the position of the substituent is preferably located at the end of the hydrophobic group.
As such a hydrophobic group having a non-hydrophobic substituent in part, any hydrophobic group in which the non-hydrophobic substituent is introduced into the above-described alkyl group or the like can be used without particular limitation. The 1-amido-alkyl group, 1-hydroxy-alkyl group, 1-thymine-alkyl group and the like shown in the following chemical formula can be particularly preferably mentioned.
Thereby, a more stable complex can be formed with the nucleic acid.
Specifically, the following hydrophobic groups can be mentioned.
＜含窒素基＞
上記含窒素基は、分子中に１つ以上の窒素を含有する基であり、具体的には、アミド基、イミド基、ウレア基、イミダゾリウム基などが挙げられ、アミド基、イミダゾリウム基が好ましく挙げられる。
これにより、ＤＮＡグルーブの間と水素結合することができ、ＤＮＡと安定した複合体を形成することができる。
また、上記含窒素基の上記特定個数は、１〜５個であり、１個であるのが好ましい。この範囲内とすることにより、生体への毒性が低く、小粒子径な核酸複合体を形成することができる。
アミド基の具体例としては、下記する置換基などが挙げられる。
The nitrogen-containing group is a group containing one or more nitrogen atoms in the molecule, and specific examples include an amide group, an imide group, a urea group, an imidazolium group, and the like. Preferably mentioned.
Thereby, hydrogen bonds can be formed between the DNA grooves, and a stable complex with DNA can be formed.
Moreover, the said specific number of the said nitrogen-containing group is 1-5 pieces, and it is preferable that it is one piece. By setting it within this range, it is possible to form a nucleic acid complex having a small particle diameter with low toxicity to a living body.
Specific examples of the amide group include the following substituents.
＜カチオン性基＞
また、本発明の界面活性剤様化合物は、上記疎水性基と上記含窒素基との間にカチオン性基を有するのが好ましい。
上記カチオン性基としては、イミダゾリウム基、４級アミノ基、グアニジウム基、下記の置換基などが挙げられ、イミダゾリウム基が好ましく挙げられる。
これにより、核酸のリン酸基と静電相互作用で核酸同士の架橋を抑制し結合することができ、安定した複合体を形成することができる。また、モノカチオンであるためカチオン過剰による毒性がない複合体を形成できる。
上記カチオン性基の具体例としては、下記に示す基（イミダゾリウム基）等を好ましく挙げることができる。<Cationic group>
The surfactant-like compound of the present invention preferably has a cationic group between the hydrophobic group and the nitrogen-containing group.
Examples of the cationic group include an imidazolium group, a quaternary amino group, a guanidinium group, and the following substituents, and an imidazolium group is preferable.
Thereby, the nucleic acid phosphate group and the electrostatic interaction can suppress the cross-linking between the nucleic acids, and a stable complex can be formed. Moreover, since it is a monocation, a complex free from toxicity due to cation excess can be formed.
Specific examples of the cationic group include the following groups (imidazolium groups) and the like.
本発明の界面活性剤様化合物の具体例としては、以下の化合物などが挙げられる。
なお、本発明の界面活性剤様化合物は、界面活性剤と同様に親水性基と疎水性基とを分子の両末端に有する化合物という意味であり、必ずしも界面活性剤としての機能を有する意味ではない。
以下の式中ｎは上述の範囲のとおりである。
In addition, the surfactant-like compound of the present invention means a compound having a hydrophilic group and a hydrophobic group at both ends of the molecule in the same manner as the surfactant, and does not necessarily have a function as a surfactant. Absent.
In the following formula, n is as described above.
＜製造方法＞
本発明の界面活性剤様化合物の製造方法を説明する。
本発明の界面活性剤様化合物は、上記カチオン性基を具備しない場合、上記親水性基の末端をアミノ化した化合物と、上記疎水性基を具備するカルボン酸無水物、カルボン酸ハロゲン化物、カルボン酸アジドまたは活性エステルなどとを反応させ、アミド結合を形成させることなどにより製造することができる。
また、上記カチオン性基を具備する場合、上記カチオン性基のカルボン酸化合物をＮ−ヒドロキシスクシンイミド（ＮＨＳ）と反応させることで活性化エステルを合成し、該活性化エステルと上記親水性基の末端をアミノ化した化合物とを反応させることでアミド結合させた化合物を合成し、該化合物に上記疎水性基を具備するハロゲン化物を反応させることなどで上記疎水性基を結合させるなどして製造することができる。なお、当該ハロゲン化物としては市販品を用いることもできる。<Manufacturing method>
A method for producing the surfactant-like compound of the present invention will be described.
In the case where the surfactant-like compound of the present invention does not have the cationic group, the hydrophilic group-containing compound is aminated, and the hydrophobic group-containing carboxylic acid anhydride, carboxylic acid halide, carboxylic acid, and the like. It can be produced by reacting an acid azide or an active ester to form an amide bond.
When the cationic group is included, an activated ester is synthesized by reacting the carboxylic acid compound of the cationic group with N-hydroxysuccinimide (NHS), and the activated ester and the end of the hydrophilic group are synthesized. It is produced by synthesizing an amide-bonded compound by reacting with an aminated compound, and by bonding the hydrophobic group by reacting the compound with a halide having the hydrophobic group. be able to. In addition, a commercial item can also be used as the said halide.
本発明の界面活性剤様化合物は、核酸複合体形成用として好ましく用いることができる。
ここで、本発明の界面活性剤様化合物が複合体を形成しうる「核酸」としては、各種疾患の治療や予防の効果をもつＤＮＡやＲＮＡ、例えば、タンパク質をコードするＤＮＡやＲＮＡ、アンチセンスＤＮＡやＲＮＡ等のＤＮＡやＲＮＡ及びいわゆる人工核酸などが挙げられ、部分的に修飾されたものや、これらのキメラなどでもよい。
核酸複合体形成用として用いる場合、本発明の界面活性剤様化合物とＤＮＡやＲＮＡ等とを混合することで、本発明の界面活性剤様化合物とＤＮＡやＲＮＡ等の核酸との複合体を調製することができる。
本発明の界面活性剤様化合物と核酸との複合体は、粒子径が小さく且つ安定した複合体となる。これにより、体内における対象核酸の標的部位への輸送効率及び遺伝子発現効率が高くなるので、ドラッグデリバリーシステムとして好適である。
上記複合体を形成する際の本発明の界面活性剤様化合物と上記核酸との配合比は、核酸リン酸基の数に対する界面活性剤様化合物の分子数の比で界面活性剤様化合物：核酸＝０．１〜６４：１とするのが好ましく、０．１〜２：１とするのがさらに好ましい。
上記複合体を形成する際の上記核酸の鎖長は、２本鎖ＤＮＡの場合４０００〜６０００ｂｐ、１本鎖ＤＮＡまたはＲＮＡの場合２０〜３０ｂａｓｅであるのが好ましい。
また、複合体は、各種溶媒の存在下で形成することができ、この際用いることができる溶媒としては、任意の緩衝液（ＰＢＳ、ＨＥＰＥＳ等）等を挙げることができる。また、複合体の形成は、公知の手法を特に制限なく用いて行うことができる。
また、本発明の界面活性剤様化合物を用いて形成された上記複合体はカチオン過剰による毒性がなく、且つ、ポリエチレングリコール基など生体適合性を有する親水性基を具備することで生体適合性及び血液中での安定性が高く、ドラッグデリバリー用として好ましい。
ドラッグデリバリー用の用途としては、核酸を用いた遺伝子治療、ＲＮＡ干渉、ｍＲＮＡデリバリーなどの用途を挙げることができ、これらの用途には上記複合体を投与することで対応することができる。
上記複合体の投与は、皮下、皮内、静脈、動脈または筋肉内への注射、経粘膜（口腔、鼻、肺、眼、直腸、子宮など）投与、脳内投与、腫瘍内投与などにより行うことができる。その際、必要に応じて、希釈剤やゲルなどの添加物も用いることができる。
上記複合体の投与量および期間などは特に限定されず、有効成分としての核酸の種類や投与対象の体重、年齢など種々の条件により適宜選択することが可能である。
また、投与に際しては、物理的エネルギーとの併用により、デリバリー効率や標的組織の選択性を与えることも可能である。この際の物理的エネルギーとは、磁場、光、超音波、電気、圧力を意味する。The surfactant-like compound of the present invention can be preferably used for nucleic acid complex formation.
Here, the “nucleic acid” that can form a complex with the surfactant-like compound of the present invention is DNA or RNA having an effect of treating or preventing various diseases, for example, DNA or RNA encoding a protein, antisense. Examples thereof include DNA and RNA such as DNA and RNA, and so-called artificial nucleic acids, which may be partially modified, or chimeras thereof.
