EP1663152A2 - Verfahren zur herstellung von homogenen liposomen und lipoplexen - Google Patents
Verfahren zur herstellung von homogenen liposomen und lipoplexenInfo
- Publication number
- EP1663152A2 EP1663152A2 EP04764641A EP04764641A EP1663152A2 EP 1663152 A2 EP1663152 A2 EP 1663152A2 EP 04764641 A EP04764641 A EP 04764641A EP 04764641 A EP04764641 A EP 04764641A EP 1663152 A2 EP1663152 A2 EP 1663152A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- liposomes
- lipoplexes
- lipid
- mixture
- lipoplex
- 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.)
- Withdrawn
Links
- 239000002502 liposome Substances 0.000 title claims abstract description 246
- 238000000034 method Methods 0.000 title claims abstract description 128
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 52
- 230000008569 process Effects 0.000 title claims description 59
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 81
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 81
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 81
- 238000001125 extrusion Methods 0.000 claims abstract description 80
- 150000002632 lipids Chemical class 0.000 claims description 119
- 239000000203 mixture Substances 0.000 claims description 104
- 239000000725 suspension Substances 0.000 claims description 70
- 239000002479 lipoplex Substances 0.000 claims description 65
- -1 cationic lipid Chemical class 0.000 claims description 59
- 239000012528 membrane Substances 0.000 claims description 55
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 claims description 47
- 238000004108 freeze drying Methods 0.000 claims description 32
- 230000007935 neutral effect Effects 0.000 claims description 29
- OGQYPPBGSLZBEG-UHFFFAOYSA-N dimethyl(dioctadecyl)azanium Chemical compound CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC OGQYPPBGSLZBEG-UHFFFAOYSA-N 0.000 claims description 28
- 238000002156 mixing Methods 0.000 claims description 27
- 239000011148 porous material Substances 0.000 claims description 22
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Natural products C1C=C2C[C@@H](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 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 20
- 238000001035 drying Methods 0.000 claims description 18
- 238000001415 gene therapy Methods 0.000 claims description 15
- 238000010438 heat treatment Methods 0.000 claims description 12
- 239000004417 polycarbonate Substances 0.000 claims description 12
- 229920000515 polycarbonate Polymers 0.000 claims description 12
- 235000012000 cholesterol Nutrition 0.000 claims description 11
- 150000003839 salts Chemical class 0.000 claims description 11
- 239000003381 stabilizer Substances 0.000 claims description 10
- 235000000346 sugar Nutrition 0.000 claims description 9
- 238000010924 continuous production Methods 0.000 claims description 8
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 claims description 7
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 claims description 7
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 claims description 7
- 229920000642 polymer Polymers 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- CITHEXJVPOWHKC-UUWRZZSWSA-N 1,2-di-O-myristoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCC CITHEXJVPOWHKC-UUWRZZSWSA-N 0.000 claims description 5
- HIHOWBSBBDRPDW-PTHRTHQKSA-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] n-[2-(dimethylamino)ethyl]carbamate Chemical compound C1C=C2C[C@@H](OC(=O)NCCN(C)C)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 HIHOWBSBBDRPDW-PTHRTHQKSA-N 0.000 claims description 5
- 229960003724 dimyristoylphosphatidylcholine Drugs 0.000 claims description 5
- 230000009467 reduction Effects 0.000 claims description 5
- 150000002169 ethanolamines Chemical class 0.000 claims description 4
- 150000001841 cholesterols Chemical class 0.000 claims description 3
- 150000003248 quinolines Chemical class 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- HKOOXMFOFWEVGF-UHFFFAOYSA-N phenylhydrazine Chemical compound NNC1=CC=CC=C1 HKOOXMFOFWEVGF-UHFFFAOYSA-N 0.000 claims 3
- NVTPMUHPCAUGCB-UHFFFAOYSA-N pentyl dihydrogen phosphate Chemical compound CCCCCOP(O)(O)=O NVTPMUHPCAUGCB-UHFFFAOYSA-N 0.000 claims 2
- QFMZQPDHXULLKC-UHFFFAOYSA-N 1,2-bis(diphenylphosphino)ethane Chemical compound C=1C=CC=CC=1P(C=1C=CC=CC=1)CCP(C=1C=CC=CC=1)C1=CC=CC=C1 QFMZQPDHXULLKC-UHFFFAOYSA-N 0.000 claims 1
- GOJFAKBEASOYNM-UHFFFAOYSA-N 2-(2-aminophenoxy)aniline Chemical compound NC1=CC=CC=C1OC1=CC=CC=C1N GOJFAKBEASOYNM-UHFFFAOYSA-N 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- 238000003860 storage Methods 0.000 abstract description 22
- 238000001890 transfection Methods 0.000 description 41
- 239000000243 solution Substances 0.000 description 40
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 36
- 108020004414 DNA Proteins 0.000 description 32
- ISXSJGHXHUZXNF-LXZPIJOJSA-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] n-[2-(dimethylamino)ethyl]carbamate;hydrochloride Chemical compound Cl.C1C=C2C[C@@H](OC(=O)NCCN(C)C)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 ISXSJGHXHUZXNF-LXZPIJOJSA-N 0.000 description 24
- 125000002091 cationic group Chemical group 0.000 description 18
- 239000011780 sodium chloride Substances 0.000 description 18
- NEZDNQCXEZDCBI-UHFFFAOYSA-N 2-azaniumylethyl 2,3-di(tetradecanoyloxy)propyl phosphate Chemical compound CCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCC NEZDNQCXEZDCBI-UHFFFAOYSA-N 0.000 description 17
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 17
- 229930006000 Sucrose Natural products 0.000 description 17
- 239000005720 sucrose Substances 0.000 description 17
- 238000002360 preparation method Methods 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 10
- 239000002245 particle Substances 0.000 description 10
- 238000011049 filling Methods 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 150000008163 sugars Chemical class 0.000 description 7
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000009826 distribution Methods 0.000 description 6
- 230000002572 peristaltic effect Effects 0.000 description 6
- 238000004626 scanning electron microscopy Methods 0.000 description 6
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000006185 dispersion Substances 0.000 description 5
- 238000002296 dynamic light scattering Methods 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 150000005846 sugar alcohols Chemical class 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 4
- PFNFFQXMRSDOHW-UHFFFAOYSA-N Spermine Natural products NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 229920002246 poly[2-(dimethylamino)ethyl methacrylate] polymer Polymers 0.000 description 4
- 229920001296 polysiloxane Polymers 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 3
- KWVJHCQQUFDPLU-YEUCEMRASA-N 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KWVJHCQQUFDPLU-YEUCEMRASA-N 0.000 description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 3
- XULFJDKZVHTRLG-JDVCJPALSA-N DOSPA trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F.CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)CCNC(=O)C(CCCNCCCN)NCCCN)OCCCCCCCC\C=C/CCCCCCCC XULFJDKZVHTRLG-JDVCJPALSA-N 0.000 description 3
- 229930195725 Mannitol Natural products 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000004698 Polyethylene Substances 0.000 description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 3
- 238000010668 complexation reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000594 mannitol Substances 0.000 description 3
- 235000010355 mannitol Nutrition 0.000 description 3
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 3
- 239000000902 placebo Substances 0.000 description 3
- 229940068196 placebo Drugs 0.000 description 3
- 229920000573 polyethylene Polymers 0.000 description 3
- 229940063675 spermine Drugs 0.000 description 3
- 238000011146 sterile filtration Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 239000008215 water for injection Substances 0.000 description 3
- QPYIWAXVQDAFLS-UHFFFAOYSA-M 1,2-di(tetradecanoyloxy)propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCC(=O)OC(C)C([N+](C)(C)CCO)OC(=O)CCCCCCCCCCCCC QPYIWAXVQDAFLS-UHFFFAOYSA-M 0.000 description 2
- YNGDWRXWKFWCJY-UHFFFAOYSA-N 1,4-Dihydropyridine Chemical class C1C=CNC=C1 YNGDWRXWKFWCJY-UHFFFAOYSA-N 0.000 description 2
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 2
- WALUVDCNGPQPOD-UHFFFAOYSA-M 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCCCCCCCC WALUVDCNGPQPOD-UHFFFAOYSA-M 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- GAWIXWVDTYZWAW-UHFFFAOYSA-N C[CH]O Chemical group C[CH]O GAWIXWVDTYZWAW-UHFFFAOYSA-N 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 239000004375 Dextrin Substances 0.000 description 2
- 229920001353 Dextrin Polymers 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 102000003951 Erythropoietin Human genes 0.000 description 2
- 108090000394 Erythropoietin Proteins 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 238000003109 Karl Fischer titration Methods 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 2
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 2
- 108700005077 Viral Genes Proteins 0.000 description 2
- 241001441550 Zeiformes Species 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 235000019425 dextrin Nutrition 0.000 description 2
- UMGXUWVIJIQANV-UHFFFAOYSA-M didecyl(dimethyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCC[N+](C)(C)CCCCCCCCCC UMGXUWVIJIQANV-UHFFFAOYSA-M 0.000 description 2
- 150000002016 disaccharides Chemical class 0.000 description 2
- 229940105423 erythropoietin Drugs 0.000 description 2
- 230000009477 glass transition Effects 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 2
- 229960000367 inositol Drugs 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 229940047122 interleukins Drugs 0.000 description 2
- 239000012669 liquid formulation Substances 0.000 description 2
- 238000012792 lyophilization process Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000002304 perfume Substances 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 2
- 238000009516 primary packaging Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000001294 propane Substances 0.000 description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 229960000187 tissue plasminogen activator Drugs 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 102000003390 tumor necrosis factor Human genes 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- LOLKAJARZKDJTD-UHFFFAOYSA-N 4-Ethoxy-4-oxobutanoic acid Chemical class CCOC(=O)CCC(O)=O LOLKAJARZKDJTD-UHFFFAOYSA-N 0.000 description 1
- 241000242764 Aequorea victoria Species 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- HEBKCHPVOIAQTA-QWWZWVQMSA-N D-arabinitol Chemical compound OC[C@@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-QWWZWVQMSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 208000036993 Frustration Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 108010018924 Heme Oxygenase-1 Proteins 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000897480 Homo sapiens C-C motif chemokine 2 Proteins 0.000 description 1
- 101000898034 Homo sapiens Hepatocyte growth factor Proteins 0.000 description 1
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 1
- 101000868152 Homo sapiens Son of sevenless homolog 1 Proteins 0.000 description 1
- 101100369992 Homo sapiens TNFSF10 gene Proteins 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical class Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000014429 Insulin-like growth factor Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- 108090000171 Interleukin-18 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 101000978374 Mus musculus C-C motif chemokine 12 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101710090055 Nitric oxide synthase, endothelial Proteins 0.000 description 1
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 1
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- ZYFVNVRFVHJEIU-UHFFFAOYSA-N PicoGreen Chemical compound CN(C)CCCN(CCCN(C)C)C1=CC(=CC2=[N+](C3=CC=CC=C3S2)C)C2=CC=CC=C2N1C1=CC=CC=C1 ZYFVNVRFVHJEIU-UHFFFAOYSA-N 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 101001079591 Rattus norvegicus Putative heme oxygenase 3 Proteins 0.000 description 1
- 102000046283 TNF-Related Apoptosis-Inducing Ligand Human genes 0.000 description 1
- 108700012411 TNFSF10 Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 125000003289 ascorbyl group Chemical class [H]O[C@@]([H])(C([H])([H])O*)[C@@]1([H])OC(=O)C(O*)=C1O* 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- JPNZKPRONVOMLL-UHFFFAOYSA-N azane;octadecanoic acid Chemical class [NH4+].CCCCCCCCCCCCCCCCCC([O-])=O JPNZKPRONVOMLL-UHFFFAOYSA-N 0.000 description 1
- 150000001558 benzoic acid derivatives Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000009529 body temperature measurement Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000007707 calorimetry Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002800 charge carrier Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 150000001860 citric acid derivatives Chemical class 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 229940097362 cyclodextrins Drugs 0.000 description 1
- 230000000368 destabilizing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 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 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 239000012537 formulation buffer Substances 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-L fumarate(2-) Chemical class [O-]C(=O)\C=C\C([O-])=O VZCYOOQTPOCHFL-OWOJBTEDSA-L 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- FBPFZTCFMRRESA-GUCUJZIJSA-N galactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-GUCUJZIJSA-N 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 108010031102 heme oxygenase-2 Proteins 0.000 description 1
- 102000046768 human CCL2 Human genes 0.000 description 1
- 210000005119 human aortic smooth muscle cell Anatomy 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 239000001341 hydroxy propyl starch Substances 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 235000013828 hydroxypropyl starch Nutrition 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 150000003893 lactate salts Chemical class 0.000 description 1
- 239000000832 lactitol Substances 0.000 description 1
- 235000010448 lactitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-JVCRWLNRSA-N lactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-JVCRWLNRSA-N 0.000 description 1
- 229960003451 lactitol Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 150000002688 maleic acid derivatives Chemical class 0.000 description 1
- 150000004701 malic acid derivatives Chemical class 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 229960001855 mannitol Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229940126601 medicinal product Drugs 0.000 description 1
- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-M methanesulfonate group Chemical class CS(=O)(=O)[O-] AFVFQIVMOAPDHO-UHFFFAOYSA-M 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229940112042 peripherally acting choline derivative muscle relaxants Drugs 0.000 description 1
- 125000005342 perphosphate group Chemical group 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- IDOWTHOLJBTAFI-UHFFFAOYSA-N phenmedipham Chemical compound COC(=O)NC1=CC=CC(OC(=O)NC=2C=C(C)C=CC=2)=C1 IDOWTHOLJBTAFI-UHFFFAOYSA-N 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 108700038606 rat Smooth muscle Proteins 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- HEBKCHPVOIAQTA-ZXFHETKHSA-N ribitol Chemical compound OC[C@H](O)[C@H](O)[C@H](O)CO HEBKCHPVOIAQTA-ZXFHETKHSA-N 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 150000003873 salicylate salts Chemical class 0.000 description 1
- 235000021309 simple sugar Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 150000003900 succinic acid esters Chemical class 0.000 description 1
- 229940097346 sulfobutylether-beta-cyclodextrin Drugs 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 150000003892 tartrate salts Chemical class 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- ODLHGICHYURWBS-LKONHMLTSA-N trappsol cyclo Chemical compound CC(O)COC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)COCC(O)C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1COCC(C)O ODLHGICHYURWBS-LKONHMLTSA-N 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Classifications
-
- 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
- A61K9/1277—Processes for preparing; Proliposomes
-
- 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
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
Definitions
- the present invention relates to processes for the production of liposomes and complexes consisting of liposomes and nucleic acid molecules (lipoplexes), processes for the stable storage of corresponding lipoplexes, and correspondingly prepared liposomes and lipoplexes.
- liposomes have been recognized as a promising alternative to viral gene transfer systems.
- complexation of nucleic acids in / with liposomes and the use of appropriate complexes (lipoplexes) for gene therapy approaches present new challenges to liposome technology.
- lipoplexes complexes
- the process must be able to be carried out under aseptic conditions and must meet the strict manufacturing regulations for pharmaceuticals.
- Cationic liposomes are either produced from a single cationic lipid or, more frequently, from a combination of a cationic lipid with a neutral amphiphile (helper lipid, co-lipid) .
- the first reagent of this type DOTMA ([N-1- (2,3-dioleyloxy) propyl] -N, N, N, -triemethylammonium chloride), is capable of being found in mammalian cells after mixing with an equimolar amount of DOPE (dioleoylphosphatidylethanolamine) to be transfected in vitro and in vivo (Feigner et al.
- DOPE dioleoylphosphatidylethanolamine
- cationic lipids are now known which are used either directly or in conjunction with neutral amphiphiles for gene transfer. These include, for example, DORI (1, 2-dioleoyloxycarbonylpropyl-3-dimethylhydroxyethylammonium bromide, DORIE (1, 2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide), DOTAP (dioleoyltrimethylammonium propane (N- [1- (2,3-dioleoyloxy) propyl , N, N-trimethylammonium methyl sulfate)), DMRIE (N- (1,2-dimyristoyloxypropyl) -N, N-dimethyl-N-hydroxyethylammonium bromide)), DOGS (di-octadecylamidologycylspermine), DOSPA (2,3- dioleyloxy-
- spermine cholesteryl carbamates as described, for example, in WO96 / 18372 or 1,4-dihydropyridine derivatives, as described, for example, in WO01 / 62946.