When used for forming a nucleic acid complex, a complex of the surfactant-like compound of the present invention and a nucleic acid such as DNA or RNA is prepared by mixing the surfactant-like compound of the present invention with DNA or RNA. can do.
The complex of the surfactant-like compound of the present invention and the nucleic acid is a stable complex having a small particle size. As a result, the efficiency of transporting the target nucleic acid to the target site and the gene expression efficiency in the body are high, which is suitable as a drug delivery system.
The compounding ratio of the surfactant-like compound of the present invention and the nucleic acid in forming the complex is the ratio of the number of molecules of the surfactant-like compound to the number of nucleic acid phosphate groups. = 0.1 to 64: 1 is preferable, and 0.1 to 2: 1 is more preferable.
The chain length of the nucleic acid when forming the complex is preferably 4000 to 6000 bp for double-stranded DNA and 20 to 30 base for single-stranded DNA or RNA.
The complex can be formed in the presence of various solvents, and examples of the solvent that can be used in this case include arbitrary buffer solutions (PBS, HEPES, etc.). The formation of the complex can be performed using a known technique without any particular limitation.
In addition, the complex formed using the surfactant-like compound of the present invention has no toxicity due to cation excess, and has a biocompatible hydrophilic group such as a polyethylene glycol group, thereby providing biocompatibility and It is highly stable in blood and is preferable for drug delivery.
Examples of uses for drug delivery include uses such as gene therapy using nucleic acids, RNA interference, and mRNA delivery, and these uses can be dealt with by administering the complex.
The complex is administered by subcutaneous, intradermal, intravenous, arterial or intramuscular injection, transmucosal (oral, nose, lung, eye, rectum, uterus, etc.) administration, intracerebral administration, intratumoral administration, etc. be able to. In that case, additives, such as a diluent and a gel, can also be used as needed.
The dose and period of the complex are not particularly limited, and can be appropriately selected according to various conditions such as the type of nucleic acid as an active ingredient, the body weight and age of the administration target.
In administration, it is also possible to give delivery efficiency and target tissue selectivity by combination with physical energy. The physical energy at this time means a magnetic field, light, ultrasonic waves, electricity, and pressure.
以下、本発明について実施例及び比較例を示してさらに具体的に説明するが本発明はこれらに何ら制限されるものではない。 EXAMPLES Hereinafter, although an Example and a comparative example are shown and this invention is demonstrated more concretely, this invention is not restrict | limited to these at all.
〔実施例１〕ＢｕＩｍ−ＰＥＧの合成
（イミダゾールＮＨＳ活性化エステルの合成）
１−イミダゾール酢酸１２６ｍｇと、Ｎ−ヒドロキシスクシンイミド（ＮＨＳ）１１５ｍｇとをＮ，Ｎ−ジメチルホルムアミド（ＤＭＦ）１０ｍＬで溶解させて溶液を得た。該溶液に、Ｎ，Ｎ’−ジシクロヘキシルカルボジイミド（ＤＣＣ）２０６ｍｇをＤＭＦ３ｍＬに溶解させたＤＣＣ溶液３ｍＬを加え、混合し、５０℃で１６時間反応させＮＨＳエステル化して活性化エステルを得た。
反応の説明図を以下に示す。
126 mg of 1-imidazole acetic acid and 115 mg of N-hydroxysuccinimide (NHS) were dissolved in 10 mL of N, N-dimethylformamide (DMF) to obtain a solution. To this solution, 3 mL of a DCC solution in which 206 mg of N, N′-dicyclohexylcarbodiimide (DCC) was dissolved in 3 mL of DMF was added, mixed, reacted at 50 ° C. for 16 hours, and NHS esterified to obtain an activated ester.
An explanatory diagram of the reaction is shown below.
（ＮＨＳエステルとＰＥＧ−ＮＨ２のカップリング反応によるＩｍ−ＰＥＧの合成）
ＮＨＳエステル化反応後の反応液に、以下に示すＰＥＧ−ＮＨ２（ＭＷ２０００、日油社製）４００ｍｇを直接加え混合し、５０℃で２４時間反応させた。反応を完全に行うため、該溶液に上記のＮＨＳ活性化エステル８９．２ｍｇを直接加え混合し、５０℃、２４時間の反応を繰り返し行いＩｍ−ＰＥＧ（アミド結合させた化合物）を得た。
反応の説明図を以下に示す。
To the reaction solution after the NHS esterification reaction, 400 mg of PEG-NH 2 (MW2000, manufactured by NOF Corporation) shown below was directly added and mixed, and reacted at 50 ° C. for 24 hours. In order to complete the reaction, 89.2 mg of the NHS-activated ester was directly added to the solution and mixed, and the reaction was repeated at 50 ° C. for 24 hours to obtain Im-PEG (an amide-bonded compound).
An explanatory diagram of the reaction is shown below.
（Ｉｍ−ＰＥＧの部分アルキル化によるＢｕＩｍ−ＰＥＧの合成
上記のカップリング反応後の反応後、単離したＩｍ−ＰＥＧ１００ｍｇをＤＭＦ７ｍＬで溶解させ、１−ブロモブタン（疎水性基を有するハロゲン化物）５３．７μＬ（６８．５ｍｇ）を加え混合し、２４時間、５０℃で反応させた。この１−ブロモブタンの添加を２回繰り返し行った。反応後の生成物を再沈殿及び透析により精製し、本発明の界面活性剤様化合物としてのＢｕＩｍ−ＰＥＧ５９．６ｍｇ（収率５５％）を得た。
反応の説明図を以下に示す。
An explanatory diagram of the reaction is shown below.
〔実施例２〕ＯｃＩｍ−ＰＥＧの合成
実施例１のＩｍ−ＰＥＧの部分アルキル化に用いた１−ブロモブタンを１−ブロモオクタン８６．４μＬ（９６．６ｍｇ）に変えた以外は実施例１と同様にして、本発明の界面活性剤様化合物としてのＯｃＩｍ−ＰＥＧ７０．０ｍｇ（収率６３％）を得た。
反応の説明図を以下に示す。
An explanatory diagram of the reaction is shown below.
〔実施例３〕Ｃｈｏｌ−ＰＥＧの合成
ＰＥＧ−ＮＨ２（ＭＷ２０００、日油社製、親水性基の末端をアミノ化した化合物）１２０ｍｇ及びクロロぎ酸コレステリル（疎水性基にコレステロール基が導入されたもののカルボン酸ハロゲン化物）５３．９ｍｇ、をクロロフォルム１０ｍＬに溶解させ、該溶液にトリエチルアミン（ＴＥＡ）８．３６μＬ（６．０７ｍｇ）を加え、４０℃で４８時間反応させた。反応後の生成物をろ過及び透析により精製し、本発明の界面活性剤様化合物としての下記式に示すＣｈｏｌ−ＰＥＧ１４３ｍｇ（収率９９％）を得た。
反応の説明図を以下に示す。
An explanatory diagram of the reaction is shown below.
〔実施例４〕Ｏｃ−ＰＥＧの合成
ＰＥＧ−ＮＨ２（ＭＷ２０００、日油社製）１２０ｍｇ、ノナン酸無水物３９．４μＬ（３５．８ｍｇ）、をＤＭＦ１０ｍＬに溶解させ、該溶液にトリエチルアミン（ＴＥＡ）８．３６μＬ（６．０７ｍｇ）を加え、５０℃で４８時間反応させた。反応後の生成物を透析により精製し、本発明の界面活性剤様化合物としてのＯｃ−ＰＥＧ４２ｍｇ（収率３５％）を得た。
反応の説明図を以下に示す。
An explanatory diagram of the reaction is shown below.
〔実施例５〕Ｓｔ−ＰＥＧの合成
実施例４のノナン酸無水物をステアリン酸無水物１００ｍｇ、及び、反応温度を４０℃に変えた以外は実施例４と同様にして、本発明の界面活性剤様化合物としてのＳｔ−ＰＥＧ６６．８ｍｇ（収率５９％）を得た。
反応の説明図を以下に示す。
An explanatory diagram of the reaction is shown below.
〔実施例６〕ＨｅＡ−Ｉｍ−ＰＥＧ（「ＡＰｅ−Ｉｍ−ＰＥＧ」と略記することもある）の合成
実施例１のＩｍ−ＰＥＧの部分アルキル化に用いた１−ブロモブタンを６‐ブロモヘキサンアミド５４．３ｍｇに変えた以外は実施例１と同様にして、疎水性基としてヘキサンアミド基（ＨｅＡ）（「１-アミド-ペンチル基（ＡＰｅ）」と同義）を有する本発明の界面活性剤様化合物としてのＨｅＡ−Ｉｍ−ＰＥＧ（ＡＰｅ−Ｉｍ−ＰＥＧ）５０ｍｇ（収率８０％）を得た。
反応の説明図を以下に示す。
なお、得られたＨｅＡ−Ｉｍ−ＰＥＧは、ＮＭＲ装置（装置名：ＡＶ５００、Ｂｒｕｋｅｒ社製）を用い、１Ｈ−ＮＭＲスペクトルを測定して合成の確認を行った。
その結果を図９に示す。[Example 6] Synthesis of HeA-Im-PEG (may be abbreviated as "APe-Im-PEG") 1-Bromobutane used for partial alkylation of Im-PEG of Example 1 is 6-bromohexanamide The surfactant-like surfactant of the present invention having a hexaneamide group (HeA) (synonymous with “1-amido-pentyl group (APe)”) as a hydrophobic group in the same manner as in Example 1 except that the amount was changed to 54.3 mg. 50 mg (yield 80%) of HeA-Im-PEG (APe-Im-PEG) as a compound was obtained.