- a non-exhaustive summary of relevant cationic lipids can also be found, for example, in the publication by Miller (1998), Angew. Chem. 110, 1862-1880, to which express reference is made here.
- cationic lipids and mixtures are also commercially available such as Effectene TM and SuperFect TM (Qiagen, Hilden, Germany), FuGene 6 TM (Röche, Mannheim, Germany), LipoFectin TM, LipoFectin2000 TM, LipoFECTAMINE Plus TM (Invitrogen, Düsseldorf, Germany) ,
- lipofection i.e. the transfection of nucleic acid complexed in / with liposomes, has been promoted and successfully tested on many cell types and cell lines.
- lipofection i.e. the transfection of nucleic acid complexed in / with liposomes
- the lipoplexes for clinical applications are freshly prepared by the doctor in the hospital ("bed-side") or stored frozen (Galanis (2002) supra; Stopeck et al. (2001) supra; Voges et al. (2002) supra; Jacobs et al. (2001) supra; Morgtan (2002) supra, Gao & Huang (1995) Gene Therapy 2: 710-722; Feigner et al. (1995) supra; navel et al. (1994) Hum. Gene Therapy 5: 57-77 and 1089-1094), so it is very important to develop procedures that provide reproducible lipoplex quality.
- liposomes can be produced by sonicating lipid-containing solutions using an ultrasound bath or a vibrating rod.
- Vibrating rod contaminate the liposomes with small metal particles.
- liposomes can be formed using a dehydration-hydration method. Active ingredients can also be included in the liposomes.
- the lipid suspensions formed show a high inhomogeneity in terms of size distribution and polydispersity. More homogeneous lipid suspensions are only obtained after an additional process step, microfluidization (Washington & Davis, (1988) Int. J. Pharmaceutics 44: 169-176; Vuillemard JC. (1991) J. Microencapsulation 8: 547-562). However, this method works with very high pressures.
- Another object of the present invention was to find a corresponding process for the production of liposomes or lipoplexes, in which liposomes or lipoplexes can be produced under GMP conditions. This means the production of reproducibly homogeneous liposome / lipoplex batches under aseptic conditions on a larger scale.
- Another object of the present invention was to provide corresponding homogeneous and storage-stable liposomes and lipoplexes, which have sufficient transfection efficiency with good stability and GMP quality.
- the present invention relates to a process for the production of homogeneous liposomes, in which a lipid suspension is extruded in a continuous process through a porous membrane, preferably with a pore size between 600-900 nm under low pressure conditions at less than 3 x 10 5 Pa.
- the concentration of the liposomes in the lipid suspension is between 0.04 and 5 mg / ml, preferably between 0.1-2 mg / ml, in particular between 0.1-1 mg / ml.
- Flow rates between 10-250 ml / min, preferably between 50-150 ml / min, particularly preferably between 75-120 ml / min have proven to be particularly suitable in this connection.
- the extrusion process according to the invention can also be carried out at room temperature without the quality of the liposomes being impaired.
- the corresponding method according to the invention is carried out in a closed system under aseptic conditions.
- the liposomes or the liposome suspension can be prefiltered beforehand through membranes with a pore size of up to 1000 nm.
- the present invention relates to a process for the continuous low-pressure extrusion of liposomes, preferably of liposomes which contain a cationic lipid or a mixture of a cationic lipid and a neutral amphiphile, preferably a combination of DC-Chol or DAC-Chol and DOPE, at a pressure below 3x10 5 Pa, a flow rate between 10 - 250 ml / min and a lipid concentration between 0.04 - 5 mg / ml, characterized in that the lipid suspension is continuously extruded through the porous membrane between 2 - 20 times.
- a neutral amphiphile preferably a combination of DC-Chol or DAC-Chol and DOPE
- the equipment consists of a mechanical or electrical pump (1), a flow measurement control (2), a manometer for measuring the filtration pressure (3), a filter holder with an extrusion membrane (pore size e.g. 600 nm / 0 e.g. 47 mm) (4) and a ventilation valve (8), a temperature measuring device (5), a reservoir (6) and an annular line system (7), which enables a continuous directional flow through the extrusion membrane and the backflow into the reservoir.
- the present invention also relates to liposomes or liposome mixtures which contain liposomes which are produced by one of the methods according to the invention described here.
- the present invention relates in particular to liposome mixtures consisting of liposomes with a defined size between 250 and 800 nm, preferably between 250 and 600 nm, the liposomes containing a cationic lipid and a neutral amphiphile and are characterized in that the polydispersity index of the liposome mixture has a value ⁇ 0.60, preferably ⁇ 0.50, particularly preferably ⁇ 0.4.
- the liposome mixture is characterized in that the cationic lipid is a cholesterol such as, for example, DC chol ((3-beta [N (N ', N'-dimethylaminoethane) carbamoyl] cholesterol)) or DAC -Chol ((3-beta [N (N, N'-dimethylaminoethane) carbamoyl] cholesterol)).
- the liposomes according to the invention contain an ethanolamine derivative, for example dioleoylphosphatidylethanolamine (DOPE)).
- DOPE dioleoylphosphatidylethanolamine
- the mixture via a so-called Y-piece has proven to be particularly advantageous, which allows the liposomes and nucleic acid molecules to be brought together evenly and continuously.
- a liposome-nucleic acid charge ratio +/- between 4 - 0.01, preferably between +/- 1.25 - 0.75 leads to particularly stable and homogeneous lipoplexes.
- the combination of equal volumes of a suspension containing liposomes and the solution containing nucleic acid has proven to be particularly advantageous.
- the concentration of the liposomes when the liposomes and nucleic acid are mixed is between 0.02-1 mg / ml. Flow rates between 100 and 500 ml / min have been found to be advantageous when mixing liposomes and nucleic acids.
- the corresponding process can be carried out in a closed apparatus, so that the lipoplexes can be produced under aseptic conditions.
- the corresponding method according to the invention enables the production of a lipoplex mixture consisting of lipoplexes with a defined size between 250-600 nm, preferably between 275-500 nm, particularly preferably between 275-400 nm, and with a polydispersity index of ⁇ 0.50, preferably of ⁇ 0.40.
- the present invention consequently relates to lipoplexes which are produced by the process according to the invention described here, in particular those with a defined size between 250-600 nm, preferably between 275-500 nm, particularly preferably between 275-400 nm, and with a polydispersity index of ⁇ 0.50, preferably ⁇ 0.40.
- the present invention relates to a process for the long-term storage of appropriately prepared lipoplexes, for example by lyophilization in the presence of a suitable stabilizer, comprising the steps (a) freezing the lipoplex mixture to a temperature of ⁇ -50 ° C .; (b) drying the lipoplex mixture at approximately -20 ° C for at least 35 hours (c) post-drying the lipoplex mixture at approximately 20 ° C for at least 10 hours.
- the method for lyophilizing the lipoplex mixture according to the invention in the presence of a suitable one Stabilizer the following steps: (a) freezing the lipoplex mixture to a temperature of ⁇ - 50 ° C with a temperature reduction rate of approximately ⁇ 1 ° C / min; (b) incubating the Lipoplex mixture at ⁇ - 50 ° C for at least 2 hours; (c) heating the lipoplex mixture to approximately -20 ° C at a heating rate of approximately ⁇ 0.3 ° C / min; (d) drying the lipoplex mixture at about -20 ° C for at least 35 hours; (e) heating the lipoplex mixture from about -20 ° C to about 20 ° C with a heating rate of about ⁇ 0.44 ° C / min; and (f) post-drying the lipoplex mixture at about 20 ° C for at least 10 hours. Drying under point (d) at a pressure between 0.01 and 0.1 mbar, preferably between 0.025 and 0.05 m
- the present invention also relates to lipoplex lyophilisates obtainable by one of the methods according to the invention described here, and the use of the homogeneous lipoplexes or lipoplex lyophilisates described here for the production of or as a medicament in gene therapy for transfection of mammalian cells.
- Figure 1 Schematic structure of a continuous extrusion apparatus consisting of a peristaltic pump (1), flow measurement control (2), manometer for measuring the filtration pressure (3), filter holder with extrusion membrane of a defined pore size, e.g. 600 nm 0 47 mm (4) and ventilation valve (8), temperature measurement (5), a storage vessel (6) and an annular line system (7).