An explanatory diagram of the reaction is shown below.
In addition, the obtained HeA-Im-PEG was confirmed for synthesis by measuring a 1 H-NMR spectrum using an NMR apparatus (apparatus name: AV500, manufactured by Bruker).
The result is shown in FIG.
〔試験例１〕ＢｕＩｍ−ＰＥＧ・ＤＮＡ複合体の評価
（界面活性剤様化合物・ＤＮＡ複合体の調製）
ＤＮＡとして市販のプラスミドであるｐＧＬ３（プロメガ社）０．３μｇと、実施例１で得られた本発明の界面活性剤様化合物としてのＢｕＩｍ−ＰＥＧとをＤＮＡのリン酸基の数１に対しＢｕＩｍ−ＰＥＧの分子数が０．１，０．５，１，２，４，８，１６，３２及び６４の量になるようにリン酸緩衝液（組成５０ｍＭ ＮａＨ２ＰＯ４＋５０ｍＭ Ｎａ２ＨＰＯ４）で最終液量が１５μＬになるように用いて溶解させた。溶解後、３７℃で２４時間インキュベーションし、界面活性剤様化合物・ＤＮＡ複合体としてのＢｕＩｍ−ＰＥＧ・ＤＮＡ複合体を調製した。[Test Example 1] Evaluation of BuIm-PEG / DNA complex (preparation of surfactant-like compound / DNA complex)
0.3 μg of pGL3 (Promega), a commercially available plasmid as DNA, and BuIm-PEG as the surfactant-like compound of the present invention obtained in Example 1 were combined with BuIm for the number of phosphate groups of DNA. -Phosphate buffer solution (composition 50 mM NaH 2 PO 4 +50 mM Na 2 HPO 4 ) so that the number of PEG molecules is 0.1, 0.5, 1, 2, 4, 8, 16, 32 and 64 The final solution volume was 15 μL. After dissolution, the mixture was incubated at 37 ° C. for 24 hours to prepare a BuIm-PEG / DNA complex as a surfactant-like compound / DNA complex.
（界面活性剤様化合物・ＤＮＡ複合体のアガロースゲル電気泳動による分析）
ＢｕＩｍ−ＰＥＧ・ＤＮＡ複合体をアガロースゲル電気泳動により分析し、ＤＮＡ複合体形成を確認した。
得られたＢｕＩｍ−ＰＥＧ・ＤＮＡ複合体溶液を、ピペッティングにより混合し、全量をエチジウムブロマイド１μｇ／ｍＬ含有アガロースゲルにロードし、下記の条件で電気泳動を行った。
条件：
アガロースゲル： アガロース１重量％、緩衝液リン酸緩衝液（組成５０ｍＭ ＮａＨ２ＰＯ４＋５０ｍＭ Ｎａ２ＨＰＯ４）
電圧：５０ｍＶ
電気泳動時間：３０分
電気泳動終了後のアガロースゲルを、ＵＶトランスイルミネーターで発色させ、ゲル撮影装置（装置名ＧｅｌＤｏｃ２０００、ＢＩＯ−ＲＡＤ社製）にて画像を取得した。
なお、比較サンプルとして、界面活性剤様化合物を含まないＤＮＡのみのサンプルも合わせて試験した。
その結果を図１に示す。
なお、図中のＰＥＧ／Ｐは界面活性剤様化合物・ＤＮＡ複合体におけるＤＮＡのリン酸基とＰＥＧの分子数の比を意味し、ＤＮＡのリン酸基の数を１とした場合のＰＥＧの分子数を表す。また、図中のＤＮＡは界面活性剤様化合物を含まないＤＮＡのみのサンプルを意味する。(Anarose gel electrophoresis analysis of surfactant-like compound and DNA complex)
The BuIm-PEG • DNA complex was analyzed by agarose gel electrophoresis to confirm the formation of the DNA complex.
The resulting BuIm-PEG / DNA complex solution was mixed by pipetting, and the entire amount was loaded on an agarose gel containing 1 μg / mL ethidium bromide, and electrophoresis was performed under the following conditions.
conditions:
Agarose gel: 1% by weight of agarose, buffer phosphate buffer (composition 50 mM NaH 2 PO 4 +50 mM Na 2 HPO 4 )
Voltage: 50mV
Electrophoresis time: 30 minutes The agarose gel after the completion of electrophoresis was developed with a UV transilluminator, and an image was obtained with a gel imaging device (device name: GelDoc2000, manufactured by BIO-RAD).
In addition, as a comparative sample, a sample containing only DNA containing no surfactant-like compound was also tested.
The result is shown in FIG.
In the figure, PEG / P means the ratio of the number of DNA phosphate groups to the number of PEG molecules in the surfactant-like compound / DNA complex. Represents the number of molecules. Moreover, DNA in the figure means a sample containing only DNA that does not contain a surfactant-like compound.
（結果及び考察）
図１に示す結果からＰＥＧ／Ｐが０．１以上において矢印で示すように陰極方向へのバンドのシフトがみられ、８及び１６ではシフトしたバンドの量が多くなり、３２及び６４においてはバンドの消失が見られた。
バンドのシフトは、ＢｕＩｍ−ＰＥＧとＤＮＡとが複合体を形成し分子量が大きくなり、且つ、ＤＮＡ電荷の部分的な打消しが起こり、泳動距離が短い方向にシフトすることを意味する。
バンドの消失は、ＢｕＩｍ−ＰＥＧとＤＮＡとが複合体を形成し且つＤＮＡが凝集状態になることでエチジウムブロマイドをインターカレーションできずＤＮＡが染色されないことを意味する。
これらの結果から、ＢｕＩｍ−ＰＥＧとＤＮＡとは、複合体を形成し、ＤＮＡが凝集しており特に３２及び６４においてはＢｕＩｍ−ＰＥＧ・ＤＮＡ複合体の安定性が高いことが判る。(Results and discussion)
From the results shown in FIG. 1, a band shift toward the cathode is observed as indicated by an arrow when PEG / P is 0.1 or more, the amount of the shifted band increases in 8 and 16, and the band in 32 and 64 Disappearance was observed.
The band shift means that BuIm-PEG and DNA form a complex to increase the molecular weight, and the DNA charge is partially canceled and the migration distance is shifted in a shorter direction.
The disappearance of the band means that BuIm-PEG and DNA form a complex and the DNA is in an aggregated state, so that ethidium bromide cannot be intercalated and DNA is not stained.
From these results, it can be seen that BuIm-PEG and DNA form a complex, and the DNA is aggregated. In particular, in 32 and 64, the stability of the BuIm-PEG • DNA complex is high.
〔試験例２〕ＯｃＩｍ−ＰＥＧ・ＤＮＡ複合体の評価
（界面活性剤様化合物・ＤＮＡ複合体のアガロースゲル電気泳動による分析）
界面活性剤様化合物として実施例２で得られたＯｃＩｍ−ＰＥＧを用いた以外は、試験例１と同様にして、ＯｃＩｍ−ＰＥＧ・ＤＮＡ複合体を調製し、アガロースゲル電気泳動により分析した。
その結果を図２に示す。[Test Example 2] Evaluation of OcIm-PEG / DNA complex (analysis of surfactant-like compound / DNA complex by agarose gel electrophoresis)
An OcIm-PEG / DNA complex was prepared and analyzed by agarose gel electrophoresis in the same manner as in Test Example 1 except that the OcIm-PEG obtained in Example 2 was used as the surfactant-like compound.
The result is shown in FIG.
（結果及び考察）
図２に示す結果からＰＥＧ／Ｐが０．１以上において矢印で示すように陰極方向へのバンドのシフトがみられ、８以上でシフトしたバンドの量が多くなった。
このことから、ＯｃＩｍ−ＰＥＧとＤＮＡとは、複合体を形成しているのがわかる。(Results and discussion)
From the results shown in FIG. 2, a band shift toward the cathode was observed as indicated by an arrow when PEG / P was 0.1 or more, and the amount of the band shifted when it was 8 or more increased.
From this, it can be seen that OcIm-PEG and DNA form a complex.
〔試験例３〕Ｃｈｏｌ−ＰＥＧ・ＤＮＡ複合体の評価
界面活性剤様化合物として実施例３で得られたＣｈｏｌ−ＰＥＧを用い、Ｃｈｏｌ−ＰＥＧをＤＮＡのリン酸基の数１に対しＰＥＧの分子数が１，４，８，１６，３２，６４及び９６の量になるようにリン酸緩衝液（組成５０ｍＭ ＮａＨ２ＰＯ４＋５０ｍＭ Ｎａ２ＨＰＯ４）で最終液量が１５μＬになるように溶解させた以外は、試験例１と同様にして、Ｃｈｏｌ−ＰＥＧ・ＤＮＡ複合体を調製し、アガロースゲル電気泳動により分析した。
その結果を図３に示す。[Test Example 3] Evaluation of Chol-PEG / DNA Complex Using the Chol-PEG obtained in Example 3 as a surfactant-like compound, Chol-PEG was converted into a PEG molecule with respect to the number of phosphate groups in DNA. Solubilize so that the final volume is 15 μL with phosphate buffer (composition 50 mM NaH 2 PO 4 +50 mM Na 2 HPO 4 ) so that the number is 1, 4, 8, 16, 32, 64 and 96. Except for the above, in the same manner as in Test Example 1, a Chol-PEG • DNA complex was prepared and analyzed by agarose gel electrophoresis.
The result is shown in FIG.
（結果及び考察）
図３に示す結果からＰＥＧ／Ｐが１以上において矢印で示すように、ＤＮＡの電荷の打消しが起こらないにもかかわらず、陰極方向へのバンドのシフトがみられ、８、１６及び３２においてシフトしたバンドの量が多くなり、６４及び９６ではバンドの消失が見られた。
このことから、Ｃｈｏｌ−ＰＥＧとＤＮＡとは複合体を形成し、ＤＮＡが凝集しており、特に６４及び９６ではＣｈｏｌ−ＰＥＧ・ＤＮＡ複合体の安定性が高いことが判る。(Results and discussion)
From the results shown in FIG. 3, as indicated by an arrow when PEG / P is 1 or more, a band shift toward the cathode is observed despite the absence of DNA charge cancellation. The amount of shifted bands increased, and disappearance of bands was seen at 64 and 96.
From this, it can be seen that Chol-PEG and DNA form a complex, and the DNA is aggregated. In particular, in 64 and 96, the stability of the Chol-PEG • DNA complex is high.
〔試験例４〕Ｏｃ−ＰＥＧ・ＤＮＡ複合体の評価
界面活性剤様化合物として実施例４で得られたＯｃ−ＰＥＧを用いた以外は、試験例３と同様にして、Ｏｃ−ＰＥＧ・ＤＮＡ複合体を調製し、アガロースゲル電気泳動により分析した。
その結果を図４に示す。[Test Example 4] Evaluation of Oc-PEG / DNA complex Oc-PEG / DNA complex as in Test Example 3 except that the Oc-PEG obtained in Example 4 was used as the surfactant-like compound. Body was prepared and analyzed by agarose gel electrophoresis.
The result is shown in FIG.
（結果及び考察）
図４に示す結果からＰＥＧ／Ｐが１以上において矢印で示すように陰極方向へのバンドのシフトがみられ、６４以上においてシフトしたバンドの量が多くなった。
このことから、Ｏｃ−ＰＥＧとＤＮＡとは、複合体を形成していることが分かる。(Results and discussion)
From the results shown in FIG. 4, when PEG / P is 1 or more, a band shift toward the cathode is observed as indicated by an arrow, and when it is 64 or more, the amount of the shifted band increases.
From this, it can be seen that Oc-PEG and DNA form a complex.
〔試験例５〕Ｓｔ−ＰＥＧ・ＤＮＡ複合体の評価
界面活性剤様化合物として実施例５で得られたＳｔ−ＰＥＧを用いた以外は、試験例３と同様にして、Ｓｔ−ＰＥＧ・ＤＮＡ複合体を調製し、アガロースゲル電気泳動により分析した。
その結果を図５に示す。[Test Example 5] Evaluation of St-PEG / DNA complex A St-PEG / DNA complex was prepared in the same manner as in Test Example 3 except that St-PEG obtained in Example 5 was used as the surfactant-like compound. Body was prepared and analyzed by agarose gel electrophoresis.
The result is shown in FIG.
（結果及び考察）
図５に示す結果からＰＥＧ／Ｐが１以上において矢印で示すように陰極方向へのバンドのシフトがみられ、１６以上においてシフトしたバンドの量が多くなった。
このことから、Ｓｔ−ＰＥＧとＤＮＡとは、複合体を形成していることが分かる。(Results and discussion)
From the results shown in FIG. 5, a band shift toward the cathode was observed as indicated by an arrow when PEG / P was 1 or more, and the amount of the shifted band increased when 16 or more.
From this, it can be seen that St-PEG and DNA form a complex.
〔試験例６〕界面活性剤様化合物・ＤＮＡ複合体の粒子径測定
ＤＮＡとして市販のプラスミドであるｐＧＬ３ ０．７μｇと、実施例１〜５で得られた本発明の界面活性剤様化合物をＤＮＡのリン酸基の数１に対しＰＥＧの分子数が６４の量になるように、リン酸緩衝液（組成５０ｍＭ ＮａＨ２ＰＯ４＋５０ｍＭ Ｎａ２ＨＰＯ４＋１３０ｍＭ ＮａＣｌ）１００μＬで調製し、３７℃で２４時間インキュベーションし、界面活性剤様化合物・ＤＮＡ複合体を調製した。
得られた界面活性剤様化合物・ＤＮＡ複合体を動的光散乱法により粒子径を測定した。
動的光散乱法による粒子径の測定は、以下の条件で散乱光強度を測定し、散乱光強度から粒子径を算出することで行った。
条件：
装置：装置名ＥＬＳＺ２（大塚電子社製）
なお、比較サンプルとして、界面活性剤様化合物を含まないＤＮＡのみのサンプル、及び、界面活性剤様化合物の変わりにＰＥＧ−ＮＨ２を用いて調製したサンプルも合わせて試験した。
その結果を表１に示す。
なお、表中のＰＥＧ／Ｐは、界面活性剤様化合物・ＤＮＡ複合体におけるＤＮＡのリン酸基とＰＥＧの分子数の比を意味し、ＤＮＡのリン酸基の数を１とした場合のＰＥＧの分子数を表し、Ｎ．Ｄは粒子径の測定が不可能であったことを示す。[Test Example 6] Particle size measurement of surfactant-like compound / DNA complex: 0.7 μg of commercially available plasmid pDNA3 as DNA and the surfactant-like compound of the present invention obtained in Examples 1 to 5 as DNA Prepared with 100 μL of phosphate buffer (composition 50 mM NaH 2 PO 4 +50 mM Na 2 HPO 4 +130 mM NaCl) so that the number of PEG molecules is 64 with respect to the number of phosphate groups of 1 and 24 at 37 ° C. After incubation for a time, a surfactant-like compound / DNA complex was prepared.
The particle size of the obtained surfactant-like compound / DNA complex was measured by a dynamic light scattering method.
Measurement of the particle diameter by the dynamic light scattering method was performed by measuring the scattered light intensity under the following conditions and calculating the particle diameter from the scattered light intensity.
conditions:
Device: Device name ELSZ2 (Otsuka Electronics Co., Ltd.)
In addition, as a comparative sample, a sample containing only a DNA containing no surfactant-like compound and a sample prepared using PEG-NH 2 instead of the surfactant-like compound were also tested.
The results are shown in Table 1.
In the table, PEG / P means the ratio of the number of DNA phosphate groups to the number of PEG molecules in the surfactant-like compound / DNA complex. Represents the number of molecules of N. D indicates that the particle size could not be measured.
（結果及び考察）
実施例１〜５で得られた本発明の界面活性剤様化合物とＤＮＡとの複合体の粒子径は、１３．０±１．１〜１３７．５±２８．７ｎｍと小さい粒子径であった。
この結果は、本発明の界面活性剤様化合物とＤＮＡとの複合体は、生体投与時において血管から細胞へ移行することができる大きさであり静脈注射に適した粒子径であることを示している。
また、カチオン性基を具備する実施例１及び２の界面活性剤様化合物とＤＮＡとの複合体の粒子径は、それぞれ１３．０±１．１ｎｍ及び８７．３±１．１ｎｍであった。
この結果は、カチオン性基を具備する本発明の界面活性剤様化合物は、粒子径が小さいＤＮＡ複合体を形成でき、これにより、生体投与時における血管から細胞へ移行効率が上昇することを示す。(Results and discussion)
The particle size of the complex of the surfactant-like compound of the present invention and DNA obtained in Examples 1 to 5 was as small as 13.0 ± 1.1 to 137.5 ± 28.7 nm. .