- Figure 2 Flow diagram for the production of lipoplexes of defined particle size.
- FIG. 3 Schematic sequence for the production of storable lipoplexes.
- Storage vessel for lipid suspension (1) a first pump (2), extrusion device with a porous membrane of 600 - 900 nm (3), valves (4), branching (5), Y-piece (6), storage vessel for nucleic acids (7 ), a second pump (8) and a collecting vessel for lipoplexes (9).
- Figure 4 Flow diagram for the preparation of DC30 / Nu kl unic acid lipoplexes in the ratio 4: 1. Batch size: 3750 ml bulk, dose: 0.025 mg / ml nucleic acid.
- Figure 5 Flow diagram for the production of DAC30 / nucleic acid lipoplexes in the ratio 5: 1. Batch size: 700 ml bulk, dose: 0.025 mg / ml nucleic acid.
- Figure 6 (A) SEM (scanning electron microscopy) image of the lyophilization cake surface. (B) SEM (scanning electron microscopy) image in the lyophilization cake.
- Figure 7 Influence of the different mixing order in the lipoplex production of lipofectin and pAH7-EGFP plasmid in a mass ratio of 6: 1 (w / w) on the transfection efficiency and lipoplex (aggregate) size.
- LzD lipid to DNA
- DzL DNA to lipid.
- the lipoplex size was determined using PCS. The transfection efficiency was tested on A-10 SMC (smooth muscle cells).
- FIG. 8 Bioactivity (transfection efficiency) of DAC30 / pMCP-1 5: 1 (w / w) lipoplexes stored at 4 ° C for several months. The transfection efficiency is based on an internal standard.
- Figure 9 Y-piece for mixing nucleic acid and liposomes.
- the present invention relates to a process for the production of homogeneous liposomes, in which a lipid suspension is extruded in a continuous process through a porous membrane with a pore size of preferably 600-900 nm, characterized in that the extrusion under low pressure conditions at pressures below 3x10 5 Pa is carried out.
- the liposomes produced accordingly have an average size of 250-800 nm.
- the polydispersity index of the liposome mixture is ⁇ 0.6, preferably ⁇ 0.5 or ⁇ 0.4.
- liposomes is understood to mean an aqueous lipid-containing suspension of multilayer or multilayer (at least consisting of a lipid bilayer) mostly spherical assemblages of lipid molecules that result from mechanical mixing of a dry lipid in water.
- low pressure conditions in the sense of the invention means filtration pressures of less than 3 x 10 5 Pa, preferably less than 2 x 10 5 Pa and particularly preferably less than 1 x 10 5 Pa.
- Extrusion through a membrane with a defined pore size enables the production of liposomes of a defined size. It has been shown that not only the pore size but also other parameters such as pressure, flow rate, lipid concentration influence the chemical-physical properties of the extruded liposomes. Extrusion at high pressures (greater than 3 ⁇ 10 5 Pa) leads to liposomes with relatively small diameters of approximately less than 200 nm. When mixed with nucleic acids, corresponding liposomes show a very low transfection efficiency, which severely limits their suitability in the field of gene therapy. In addition, a process that requires very high working pressures can only be converted to an industrial scale with great technical effort.
- the present invention has succeeded in creating a process for the production of homogeneous liposomes with a defined size between 250-800 nm, preferably between 280-700 nm, particularly preferably between 280-600 nm to provide.
- the process according to the invention is characterized by a high degree of reproducibility, which in turn enables the production of homogeneous batches of liposomes for gene therapy purposes.
- the present invention also relates to a process for the preparation of homogeneous liposomes in which a lipid suspension in a continuous process' through a porous membrane having a pore size from 600 to 900 nm under low pressure conditions at low 3x10 5 Pa is extruded, characterized in that Lipid concentration is 0.04-5 mg / ml, preferably 0.1-2 mg / ml, particularly preferably 0.1-1 mg / ml, even more preferably 0.25-1 mg / ml.
- the flow rate also has an influence on the homogeneity of the liposomes / liposome mixture.
- flow rates between 10-250 ml / min, preferably between 50-150 ml / min, particularly preferably between 75-120 ml / min, led to particularly homogeneous liposomes.
- the present invention also relates to methods for producing homogeneous liposomes by low-pressure extrusion through a 600-900 nm membrane, characterized in that the lipid concentration is between 0.04-5 mg / ml, preferably between 0.1-2 mg / ml, particularly preferably between 0.1-1 mg / ml, even more preferably between 0.25-1 mg / ml and the flow rate during extrusion between 10 - 250 ml / min, preferably between 50-150 ml / min, particularly preferably between 75-120 ml / min.
- the extrusion membrane is preferably a polycarbonate membrane with a pore size of 600-900 nm, for example with a pore size of 600, 650, 750, 800, 850 or 900 nm.
- extrusion membranes made of other materials are also suitable for the purposes of the invention , e.g. from polymers with suitable properties.
- the present invention also relates to processes for the production of homogeneous liposomes by low-pressure extrusion of a lipid suspension, preferably with a lipid concentration between 0.04-5 mg / ml, preferably between 0.1-2 mg / ml, particularly preferably between 0.1-1 mg / ml preferably between 0.25
- lipid suspension is extruded through the membrane at least twice, preferably between 2 and 20 times.
- a corresponding method in which the extrusion takes place in a continuous process is particularly preferred. Even more preferred is a corresponding extrusion process which is carried out in a closed system, as shown for example in FIG. 1, under aseptic conditions.
- the process according to the invention is particularly suitable for producing homogeneous cationic liposomes / liposome mixtures or of liposomes / liposome mixtures containing a cationic lipid and a neutral amphiphile.
- liposomes which consist of a mixture of neutral amphiphile and cationic lipid, have been proven proved to be particularly homogeneous and stable if they were produced by the process according to the invention described here.
- a “cationic lipid” is understood to mean a lipid which has a positive excess charge under the given conditions.
- a neutral (zwitterionic) amphiphile is a molecule which has no excess charge under the given conditions and is therefore charge-neutral
- the present invention also relates to methods for producing homogeneous liposomes / liposome mixtures containing at least one cationic lipid or a cationic lipid and a neutral amphiphile.
- Suitable cationic lipids for the purposes of the invention are, for example, DOTMA [N-1 - (2,3-dioleyloxy) propyl] -N, N, N, -triemethylammonium chloride) DORI (1, 2-dioleoyloxycarbonylpropyl-3-dimethylhydroxyethylammonium bromide, DORIE ( 1, 2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide), DOTAP (dioloyltrimethylammonium propane (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium methylsulfate)), DMRIE (N- (1
- spermine cholesteryl carbamates as exemplified in WO96 / 18372, or 1,4-dihydropyridine derivatives, as described, for example, in WO01 / 62946.
- a non-exhaustive summary of relevant cationic lipids can be found, for example, in the publication by Miller (1998), Angew. Chem. 110, 1862-1880, to which express reference is made here.
- the process according to the invention is preferably suitable for the production of homogeneous cholesterol-containing liposomes / liposome mixtures, preferably of liposomes / liposome mixtures which contain DAC-Chol or DC-Chol as the cationic lipid.
- DAC-Chol can also be found, inter alia, in WO96 / 20208, Reszka et al., A description of DC-Chol in US Pat. No. 5,283,185, Epand et al.
- Some cationic lipids and mixtures are also commercially available, such as Effectene TM and SuperFect TM (Qiagen, Hilden, Germany), FuGene 6 TM (Röche, Mannheim, Germany), LipoFectin TM, LipoFectin2000 TM, LipoFECTAMINE Plus TM (Invitrogen, Düsseldorf, Germany).
- Suitable neutral amphiphiles within the meaning of the invention are, for example, choline derivatives such as dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC) or ethanolamine derivatives such as dimyristoylphosphatidylethanolamine (DMPE), dipalethanolamine (DMPE), dipalethylamine (PEPE), dipalethanolamine (DPPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE), dipalethanolamine (DMPE
- lipid suspension to be extruded contains DOPE as a neutral amphiphile and DC-Chol, preferably DAC-Chol, as a cationic lipid.
- Cationic lipid and neutral amphiphile can be present in a weight ratio of 1:99 to 99: 1, which is intended to encompass all weight ratios between these values. Particularly stable and homogeneous liposomes are obtained when the cationic lipid and the neutral amphiphile are used in a weight ratio of 10:90 to 40:60, for example 10:90, 15:85, 20:80, 25:75, 30:70 , 35:65, 40:60 mixes.
- DC-Chol: DOPE or DAC-Chol: DOPE are also referred to below as DC30 in the case of DC-Chol: DOPE and as DAC30 in the case of DAC-Chol: DOPE.
- the present invention relates to the production of homogeneous liposomes with a low polydispersity index of preferably 0.6 0.6, more preferably of ⁇ 0.5, and even more preferably of ⁇ 0.4, consisting of DC-Chol: DOPE, preferably DAC-Chol: DOPE in a weight ratio of 30:70 (DC30 or DAC30).
- the lipid suspension to be extruded is a suspension of a cationic lipid and a neutral amphiphile in an aqueous solution.
- the lipid suspension can also contain other substances, for example salts, polymers, sugars or sugar alcohols.
- auxiliary substances can further improve the stability of the liposomes to be produced and of the liposome-nucleic acid complexes produced therefrom.
- polymers examples include polyvinyl pyrrolidones, derivatized celluloses such as e.g. Hydroxymethyl, hydroxyethyl or hydroxypropyl ethyl cellulose, polymeric sugars such as e.g. Ficoll or dextran, starch such as Hydroxyethyl or hydroxypropyl starch, dextrins such as e.g. Cyclodextrins (2-hydroxypropyl-ß-cyclodextrin, sulfobutyl ether-ß-cyclodextrin), polyethylenes, glycols, chitosan, collagen, hyaluronic acid, polyacrylates, polyvinyl alcohols and / or pectins.
- polyvinyl pyrrolidones derivatized celluloses such as e.g. Hydroxymethyl, hydroxyethyl or hydroxypropyl ethyl cellulose
- polymeric sugars such as e.g.
- sugars can be mono-, di-, oligo- or polysaccharides or a combination thereof.
- simple sugar are fructose, maltose, galactose, glucose, D-mannose, sorbose and the like.
- Double sugars are, for example, lactose, sucrose, trehalose, cellobiose, and the like.
- raffinose, melezitose, dextrin, starch and the like are particularly suitable.
- sugar alcohols are also suitable.
- Particularly suitable salts are pharmaceutically acceptable salts, such as, for example, inorganic salts such as chlorides, sulfates, phosphates, di-phosphates, hydrobromides and / or nitrate salts.
- the lipid suspension can also contain organic salts, such as, for example, malates, maleates, fumarates, tartrates, succinates, ethyl succinates, citrates, acetates, lactates, methanesulfonates, benzoates, ascorbates, paratoluenesulfonates, palmoates, salicylates, stearates, labolate salts or glucept salts ,
- the production process according to the invention makes it possible to provide homogeneous liposome mixtures consisting of liposomes with a defined size between 250-800 nm, preferably between 280-600 nm, particularly preferably between 280-500 nm, further preferably between 280-400 n
- the present invention also relates to corresponding liposome mixtures consisting of liposomes with a defined size between 250 and 800 nm, preferably between 280-600 nm, particularly preferably between 280-500 nm, more preferably between 280-400 nm, the liposomes being a cationic lipid and contain a neutral amphiphile, characterized in that the polydispersity index of the liposome mixture has a value of ⁇ 0.60, preferably ⁇ 0.50, more preferably ⁇ 0.40.
- the liposomes according to the invention preferably contain a cholesterol derivative such as DC-Chol or DAC-Chol in combination with a neutral amphiphile selected from DMPC, DPPC, DOPC, DMPE, DPPE, or preferably in combination with DOPE.
- a cholesterol derivative such as DC-Chol or DAC-Chol in combination with a neutral amphiphile selected from DMPC, DPPC, DOPC, DMPE, DPPE, or preferably in combination with DOPE.
- Corresponding liposome I liposome mixtures which contain or consist of DOPE as a neutral amphiphile and DC-Chol and / or DAC-Chol as a cationic lipid have proven to be particularly advantageous, the mass ratio of DOPE to the cationic lipid being 70:30 (DC30 or . DAC30).
- the process according to the invention can be used to prepare liposomes / liposome mixtures which contain or consist of the above-mentioned cationic lipids, neutral amphiphiles, salts, polymers, sugars, sugar alcohols or a combination thereof.
- the liposomes according to the invention and described here are suitable for producing homogeneous liposome-nucleic acid complexes, so-called lipoplexes, by simply mixing the corresponding liposomes with nucleic acid molecules.
- the nucleic acid molecules are usually genomic DNA, cDNA, synthetic DNA, RNA, mRNA, ribozymes, antisense-RNA, synthetic peptide nucleotides and single-stranded ones Oligonucleotides, preferably around cDNA.
- the nucleic acid can, for example, be contained in a DNA expression vector or in an expression cassette and thus enable the recombinant expression of a gene of interest after transfection in a target cell.
- therapeutic genes can be introduced into a target cell and expressed there.
- therapeutic genes are, for example, insulin, insulin-like growth factor, human growth hormone (hGH) and other growth factors, tissue plasminogen activator (tPA), erythropoietin (EPO), cytokines, for example interleukins (IL) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL- 16, IL-17, IL-18, interferon (IFN) - alpha, beta, gamma, omega or tau, tumor necrosis factor (TNF) such as TNF-alpha, beta or gamma, TRAIL, G-CSF, GM-CSF, M -CSF, MCP-1 to MCP-5, eNOS, iNOS, HO-1,
- TNF tumor necrosis factor
- Lipoplex production is usually carried out by adding nucleic acid molecules to liposomes or vice versa by adding liposomes to nucleic acid molecules.
- the uniform merging of liposomes and nucleic acids has proven to be particularly advantageous, for example via a so-called Y-piece.
- a Y-piece is understood to mean a 3-leg pipe, as shown by way of example in FIG. 9. It consists of two inlet tubes which are brought together in an outlet tube at an acute angle. The continuous mixing process results in lipoplexes with a constant nucleic acid content.
- the present invention relates to a method for producing lipoplexes, characterized in that the liposomes and nucleic acid molecules are mixed via a Y-piece, which allows the liposomes and nucleic acid molecules to be brought together evenly and continuously.
- a Y-piece in which the two inlet tubes via which the liposome suspension and the nucleic acids are brought together at an acute angle of ⁇ 80, preferably ⁇ 70, more preferably ⁇ 60, even more preferred of ⁇ 50 or ⁇ 40 degrees to each other (see FIG.
- homogeneous lipoplexes with a size of 250-600 nm and a polydispersity of ⁇ 0.5, preferably of ⁇ 0.4 can be produced.
- the inner tube diameter depends on the volumes of liposomes and nucleic acid that are brought together by the Y-piece. In the context of the present invention, Y-pieces with an inner tube diameter of 3 to 5 mm have proven to be advantageous. At flow rates of 20-800 ml / min, preferably at 100-500 ml / min, corresponding Y-pieces enable a continuous complexation of liposomes and nucleic acid to form homogeneous lipoplexes of the appropriate size.
- the lipoplexes shown in the exemplary embodiments were mixed, for example, at a flow rate of approximately 150-170 ml / min or approximately 400 ml / min.
- cationic liposomes or liposome mixtures consisting of a neutral amphiphile and a cationic lipid, such as DC30 or DAC30, were used to produce the lipoplexes.
- the corresponding liposomes thus carry positive charges on their surface, while the nucleic acids due to their Phosphate structure are negatively charged.
- WO98 / 01030 it was known that a charge ratio of positively charged liposomes to negatively charged nucleic acid of 1:20 (+/-), preferably 2:10 (+/-) has a stabilizing influence on the lipoplexes formed.
- the liposome-nucleic acid charge ratio (+/-) reflects the ratio of the positive charge of the cationic lipid used to the negative charge of the nucleic acid.
- the charge carriers of the negative charge on the nucleic acid are the phosphate groups (one negative charge per phosphate group).
- a liposome-nucleic acid charge ratio (+/-) of 1.25-0.75 has proven to be particularly advantageous, in particular in connection with the use of DC30 liposomes or preferably with the use of DAC30 liposomes.