This result shows that the complex of the surfactant-like compound of the present invention and DNA has a size that can be transferred from a blood vessel to a cell at the time of biological administration and has a particle size suitable for intravenous injection. Yes.
Moreover, the particle diameters of the complex of the surfactant-like compound of Example 1 and 2 having a cationic group and DNA were 13.0 ± 1.1 nm and 87.3 ± 1.1 nm, respectively.
This result shows that the surfactant-like compound of the present invention having a cationic group can form a DNA complex having a small particle size, thereby increasing the transfer efficiency from blood vessels to cells at the time of biological administration. .
〔試験例７〕界面活性剤様化合物・ＤＮＡ複合体の円偏光二色性スペクトル測定
実施例１で得られた本発明の界面活性剤様化合物（ＢｕＩｍ−ＰＥＧ）をＤＮＡ（ｐＧＬ３）のリン酸基の数１に対しＰＥＧの分子数が６４の量になるように、純水１０００μＬで調製し、３７℃で２４時間インキュベーションし、界面活性剤様化合物・ＤＮＡ複合体を調製し、以下の条件で円偏光二色性スペクトル（ＣＤスペクトル測定）を測定した。
条件：ＤＮＡ量：１２μｇ
装置：Ｊ−８２０（日本分光社製）
なお、比較サンプルとして、界面活性剤様化合物を含まないＤＮＡのみのサンプルも合わせて試験した。
その結果を図６（チャート図）及び、表２（最大モル楕円率を示す波長（λｍａｘ）および３００ｎｍにおけるモル楕円率（［θ］３００））に示す。[Test Example 7] Circular dichroism spectrum measurement of surfactant-like compound / DNA complex The surfactant-like compound (BuIm-PEG) of the present invention obtained in Example 1 was converted to DNA (pGL3) phosphate. Prepare a surfactant-like compound / DNA complex by preparing 1000 μL of pure water so that the number of PEG molecules is 64 per group of 1 and incubating at 37 ° C. for 24 hours. The circular dichroism spectrum (CD spectrum measurement) was measured.
Condition: DNA amount: 12 μg
Apparatus: J-820 (manufactured by JASCO Corporation)
In addition, as a comparative sample, a sample containing only DNA containing no surfactant-like compound was also tested.
The results are shown in FIG. 6 (chart diagram) and Table 2 (wavelength (λ max ) indicating maximum molar ellipticity and molar ellipticity at 300 nm ([θ] 300 )).
（結果及び考察）
表２の［θ］３００と図６の図中の１で示した部分の結果から界面活性剤様化合物・ＤＮＡ複合体中のＤＮＡに負のコットン効果が出現するのがわかり、表２のλｍａｘと図６の図中の２で示した部分の結果からブルーシフトが見られることがわかる。
これらの結果から、界面活性剤様化合物・ＤＮＡ複合体中のＤＮＡが脱水和によりコンフォーメーションの変化を起こしているのが判る。(Results and discussion)
From the results of [θ] 300 in Table 2 and the part indicated by 1 in FIG. 6, it can be seen that a negative cotton effect appears in DNA in the surfactant-like compound / DNA complex. It can be seen that a blue shift can be seen from the results of max and the part indicated by 2 in FIG.
From these results, it can be seen that the DNA in the surfactant-like compound / DNA complex undergoes a conformational change due to dehydration.
〔試験例８〕遺伝子導入効率の評価試験１
実施例３で得られた本発明の界面活性剤様化合物（Ｃｈｏｌ−ＰＥＧ）を用いて作成した界面活性剤様化合物・ＤＮＡ複合体の遺伝子導入効率の評価試験を行った。
（界面活性剤様化合物・ＤＮＡ複合体の調製）
ＤＮＡとして市販のプラスミドでＳＶ４０プロモーターの下流に改変型ルシフェラーゼ遺伝子を連結させたｐＧＬ３（プロメガ社）３．５μｇと、実施例３で得られた本発明の界面活性剤様化合物としてのＣｈｏｌ−ＰＥＧをＤＮＡのリン酸基の数１に対しコレステロール基の分子数が０．１，０．５，１．０及び２．０の量になるようにリン酸緩衝生理食塩水（ＰＢＳ、組成１３７ｍＭ ＮａＣｌ＋２．７ｍＭ ＫＣｌ＋１０ｍＭ Ｎａ２ＨＰＯ４＋１．７６ｍＭ ＫＨ２ＰＯ４）で最終液量が３５μＬになるように溶解させた。溶解後、３７℃で２４時間インキュベーションし、界面活性剤様化合物・ＤＮＡ複合体としてのＣｈｏｌ−ＰＥＧ・ＤＮＡ複合体を調製した。
（マウスへの投与）
調製したＣｈｏｌ−ＰＥＧ・ＤＮＡ複合体をろ過滅菌し、３５μＬをマウス（系統ＩＣＲ、５週令、体重２０〜２５ｇ）の背皮内部位に皮内注射した。
なお、比較サンプルとして、ＰＢＳのみを注射したサンプル、界面活性剤様化合物を含まないＤＮＡのみのサンプルの試験も行った。
（組織の摘出）
投与から２日後、動物実験の倫理指針に従ってマウスを麻酔した後に解剖し、皮膚を摘出した。
摘出後の皮膚は、ルシフェラーゼアッセイまで−８０℃で保存した。
（タンパク質抽出〜ルシフェラーゼアッセイ）
皮膚からのタンパク質抽出は、ホモジネートにより行った。
ルシフェラーゼアッセイは、Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｓｙｓｔｅｍ（プロメガ社製）を用いて、ルミノメータ−（装置名ＬＢ９６Ｖ、ベルトールド社製）で発光の定量をすることで行い、発光量は、サンプルのタンパク質量で標準化した。
なお、サンプルのタンパク質量の測定は、ＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔ（Ｐｉｅｒｃｅ社製）を用いてＢＣＡ法により行った。
その結果を図７に示す。
なお、図中Ｃｈｏｌ／Ｐは、界面活性剤様化合物・ＤＮＡ複合体におけるＤＮＡのリン酸基とコレステロール基の分子数の比を意味し、ＤＮＡのリン酸基の数を１とした場合のコレステロール基の分子数を表し、ＰＢＳはＰＢＳのみを投与したサンプルを表し、ＤＮＡは界面活性剤様化合物を含まないＤＮＡのみのサンプルを表す。
（結果及び考察）
図７から、本発明の界面活性剤様化合物（Ｃｈｏｌ−ＰＥＧ）を用いて作成したＣｈｏｌ−ＰＥＧ・ＤＮＡ複合体の投与はＰＥＧ／Ｐが１において、ＤＮＡのみの投与よりも、生体内のルシフェラーゼによる発光が高かった。
この結果から、本発明の界面活性剤様化合物（Ｃｈｏｌ−ＰＥＧ）を用いて作製したＣｈｏｌ−ＰＥＧ・ＤＮＡ複合体の投与は、臨床応用例があるＤＮＡのみの投与より、生体内での遺伝子発現が高いことがわかる。[Test Example 8] Gene transfer efficiency evaluation test 1
An evaluation test of gene transfer efficiency of a surfactant-like compound / DNA complex prepared using the surfactant-like compound (Chol-PEG) of the present invention obtained in Example 3 was performed.
(Preparation of surfactant-like compound / DNA complex)
PGL3 (Promega) 3.5 μg obtained by ligating a modified luciferase gene downstream of the SV40 promoter with a commercially available plasmid as DNA, and Chol-PEG as the surfactant-like compound of the present invention obtained in Example 3 Phosphate buffered saline (PBS, composition 137 mM NaCl + 2.P) so that the number of cholesterol groups is 0.1, 0.5, 1.0 and 2.0 with respect to the number of phosphate groups in DNA. 7 mM KCl + 10 mM Na 2 HPO 4 +1.76 mM KH 2 PO 4 ) so that the final volume was 35 μL. After dissolution, the mixture was incubated at 37 ° C. for 24 hours to prepare a Chol-PEG / DNA complex as a surfactant-like compound / DNA complex.
(Administration to mice)
The prepared Chol-PEG / DNA complex was sterilized by filtration, and 35 μL was injected intradermally into the intradermal region of the back skin of a mouse (line ICR, 5 weeks old, body weight 20-25 g).
In addition, as a comparative sample, a sample injected with PBS alone and a sample containing only DNA not containing a surfactant-like compound were also tested.
(Tissue extraction)
Two days after the administration, the mice were anesthetized according to the ethical guidelines for animal experiments, dissected, and the skin was removed.
The removed skin was stored at −80 ° C. until luciferase assay.
(Protein extraction-Luciferase assay)
Protein extraction from the skin was performed with a homogenate.