- the present invention also relates to a method for producing homogeneous lipoplexes from cationic liposomes, or from liposomes which contain a cationic lipid and a neutral amphiphile, such as DC30 or DAC30, via a Y-piece, characterized in that liposome nucleic acid Charge ratio (+/-) 4 - 0.01, preferably 2 - 0.1, particularly preferably 1.5 - 0.5 and even more preferably 1.25 - 0.75.
- the stability of the lipoplexes during the mixing process can be positively influenced by the liposome concentration used. It has been found that in the continuous mixing of cationic liposomes, for example DC30 or preferably DAC30, with nucleic acids via a Y-piece with a continuous flow rate of 20-800 ml / min, preferably of 100 - 500 ml / min and a liposome-nucleic acid charge ratio (+/-) of 4
- the present invention relates to a process for the preparation of lipoplex mixtures by continuous mixing of cationic liposomes, e.g.
- DC30 or preferably DAC30 with nucleic acids via a Y-piece with a continuous flow rate of 20-800 ml / min, preferably 100-500 ml / min and a liposome-nucleic acid charge ratio (+/-) of 4-0.01, preferably from 2 to 0.1, particularly preferably from 1.5 to 0.5 and even more preferably from 1.25 to 0.75, characterized in that a homogeneous liposome suspension with a liposome concentration of approximately 0.02-1 mg / ml, preferably approximately 0.1-0.5 mg / ml, is used.
- the processes for producing the liposomes and the mixture of liposomes and nucleic acids can be coupled directly to one another.
- the present invention relates to a process for the production of homogeneous lipoplex mixtures with lipoplexes with a size of 250-600 nm, preferably 275-500 nm, particularly preferably 275-400 nm and preferably a polydispersity index of ⁇ 0.5, particularly preferably from ⁇ 0.4, comprising the steps: (a) extrusion of a lipid suspension containing a cationic lipid or a mixture of a cationic lipid and a neutral amphiphile, for example DC30 or preferably DAC30, in a continuous process through a 600-900 nm membrane , preferably with a flow rate between 10 - 250 ml / min, particularly preferably between 50 - 150 ml / min, further preferably between 75 - 120 ml / min,
- the lipid suspension is extruded through the membrane at least once, but preferably continuously between 2 and 20 times; and (b) the correspondingly prepared liposome mixture with nucleic acid molecules which have been previously sterile filtered, preferably through a 0.2 ⁇ m filter, over a Y-piece with a continuous flow rate of 20-800 ml / min, preferably 100-500 ml / min and a liposome-nucleic acid charge ratio (+/-) of 4-0.01, preferably 2-0.1, particularly preferably 1.5-0.5 and even more preferably 1.25-0.75.
- this “combined” method is carried out in a closed system under aseptic conditions.
- An apparatus with which a correspondingly combined method can be carried out is shown by way of example in FIG. 3.
- the lipid suspension is pumped continuously by means of a pump (2) through an extrusion device with a porous membrane of 600-900 nm (3), behind the extrusion device there is a branch (5) which, on the one hand, refluxes the lipid suspension into the receptacle (1), thus allowing multiple extrusion of the lipid suspension or, alternatively, allowing the extruded lipid suspension to be passed on to the Y-piece (6), via which the mixture with the nucleic acid takes place, between the branching (5) and the receptacle (1) and between the V Branch (5) and the Y-piece (6) are each valves (4) through which the return flow or the flow in the direction of the Y-piece can be regulated.
- the sterile-filtered nucleic acid is located in a second storage container (7) and can be pumped directly to the Y-piece (6) by means of a second pump (8).
- the lipoplexes formed by mixing extruded lipid suspension and nucleic acid can be collected in a collecting vessel (9).
- the device is preferably a closed system, so that the manufacturing process can be carried out under aseptic conditions.
- a corresponding device is also the subject of the present invention.
- the lipoplex mixtures provided in the context of the present invention were characterized by lipoplexes with a size of 250-600 nm, preferably from 275-500 nm, more preferably from 275-400 nm and a low polydispersity index of ⁇ 0.5, preferably of ⁇ 0.4 and in some cases even from ⁇ 0.3. Due to the high degree of automation, homogeneous lipoplex mixtures could be generated across batches, which are particularly suitable as or for the production of medicinal products for gene therapy applications.
- the present invention also relates to lipoplex mixtures consisting of lipoplexes with a defined size between 250 and 600 nm, the lipoplexes consisting of a mixture of homogeneous liposomes according to the invention, as described above, and nucleic acid molecules characterized in that the polydispersity index of the lipoplex mixture has a value ⁇ 0.5, preferably ⁇ 0.4.
- the lipoplexes are corresponding liposome-nucleic acid complexes which consist of DC30 or preferably of DAC30-nucleic acid complexes with corresponding physical parameters.
- Another aspect of the present invention relates to a method for lyophilization of the lipoplexes according to the invention described here, preferably of lipoplexes containing a mixture of DOPE and DC-Chol or DOPE and DAC-Chol, preferably in a ratio of 70:30 (DC30 or DAC30).
- the method described below makes it possible to store the lipoplexes over longer periods, preferably over at least 8 months (cf. Table 20).
- the lipoplexes after their reconstitution, differ neither in their physical parameters nor in their bioactivity from non-lyophilized (ie “freshly” prepared) lipoplexes.
- the process according to the invention for the lyophilization of lipoplexes takes place in the presence of a suitable stabilizer, predominantly in Presence of 250 mM succrose and 25 mM sodium chloride and comprises the following steps: (a) freezing the lipoplex mixture to a temperature of ⁇ - 50 ° C; (b) drying the lipoplex mixture at approximately - 20 ° C for at least 35 hours, preferably under vacuum for 35-60 hrs. (c) post-dry the lipoplex mixture at approximately 20 ° C for at least 10 hours, preferably for 10 - 24 hours.
- the times here are to be understood as guide times.
- Various sugars, sugar alcohols or polymers can be used as stabilizers, for example. These can be used as single commons, as a mixture and / or in combination with salts. Corresponding examples of suitable sugars, sugar alcohols, polymers or salts can be found above. It is advantageous to use the stabilizing agent in the form of an isoomotic solution (approx. 290-330 mOsm), preferably in the preparation of the liposomes.
- the lipids can, for example, be suspended in an appropriate solution containing an appropriate stabilizing agent before extrusion.
- a composition which contains sucrose as the disaccharide and sodium chloride as the inorganic salt is particularly advantageous for these purposes.
- An example of such an isoosmotic composition (for example 300 mOsm) is a combination of sodium chloride in a concentration in a range from about 5 mM to about 100 mM, in particular 5, 10, 15, 20, 25, 30, 35, 40 , 45 or 50 mM, with an appropriate proportion of sucrose.
- sucrose or trehalose preferred for the aforementioned purposes.
- an isoosmotic composition e.g. 300 mOsm
- sucrose or trehalose preferred for the aforementioned purposes.
- such an isoosmotic composition e.g. 300 mOsm
- sucrose or trehalose in a concentration in a range from about 10-290 mM, in particular about 150-290 mM
- sucrose or trehalose in a concentration in a range of about 10- 290 mM, in particular about 10-150 mM.
- sucrose in combination with sodium chloride preferably 250 mM sucrose and 25 mM sodium chloride, has proven to be advantageous.
- a method for lyophilization of the lipoplexes described above has proven to be particularly advantageous, which comprises the following steps: (a) freezing the lipoplex mixture to a temperature of ⁇ -50 ° C. with a temperature reduction rate of approximately ⁇ 1 ° C./min; (b) incubation of the lipoplex mixture at ⁇ - 50 ° C for at least 2 hours; (c) heating the lipoplex mixture to approximately -20 ° C with a heating rate of approximately ⁇ 0.3 ° C / min; (d) drying the lipoplex mixture at approximately -20 ° C for at least 35 hours, preferably for 35-60 hours.
- the present invention also relates to a corresponding method.
- pressures between 0.01-0.1 mbar, preferably between 0.025-0.05 mbar have proven to be advantageous (cf. exemplary embodiments, Tables 11 and 13).
- the present invention also relates to lipoplex lyophilisates which are prepared by one of the processes described here.
- the present invention further comprises the use of the lipoplex mixtures according to the invention, directly or in lyophilized form, in gene therapy, including a combination therapy with pharmacological active ingredients. It may be useful to combine gene therapy with other therapeutic approaches, such as the application of pharmacological agents including proteins and / or peptides.
- the lipoplexes or a pharmaceutical composition containing them are usually administered with a total dose in a range from about 0.1 to about 40 ⁇ g (including all values in between), based on the total amount of nucleic acid.
- the “intermediate value” is understood to be any value between the specified upper and lower limits, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, etc .; 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc .; 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, etc .; 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, etc .; 5.0, etc .; 6.0, etc .; 7.0, etc .; 8.0, etc .; 9.0, etc .; 10.0, etc .; 11.0, etc., 12.0, etc .; 13.0
- the pharmaceutical composition is preferably administered with a total dose in a range from approximately 0.5 ⁇ g to approximately 10 ⁇ g, particularly preferably approximately 1 ⁇ g to approximately 5 ⁇ g, in each case based on the total amount of nucleic acid.
- excipients used meet the requirements for pharmaceutically approved excipients: sucrose (Südzucker AG, Kunststoff, DE), sodium chloride (Merck KG, Darmstadt, DE), WFI (water for injection) (Boehringer Ingelheim, Biberach, DE). All lipids used are commercially available: DC cholesterol and DOPE can be purchased from Avanti Polar Lipids, Inc., DAC30 from GOT Therapeutics Berlin, DE.
- the plasmids used sometimes also referred to below simply as nucleic acid, such as the plasmid encoding pMCP-1, an MCP-1 (monocyte chemoattractent protein 1), the plasmid expressing pEGFP, an EGFP (enhancing green fluorescent protein of A. victoria) were cloned and produced by Boehringer Ingelheim.
- the coding region of MCP-1 or EGFP was cloned behind a heterologous promoter (CMV promoter).
- CMV promoter heterologous promoter
- the plasmid also had a selection marker gene (neomycin phosphotransferase gene) so that positively transfected cells could be selected in the presence of a selection agent (e.g. G-418).
- the plasmids were approximately 5 kilobase pairs in size.
- the cells BHK21, COS-7, HASMC, A-10 SMC used for the transfection experiments are obtained from ATCC (American Type Culture Collection)
- a binary lipid perfume is produced according to the "Technical Information"
- lipids cationic lipid and helper lipid DOPE
- the two lipids are dissolved separately in chloroform or a chloroform: methanol mixture (2: 1 v / v).
- the two lipids are then titrated together in the desired mass ratio.
- DC30 the number after the abbreviation of the cationic lipid indicates its mass ratio in the mixture.
- DAC30 A lipid mixture consisting of 30% DAC cholesterol and 70% DOPE is referred to below as DAC30.
- the lipid mixture is then sterile filtered.
- the binary lipid mixture, dissolved in the organic solvent is transferred to a freeze-dryer, which has been pre-cooled to -20 ° C., and the sample is equilibrated until the temperature of the solution has equalized.
- the solvent is removed overnight at -20 ° C and a pressure of 0.94 mbar.
- the residual traces of organic solvents are then removed in a high vacuum (10 "3 mbar).
- the organic solvent can be used Remove by blowing a stream of nitrogen or argon over the sample (shake gently) and warm the sample to about 30 - 40 ° C.
- the residual traces of organic solvents are also removed in a high vacuum. These steps are carried out under aseptic conditions. Lipid suspensions can now be produced from the lipid films produced in this way.
- DAC30 suspension Preparation of a DAC30 suspension: To prepare a 1 mg / ml lipid suspension from DOPE / DAC-Chol 70/30 (w / w), 1 ml transfection solution (250 mM sucrose, 25 mM NaCI) and 1 mg DAC30 are mixed and mixed for 30 min Allow room temperature to swell. A 0.25 mg / ml DAC30 lipid suspension was prepared from this by appropriate dilution with transfection solution.
- the nucleic acid (1 mg / ml) stored at -20 ° C is thawed in the refrigerator at 2-8 ° C and diluted to the desired concentration with transfection solution (250 mM sucrose, 25 mM NaCl).
- the plasmid (for example pMCP-1 or pEGFP) is stirred into the transfection solution (0.05 mg / ml).
- the sterile filtration is then carried out via a 0.2 ⁇ m sterile filter (depending on the amount to be filtered, sterile filters of different sizes are used: e.g. Millipak TM 20: 100 cm 2 filter area, Millipak TM 40: 200 cm 2 filter area, Millipore). Filtration takes place via a peristaltic pump.
- the scatter of a He-Ne laser on the sample is measured at an angle of 90 ° (according to ISO 13321: 1996 (E)) and the two parameters (0 and Pl) are determined from the scattering data by evaluation with the cumulative analysis.
- the mean particle diameter P cs (hereinafter referred to only as 0) is defined according to ISO 13321: 1996 (E) as:
- the lipid concentration and the ratio of cationic lipid (eg DC-Chol) to helper lipid (eg DOPE) is determined by means of HPLC (High Performance Liquid Chromatography). See Chang CD & Harris D. (1998) J. Liqu. Chrome. & Rel. Technol. V21: 1119-1136 or Meyer O., et al. (2000) Eur. J. Pharm. Biopharm. 50: 353-356. Nukleinklaxalvtik:
- the quality of the nucleic acid used based on the assessment of ccc, oc, and linear content is determined using agarose gel (ethidium bromide staining) according to general information (cf. Ausubel, FM et al., Current protocols in molecular biology. New York: Green Publishing Associates and Wiley-Interscience. 1994 (updated), Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). The content is determined using a PicoGreen assay or UV spectroscopy.
- the residual moisture in lyophilisate samples is determined using the Karl Fischer method (European Pharmacopoeia, 2002).
- the protein expression of the transfected cells is determined using a commercially available kit according to the instructions suggested by the kit manufacturer: MCP-1: Human MCP-1 ELISA kit from BD Biosciences Pharmingen, BD Biosciences, San Diego, USA.
- MCP-1 Human MCP-1 ELISA kit from BD Biosciences Pharmingen, BD Biosciences, San Diego, USA.
- the expression of EGFP is determined using a FACS device (fluorescence activated cell sorter) from Becton Dickinson BD Biosciences, San Jose, USA in accordance with the manufacturer's instructions (filter 488 nm).
- the extrusion was carried out with a 600 nm and an 800 nm polycarbonate membrane.
- DC30 lipid suspensions with a lipid concentration of 1 mg / ml or 0.25 mg / ml were prepared as described above and continuously via a storage vessel through a polycarbonate extrusion membrane with a corresponding pore size of 600 nm or 800 nm (Millipore, supra) and pumped at a flow rate of 80 ml / min.
- the measured pressure on the membrane was less than 1 x 10 5 Pa in each case.
- the description of the samples can be found in Table 3.
- Sample 7 was prepared by diluting the previously triple extruded sample 6 to 0.25 mg / ml with transfection solution.
- liposomes with a homogeneous size distribution can be produced (see Table 4).
- the polydispersity index of the extruded liposomes decreased with the number of extrusions.
- Extrusion of a DC30 lipid suspension with 0.25 mg / ml resulted in more homogeneous Liposomes compared to extruded DC30 lipid suspensions with a lipid concentration of 1 mg / ml.
- the liposome size remains constant over a period of at least 24 hours Stable at room temperature as well as at 4 ° C and thus allows a safe and simple "further processing" of the liposomes into lipoplexes.
- the process can be implemented on an industrial scale using simple means in the sense of a continuously scalable and aseptic, validatable process.
- the quality of the DC30 liposomes remains unchanged during the extrusion process (600 nm extrusion membrane). Neither the concentration nor the ratio DC-Chol to DOPE is changed.
- the lipid content of the liposomes (Table 6) was determined by means of HPLC (high performance liquid chromatography).
- homogeneous liposomes with a size between 250 and 800 nm and a polydispersity index of ⁇ 0.6 were produced.
- DOPE / DAC-Chol 70/30 w / w a DAC30 lipid film was incubated for 30 min with transfection medium (250 mM sucrose, 25 mM NaCl) and allowed to swell for 30 min. The lipid concentration was adjusted to 1 mg / mL. The lipid suspension was transferred to the low pressure extrusion apparatus.