The luciferase assay was performed by quantifying luminescence using a luciferase assay system (manufactured by Promega) with a luminometer (device name: LB96V, manufactured by Bertrud). The amount of luminescence was standardized by the amount of protein in the sample.
In addition, the protein amount of the sample was measured by BCA method using BCA Protein Assay Kit (Pierce).
The result is shown in FIG.
In the figure, Chol / P means the ratio of the number of DNA phosphate groups to cholesterol groups in the surfactant-like compound / DNA complex, and cholesterol when the number of DNA phosphate groups is 1. Represents the number of molecules of the group, PBS represents a sample administered with PBS alone, and DNA represents a sample of DNA alone containing no surfactant-like compound.
(Results and discussion)
FIG. 7 shows that administration of the Chol-PEG / DNA complex prepared using the surfactant-like compound (Chol-PEG) of the present invention is luciferase in vivo when PEG / P is 1 and DNA alone is administered. The luminescence by was high.
From this result, administration of a chol-PEG / DNA complex prepared using the surfactant-like compound (Chol-PEG) of the present invention is more effective in gene expression in vivo than administration of DNA with clinical application examples. Is high.
〔試験例９〕遺伝子導入効率の評価試験２
実施例１及び３で得られた本発明の界面活性剤様化合物（ＢｕＩｍ−ＰＥＧ及びＣｈｏｌ−ＰＥＧ）を用いて作成した界面活性剤様化合物・ＤＮＡ複合体の遺伝子導入効率の評価試験を行った。
（界面活性剤様化合物・ＤＮＡ複合体の調製）
実施例１で得られた本発明の界面活性剤様化合物としてのＢｕＩｍ−ＰＥＧをＤＮＡ（ｐＧＬ３）の負電荷（リン酸基の電荷）数１に対しＢｕＩｍ−ＰＥＧの正電荷（カチオン基の電荷）数が０．５の量になるように混合し、また、実施例３で得られた本発明の界面活性剤様化合物としてのＣｈｏｌ−ＰＥＧをＤＮＡ（ｐＧＬ３）のリン酸基の数１に対しコレステロール基の分子数が１．０の量になるように混合した以外は、試験例８と同様にして、界面活性剤様化合物・ＤＮＡ複合体としてのＢｕＩｍ−ＰＥＧ及びＣｈｏｌ−ＰＥＧ・ＤＮＡ複合体を調製した。
（マウスへの投与）
調製したＢｕＩｍ−ＰＥＧ・ＤＮＡ複合体及びＣｈｏｌ−ＰＥＧ・ＤＮＡ複合体を試験例８と同様にしてマウスに投与した。マウスへの投与は１サンプルについて２匹のマウスに行った。
なお、比較サンプルとして、界面活性剤様化合物を含まないＤＮＡのみのサンプルの試験も行った。
（組織の摘出）
試験例８と同様にして皮膚を摘出し、保存した。
（タンパク質抽出〜ルシフェラーゼアッセイ）
試験例８と同様にして行い、発光量を測定した。
その結果を図８に示す。
（結果及び考察）
図８から、本発明の界面活性剤様化合物（ＢｕＩｍ−ＰＥＧ）を用いて作成したＢｕＩｍ−ＰＥＧ・ＤＮＡ複合体の投与は、ＤＮＡのみの投与よりも、生体内のルシフェラーゼによる発光が約７倍と顕著に高く、本発明の界面活性剤様化合物（Ｃｈｏｌ−ＰＥＧ）を用いて作成したＣｈｏｌ−ＰＥＧ・ＤＮＡ複合体の投与においても、ＤＮＡのみの投与よりも、生体内のルシフェラーゼによる発光が高かった。
この結果から、本発明の界面活性剤様化合物（ＢｕＩｍ−ＰＥＧ及びＣｈｏｌ−ＰＥＧ）を用いて作製したＢｕＩｍ−ＰＥＧ・ＤＮＡ複合体及びＣｈｏｌ−ＰＥＧ・ＤＮＡ複合体の投与は、臨床応用例があるＤＮＡのみの投与より、生体内での遺伝子発現が高いことがわかり、特にＢｕＩｍ−ＰＥＧ・ＤＮＡ複合体の投与では生体内での遺伝子発現が顕著に高いことがわかる。[Test Example 9] Evaluation test 2 of gene transfer efficiency
An evaluation test of gene transfer efficiency of a surfactant-like compound / DNA complex prepared using the surfactant-like compounds (BuIm-PEG and Chol-PEG) of the present invention obtained in Examples 1 and 3 was performed. .
(Preparation of surfactant-like compound / DNA complex)
The BuIm-PEG as the surfactant-like compound of the present invention obtained in Example 1 was replaced with a positive charge (cation group charge) of BuIm-PEG with respect to the number of negative charges (phosphate group charges) of DNA (pGL3). ) The mixture was mixed so that the number was 0.5, and Chol-PEG as the surfactant-like compound of the present invention obtained in Example 3 was changed to the number 1 of the phosphate group of DNA (pGL3). However, BuIm-PEG and Chol-PEG / DNA complex as a surfactant-like compound / DNA complex were the same as in Test Example 8 except that mixing was performed so that the number of cholesterol group molecules was 1.0. The body was prepared.
(Administration to mice)
The prepared BuIm-PEG • DNA complex and Chol-PEG • DNA complex were administered to mice in the same manner as in Test Example 8. Administration to mice was performed on 2 mice per sample.
In addition, as a comparative sample, a test using only a DNA containing no surfactant-like compound was also conducted.
(Tissue extraction)
The skin was extracted and stored in the same manner as in Test Example 8.
(Protein extraction-Luciferase assay)
The amount of luminescence was measured in the same manner as in Test Example 8.
The result is shown in FIG.
(Results and discussion)
FIG. 8 shows that administration of a BuIm-PEG / DNA complex prepared using the surfactant-like compound (BuIm-PEG) of the present invention produces about 7 times as much light emission by luciferase in vivo as that of administration of DNA alone. In the administration of the Chol-PEG / DNA complex prepared using the surfactant-like compound (Chol-PEG) of the present invention, the luminescence by luciferase in the living body is higher than that in the administration of DNA alone. It was.
From these results, administration of BuIm-PEG • DNA complex and Chol-PEG • DNA complex prepared using the surfactant-like compounds of the present invention (BuIm-PEG and Chol-PEG) has clinical application examples. It can be seen that gene expression in vivo is higher than administration of DNA alone, and that gene expression in vivo is particularly high in administration of BuIm-PEG • DNA complex.
〔試験例９〕ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体の評価１
（界面活性剤様化合物・ＤＮＡ複合体のアガロースゲル電気泳動による分析）
界面活性剤様化合物として実施例６で得られたＨｅＡ−Ｉｍ−ＰＥＧを用い、ＨｅＡ−Ｉｍ−ＰＥＧをＤＮＡのリン酸基の数１に対しＰＥＧの分子数（ＰＥＧ／Ｐ）が０．５，１，２，４，８，１６，３２，６４の量になるようにリン酸緩衝液（組成５０ｍＭ ＮａＨ２ＰＯ４＋５０ｍＭ Ｎａ２ＨＰＯ４）で最終液量が１５μＬになるように溶解させた以外は、試験例１と同様にして、ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体溶液を調製し、アガロースゲル電気泳動により分析した。
その結果を図１０に示す。
なお、ＨｅＡ−Ｉｍ−ＰＥＧをＤＮＡのリン酸基の数１に対しＰＥＧの分子数が０．５，１，２，４，８，１６，３２，６４の量に混合した場合、ＤＮＡのリン酸基の数１に対するＨｅＡ−Ｉｍ−ＰＥＧの正電荷（カチオン基の電荷）数（電荷比）も、上記リン酸基に対するＰＥＧの分子数と同じ値になる。
（結果及び考察）
図１０に示す結果からＰＥＧ／Ｐが０．５以上において矢印で示すように陰極方向へのバンドのシフトがみられ、４においては陽極方向へスメアしたバンドがみられ、８以上においてはＮａｋｅｄ ＤＮＡバンドの消失が見られた。スメアのバンドは、正味負に帯電した微小粒径のＤＮＡ複合体の形成を示唆している。さらに、８で見られたＮａｋｅｄ ＤＮＡバンドより陽極方向へシフトしたバンドは、正味負に帯電した超微小粒径のＤＮＡ複合体の形成を示唆しており、周辺研究で既存しない新概念のＤＮＡ複合体であると考えられる。
これらの結果から、ＰＥＧ／Ｐが０．５以上においてＨｅＡ−Ｉｍ−ＰＥＧはＤＮＡと複合体を形成することがわかる。特にＰＥＧ／Ｐが８以上において、ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体は、安定性が高いことがわかる。
また、ＨｅＡ−Ｉｍ−ＰＥＧにおいては、実施例１〜５の界面活性剤様化合物の結果と比較したときに、ＰＥＧ／Ｐが０．５以上という低い電荷比においてで陰極方向へのバンドのシフト（ＤＮＡとの複合体形成）が見られた。
また、ＤＮＡが凝集状態でありＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体の安定性が高いことを示すバンドの消失もＰＥＧ／Ｐが１６以上という低い電荷比でみられた。
これらの結果から、ＨｅＡ−Ｉｍ−ＰＥＧは、実施例１〜５の界面活性剤様化合物よりも、ＤＮＡとの複合体形成能、及びＤＮＡの凝集能がさらに高いものであることがわかる。[Test Example 9] Evaluation of HeA-Im-PEG / DNA complex 1
(Anarose gel electrophoresis analysis of surfactant-like compound and DNA complex)
As the surfactant-like compound, the HeA-Im-PEG obtained in Example 6 was used, and the HeA-Im-PEG had a number of PEG molecules (PEG / P) of 0.5 with respect to the number of phosphate groups of DNA. , 1, 2, 4, 8, 16, 32, 64 so that the final solution amount was 15 μL with a phosphate buffer (composition 50 mM NaH 2 PO 4 +50 mM Na 2 HPO 4 ). Except for the above, a HeA-Im-PEG • DNA complex solution was prepared in the same manner as in Test Example 1, and analyzed by agarose gel electrophoresis.
The result is shown in FIG.
In addition, when HeA-Im-PEG is mixed in an amount of 0.5, 1, 2, 4, 8, 16, 32, 64 of the number of PEG molecules with respect to the number of phosphate groups of DNA, The number of positive charges (charge of cationic groups) (charge ratio) of HeA-Im-PEG with respect to the number 1 of acid groups is also the same value as the number of molecules of PEG with respect to the phosphate groups.
(Results and discussion)
From the results shown in FIG. 10, when PEG / P is 0.5 or more, a band shift toward the cathode is observed as indicated by an arrow, when 4 is a band smeared toward the anode, and when 8 or more, Naked DNA is observed. The disappearance of the band was observed. The smear band suggests the formation of a net negatively charged microparticle DNA complex. Furthermore, the band shifted toward the anode from the Naked DNA band seen in 8 suggests the formation of a net negatively charged ultrafine particle size DNA complex. It is considered a complex.
From these results, it can be seen that HeA-Im-PEG forms a complex with DNA when PEG / P is 0.5 or more. In particular, when PEG / P is 8 or more, the HeA-Im-PEG • DNA complex is found to have high stability.
In HeA-Im-PEG, the band shift toward the cathode at a low charge ratio of PEG / P of 0.5 or higher when compared with the results of the surfactant-like compounds of Examples 1 to 5. (Complex formation with DNA) was observed.
Further, the disappearance of the band indicating that the DNA was in an aggregated state and the stability of the HeA-Im-PEG • DNA complex was high was also observed at a low charge ratio of PEG / P of 16 or more.
From these results, it can be seen that HeA-Im-PEG has higher ability to form a complex with DNA and to aggregate DNA than the surfactant-like compounds of Examples 1 to 5.
〔試験例１０〕ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体の評価２
（界面活性剤様化合物・ＤＮＡ複合体のポリアニオンに対する安定性評価）
界面活性剤様化合物・ＤＮＡ複合体の安定性の評価を行った。安定性の評価は、ポリアニオンによる、界面活性剤様化合物・ＤＮＡ複合体の置換反応に対する安定性を指標として行った。
（界面活性剤様化合物・ＤＮＡ複合体の調整）
実施例６で得られた本発明の界面活性剤様化合物としてのＨｅＡ−Ｉｍ−ＰＥＧをＤＮＡのリン酸基の数１に対しＰＥＧの分子数（ＰＥＧ／Ｐ）が１、または８の量（それぞれ電荷比：１、電荷比：８）になるようにそれぞれ混合した以外は、試験例１と同様にして、ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体溶液を調製した。
（ポリアニオン処理）
調整したＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体溶液に、ポリアニオンとしてのデキストランサルフェート（型番：Ｄ４９１１、ＳＩＧＭＡ-ＡＬＤＲＩＣＨ社製）溶液を、ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体に混合添加し、３７℃、６０分の条件でインキュベートし反応させた。なお、ＰＥＧ／Ｐが１の試料については、デキストランサルフェート溶液を最終濃度が１、２、３ｍＭになるように混合添加し、ＰＥＧ／Ｐが８の試料については、デキストランサルフェートを最終濃度が１、２、３、８ｍＭになるように混合添加した。
（アガロースゲル電気泳動による分析）
ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体とデキストランサルフェートを反応させた後、該混合物を、試験例１と同様にしてアガロース電気泳動により分析した。
その結果を図１１に示す。[Test Example 10] Evaluation 2 of HeA-Im-PEG / DNA complex
(Stability evaluation of surfactant-like compound / DNA complex against polyanion)
The stability of the surfactant-like compound / DNA complex was evaluated. The stability was evaluated using the stability of the surfactant-like compound / DNA complex with the polyanion against the substitution reaction as an index.
(Preparation of surfactant-like compound and DNA complex)
HeA-Im-PEG as the surfactant-like compound of the present invention obtained in Example 6 was used in an amount of 1 or 8 in terms of the number of PEG molecules (PEG / P) relative to the number of phosphate groups in DNA ( A HeA-Im-PEG / DNA complex solution was prepared in the same manner as in Test Example 1 except that the mixture was mixed so that the charge ratio was 1 and the charge ratio was 8), respectively.
(Polyanion treatment)
To the adjusted HeA-Im-PEG / DNA complex solution, a dextran sulfate (model number: D4911, manufactured by SIGMA-ALDRICH) solution as a polyanion was mixed and added to the HeA-Im-PEG / DNA complex, 37 ° C., The reaction was incubated for 60 minutes. For samples with PEG / P of 1, the dextran sulfate solution was mixed and added so that the final concentration was 1, 2, 3 mM, and for samples with PEG / P of 8, the final concentration of dextran sulfate was 1, The mixture was added to 2, 3, and 8 mM.
(Agarose gel electrophoresis analysis)
After the HeA-Im-PEG • DNA complex and dextran sulfate were reacted, the mixture was analyzed by agarose electrophoresis in the same manner as in Test Example 1.
The result is shown in FIG.
（結果及び考察）
図１１に示す結果から、ＰＥＧ／Ｐが１のＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体においては、デキストランサルフェートの濃度が２ｍＭ以下においてはＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体の形成を示すメインバンドがみられ、３ｍＭの濃度においてメインバンドの下（陽極側）にスーパーコイル由来のマイナーバンドがみられた。この結果は、ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体は、２ｍＭという高濃度のデキストランサルフェートに対しも安定であり、３ｍＭのデキストランサルフェートに対してはＤＮＡがリリースされることを意味する。
一方、ＰＥＧ／Ｐが８のＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体においては、デキストランサルフェートの濃度が１〜８ｍＭにおいて、バンドの変化が見られなかった。この結果は、ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体は、８ｍＭという高濃度のデキストランサルフェートに対しも安定であり、極めて安定な複合体を形成していることがわかる。さらに、この時、〔試験例１０〕で記した通り、Ｎａｋｅｄ ＤＮＡバンドより陽極方向へシフトしたバンドが維持されているため、正味負に帯電した超微小粒径のＤＮＡ複合体の安定性が示唆された。
また、これらの結果からもわかるように、本発明の界面活性剤様化合物としてのＨｅＡ−Ｉｍ−ＰＥＧとＤＮＡとの複合体は、ＰＥＧ／Ｐ比（電荷比）を変えることにより、その複合体の特性を制御することができるものである。これにより、ドラッグデリバリー特性を制御することができる。例えば、ドラッグデリバリー目的が培養細胞などに短時間で遺伝子導入する場合などにおいては、複合体の安定性を高くしすぎないように制御したり、また、生体内において消化酵素等が多くデリバリー効率が低い細胞や組織などにドラッグデリバリーを行う場合においては、複合体の安定性を高くするように制御する等の制御をすることができ、その制御はＰＥＧ／Ｐ比（電荷比）を変えるなどして簡単に行うことができる。以上から、本発明の界面活性剤様化合物は、核酸複合体形成用、ドラッグデリバリー用として好適に用いることができることがわかる。(Results and discussion)
From the results shown in FIG. 11, in the HeA-Im-PEG • DNA complex with PEG / P = 1, the main band indicating the formation of the HeA-Im-PEG • DNA complex is present at a dextran sulfate concentration of 2 mM or less. In addition, a minor band derived from the supercoil was observed under the main band (on the anode side) at a concentration of 3 mM. This result indicates that the HeA-Im-PEG • DNA complex is stable to dextran sulfate as high as 2 mM, and DNA is released to 3 mM dextran sulfate.
On the other hand, in the HeA-Im-PEG • DNA complex having PEG / P of 8, no band change was observed when the concentration of dextran sulfate was 1 to 8 mM. This result shows that the HeA-Im-PEG • DNA complex is stable against dextran sulfate at a high concentration of 8 mM and forms a very stable complex. Further, at this time, as described in [Test Example 10], since the band shifted from the Naked DNA band in the anode direction is maintained, the stability of the DNA complex having an ultrafine particle diameter charged net negative is improved. It was suggested.
Further, as can be seen from these results, the complex of HeA-Im-PEG and DNA as the surfactant-like compound of the present invention can be obtained by changing the PEG / P ratio (charge ratio). It is possible to control the characteristics. Thereby, drug delivery characteristics can be controlled. For example, when the purpose of drug delivery is to introduce a gene into cultured cells in a short period of time, the stability of the complex is controlled so as not to be too high. In the case of drug delivery to low cells or tissues, it can be controlled to increase the stability of the complex, such as changing the PEG / P ratio (charge ratio). Can be done easily. From the above, it can be seen that the surfactant-like compound of the present invention can be suitably used for nucleic acid complex formation and drug delivery.
〔試験例１１〕遺伝子導入効率の評価試験３
実施例６で得られた本発明の界面活性剤様化合物（ＨｅＡ−Ｉｍ−ＰＥＧ）を用いて作成した界面活性剤様化合物・ＤＮＡ複合体の遺伝子導入効率の評価試験を行った。
（界面活性剤様化合物・ＤＮＡ複合体の調製）
実施例６で得られた本発明の界面活性剤様化合物としてのＨｅＡ−Ｉｍ−ＰＥＧをＤＮＡのリン酸基の数１に対しＰＥＧの分子数（ＰＥＧ／Ｐ）が０．５、０．６、０．７、０．８、０．９、１．０、１．２、１．５になるようにそれぞれ混合した以外は、試験例８と同様にして、ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体溶液を調製した。
（マウスへの投与）
調製したＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体溶液を、頸骨筋内投与（３５μＬ）に変えた以外は、試験例８と同様にしてマウスに投与した。マウスへの投与は１サンプルについて２匹のマウスに行った。
なお、比較サンプルとして、界面活性剤様化合物を含まないＤＮＡのみのサンプル、リン酸緩衝液のみのサンプル、本発明の界面活性剤様化合物の代わりに陽イオン性の水溶性ポリマーである直鎖状のポリエチレンイミンをベースにした市販品のｉｎ ｖｉｖｏ用核酸導入試薬（商品名：ｊｅｔＰＥＩ、Polyplus transfection社製）を用いて調整したサンプルの試験（以下、市販品を用いた群と呼ぶこともある。）も行った。なお、ｊｅｔＰＥＩによるサンプル調整は、添付のマニュアルに従って行った。
（組織の摘出、タンパク質抽出〜ルシフェラーゼアッセイ）
投与後の組織の摘出、及びタンパク質抽出〜ルシフェラーゼアッセイの工程は、組織の摘出を投与から６日後に行った以外は、試験例８と同様にして行った。
その結果を図１２に示す。[Test Example 11] Evaluation test 3 of gene transfer efficiency
An evaluation test of the gene transfer efficiency of the surfactant-like compound / DNA complex prepared using the surfactant-like compound (HeA-Im-PEG) of the present invention obtained in Example 6 was performed.
(Preparation of surfactant-like compound / DNA complex)
HeA-Im-PEG as the surfactant-like compound of the present invention obtained in Example 6 has a number of PEG molecules (PEG / P) of 0.5, 0.6 with respect to the number of phosphate groups of DNA. , 0.7, 0.8, 0.9, 1.0, 1.2, 1.5 HeA-Im-PEG / DNA complex as in Test Example 8 A body solution was prepared.
(Administration to mice)
The prepared HeA-Im-PEG / DNA complex solution was administered to mice in the same manner as in Test Example 8, except that it was changed to intra-tibial muscle administration (35 μL). Administration to mice was performed on 2 mice per sample.
In addition, as a comparative sample, a sample containing only a DNA that does not contain a surfactant-like compound, a sample containing only a phosphate buffer, and a straight-chain that is a cationic water-soluble polymer instead of the surfactant-like compound of the present invention Tests of samples prepared using a commercially available in vivo nucleic acid introduction reagent (trade name: jetPEI, manufactured by Polyplus transfection) based on polyethyleneimine (hereinafter sometimes referred to as a group using commercially available products). ) Also went. Sample adjustment by jetPEI was performed according to the attached manual.
(Tissue extraction, protein extraction to luciferase assay)
Tissue extraction after administration and the steps of protein extraction to luciferase assay were performed in the same manner as in Test Example 8 except that tissue extraction was performed 6 days after administration.
The result is shown in FIG.
（結果及び考察）
図１２から、本発明の界面活性剤様化合物（ＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ）を用いて作成したＨｅＡ−Ｉｍ−ＰＥＧ・ＤＮＡ複合体の投与群はすべての群において、比較実験群（ＰＢＳのみの投与群、ＤＮＡのみの投与群よりも、市販品を用いた群）よりも、生体内のルシフェラーゼによる発光量が高いものであった。特に、ＰＥＧ／Ｐが０．６〜１．０において発光量が高く、中でもＰＥＧ／Ｐが０．８の群においては、ＤＮＡのみの投与群の１０倍以上、且つ市販品を用いた群より１０００倍以上の極めて高い発光量を示した。
以上の結果から、本発明の界面活性剤様化合物は、核酸複合体形成用、ドラッグデリバリー用として好適に用いることができることがわかる。(Results and discussion)
From FIG. 12, the administration group of the HeA-Im-PEG • DNA complex prepared using the surfactant-like compound (HeA-Im-PEG • DNA) of the present invention is the comparative experimental group (PBS only). The amount of luminescence due to luciferase in the living body was higher than that of the group administered with DNA and the group administered with DNA alone). In particular, the amount of luminescence is high when PEG / P is 0.6 to 1.0, and in particular, in the group where PEG / P is 0.8, more than 10 times the group administered with DNA alone, and more than the group using commercially available products An extremely high light emission amount of 1000 times or more was exhibited.
From the above results, it can be seen that the surfactant-like compound of the present invention can be suitably used for nucleic acid complex formation and drug delivery.
本発明の界面活性剤様化合物は、核酸複合体形成用として有用であり核酸治療薬のドラッグデリバリー用途に好ましく用いることができる。
本発明の界面活性剤様化合物とＤＮＡとの複合体は、粒子径が小さく且つ安定した複合体である。これにより、体内における対象ＤＮＡの標的部位への移送効率及び遺伝子発現効率が高い。
また、本発明の界面活性剤様化合物はポリエチレングリコール基など生体適合性を有する置換基を含有するため血液中での安定性が高く、且つカチオン過剰による毒性がない。これらの機能を有するためドラッグデリバリー用途に有用である。The surfactant-like compound of the present invention is useful for nucleic acid complex formation and can be preferably used for drug delivery of nucleic acid therapeutics.
The complex of the surfactant-like compound of the present invention and DNA is a stable complex having a small particle size. Thereby, the transfer efficiency and gene expression efficiency of the target DNA in the body to the target site are high.
In addition, since the surfactant-like compound of the present invention contains a biocompatible substituent such as a polyethylene glycol group, it is highly stable in blood and has no toxicity due to excessive cation. Since it has these functions, it is useful for drug delivery.
Claims (2)
（式中、ｎは４０〜１２０の整数を表す。）
A surfactant-like compound which is any compound represented by the following chemical formula.
(In the formula, n represents an integer of 40 to 120.)
Surfactant-like compound of claim 1 wherein the nucleic acid complex formation.
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| PCT/JP2014/056873 WO2014148378A1 (en) | 2013-03-19 | 2014-03-14 | Surfactant-like compound |
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| AU2015335618B2 (en) * | 2014-10-24 | 2018-02-01 | Avectas Limited | Delivery across cell plasma membranes |
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| US5730990A (en) * | 1994-06-24 | 1998-03-24 | Enzon, Inc. | Non-antigenic amine derived polymers and polymer conjugates |
| US6413507B1 (en) * | 1999-12-23 | 2002-07-02 | Shearwater Corporation | Hydrolytically degradable carbamate derivatives of poly (ethylene glycol) |
| TWI321054B (en) * | 2000-12-19 | 2010-03-01 | California Inst Of Techn | Compositions containing inclusion complexes |
| MXPA05007165A (en) * | 2002-12-31 | 2005-09-21 | Nektar Therapeutics Al Corp | Polymeric reagents comprising a ketone or a related functional group. |
| WO2005030835A1 (en) * | 2003-09-25 | 2005-04-07 | Terumo Kabushiki Kaisha | Polyethylene glycol derivatives and drug carriers containing the same as the membrane constituent |
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