- the lipid suspension was then pumped continuously and uniformly (analogously to Example 1) over a polycarbonate membrane with a pore size of 800 nm (Millipore, Billerica, MA USA) and a flow rate of 80 ml / min through the closed low-pressure extrusion apparatus (see FIG. 1) , The measured pressure at the membrane was less than 1 x 10 5 Pa. With the appropriate experimental setup, an extrusion time of 5 minutes corresponded to one (1) extrusion cycle. The experiment was carried out under aseptic conditions using sterile materials and equipment.
- Devices Peristaltic pump Filtron silicone hose (inner diameter 4 mm)
- Extrusion unit Extrusion housing Gelman Sciences 2220 800 nm polycarbonate membrane / 0 47 mm storage container: 1000 ml separating funnel
- Pl narrow polydispersity index
- Example 3 Production of homogeneous lipoplexes consisting of DOPE / DC-Chol 70/30 (DC30) and nucleic acid
- Homogeneous liposomes were produced on the basis of the findings from Example 1.
- the flow diagram (FIG. 4) shows the individual steps for the preparation of the DC30 lipoplexes.
- the mass ratio lipid to DNA was 4: 1, which corresponds to a charge ratio +/- of about 0.75.
- DNA thawed at 2-8 ° C (100 mg) at a concentration of 1 mg / ml was stirred into 1900 ml transfection solution.
- the DNA concentration after the dilution step was 0.05 mg / mL.
- the DNA solution was then sterile filtered. 1875 ml of this sterile filtered DNA solution was mixed with the DC30 liposomes in the next step.
- the initial volumes of the liposome suspension and DNA The solution was 1875 ml each and the concentrations of the liposome suspension (0.2 mg / mL) and DNA solution (0.05 mg / mL) were adjusted so that the lipid to DNA mass ratio was 4: 1.
- Stable lipoplexes were obtained using the method shown in FIGS. 3 and 4.
- the mixing process presented here must be carried out in such a way that an even and continuous combination of liposomes and nucleic acids is guaranteed.
- the respective liposome suspension and DNA solution were uniformly and over a Y-piece (eg Hibiki Y-1 0 3 mm, Hibiki Y-2 0 5 mm for larger volumes, or Y-piece Norma 0 3 mm and 0 5 mm) continuously mixed and transferred to a sterile bottle.
- Y-piece eg Hibiki Y-1 0 3 mm, Hibiki Y-2 0 5 mm for larger volumes, or Y-piece Norma 0 3 mm and 0 5 mm
- the Y-piece used had a diameter of 3 mm ( Figure 3).
- the Y-pieces can be made of polypropylene (PP), polyethylene (PE) or polyvinyl chloride (PVC) or of stainless steel or glass.
- the flow rate in the example presented was 150-170 ml / min.
- the volume of the two starting solutions was the same.
- Ismatec peristaltic pump Y-piece 0 3mm, Hibiki Y-1 (Carl Roth GmbH & Co.KG., Düsseldorf, DE) 2 silicone hoses from ITE (0 4 mm) (Intertechnik Elze, Elze, DE)
- the Lipoplex Bulk (3750 ml) was left to stand for at least 30 min at room temperature before further processing was carried out: DNA concentration: 0.025 mg / mL; Lipid concentration: 0.1 mg / ml. After 30 minutes after the preparation of the lipoplexes, the lipopolex size was generally 270-310 nm and hardly changed within 3 hours of storage at room temperature (Table 8).
- the next table shows PCS results (measured after 30 min) of liposomes (DC30) and lipoplexes (DC30 / DNA 4: 1 w / w), which were prepared by the mixing process described above.
- the liposome suspension was obtained by various extrusion cycles (600 nm extrusion membrane). It can be seen that the initial conditions have an influence on the quality of the lipoplexes. It can be seen from the data in Tables 8 and 9 that a double extrusion of the liposomes through the same extrusion membrane provides liposomes with which lipoplexes can be produced which are in the size range from 280-310 nm with Pl ⁇ 0.3.
- Example 4 Preparation of homogeneous lipoplexes consisting of DOPE / DAC-Chol 70/30 (DAC30) and nucleic acid
- the flow diagram (FIG. 5) shows the individual steps for the preparation of the DAC30 lipoplexes.
- the mass ratio lipid to DNA was 5: 1, which corresponds to a positive to negative charge ratio +/- of about 1.
- 100 ml of sterile lipid DAC30 was mixed with 100 ml of sterile transfection solution (250 mM sucrose and 25 mM NaCl), so that the lipid concentration was 1 mg / ml. After 30 min. swell at room temperature, the single extrusion was carried out over an 800 nm polycarbonate membrane.
- the mixing process presented must be carried out in such a way that an even and continuous combination of liposomes and nucleic acids is ensured (see Example 3).
- the respective liposome suspension and DNA solution were mixed uniformly and continuously via a Y-piece (FIG. 9) and transferred to a sterile bottle.
- the Y-piece used had a diameter of 3 mm.
- the two pumps for the nucleic acid solution and for the liposome suspension were "switched in the same way" so that a uniform and continuous pumping of the solutions can be carried out.
- the flow rate in the example presented was 400 ml / min.
- the volume of the two starting solutions is the same.
- Example 5 Filling of homogeneous lipoplexes consisting of DOPE / DC-Chol 70/30 (DC30) and nucleic acid
- the DC30 lipoplexes produced in Example 3 were filled into a 2 ml vial (filling quantity 1.5 ml, compare) using a standard filling machine (Bausch & Ströble, GmbH & Co. KG., Ilshofen, DE) according to the filling principle of the piston pump Figure 4). This step was also carried out with sterile materials under aseptic conditions.
- Example 6 Filling of homogeneous lipoplexes consisting of DOPE / DAC-Chol 70/30 (DAC30) and nucleic acid
- the DAC30 lipoplexes produced in Example 4 are filled using a standard filling machine (Bausch & Ströble, supra) according to the piston pump filling principle. Alternatively, for small bulk volumes, manual filling using Eppendorf pipettes is possible. This step is also carried out with sterile materials under aseptic conditions (packaging as in Example 5).
- Example 7 Lyophilization of homogeneous lipoplexes consisting of DOPE / DC-Chol 70/30 (DC30) and nucleic acid
- the lyophilizer was aerated with nitrogen and the vials were closed at 600 mbar. Based on the process parameters that are summarized in Table 12, the changes summarized in Table 13 were carried out to carry out tests 1 - 5 (V01 - V05) in order to optimize the process.
- the optical assessment of the lyophilization cake of the individual lyophilisates is summarized in Table 14. From the assessment of the optics of the lyophilization cake, it was found that the lyophilization cake from experiment V01 was unsuitable because a considerable number of lyophilization cakes had collapsed. Such lyophilization cakes had very long reconstitution times.
- the residual moisture levels, which were determined using Karl Fischer titration, are shown in Table 16. The residual moisture content of the lyophilization cake from experiment V01 was over 5%.
- Lyophilisates that had not collapsed (from V01) show a lower residual moisture.
- the tests V03 and V04 were carried out with different formulations.
- One formulation contained sodium chloride (250 mM sucrose, 25 mM NaCl), the other contained no sodium chloride (250 mM sucrose). It can be seen from the data in Table 16 that the absence of sodium chloride leads to drier lyophilisates. However, the absence of sodium chloride in the formulation leads to instabilities in the Lipoplex liquid formulation, so that sodium chloride was necessary. Longer times for post-drying enable the production of lyophilisates with residual moisture well below 3%. (V05 from table 16).
- Table 17 shows the data for the product temperature of the individual tests. Table 17:
- Tg ' The temperature of the glass transition (Tg ') of the two formulations was determined by calorimetry (DSC 821 from Mettler Toledo (Giessen, DE)): rg' turning point 10 ° C / min placebo -34 ° C placebo without NaCI -32 ° C verum - 33 to -35 ° C
- the main drying should be carried out at a temperature below the glass transition.
- FIGS. 6A and 6B show SEM (scanning electron microscopy) images of a lyophilization cake.
- Figure 6A shows the surface morphology of the lyophilization cake.
- Figure 6B shows a section of the lyophilization cake itself. This type of lyophilization cake has very short reconstitution times of a few seconds.
- the residual moisture (determined by Karl Fischer titration) of the DC30 lipoplexes of the batches from Table 9 are summarized in Table 19. 5 vials per batch were selected and their residual moisture was determined.
- the residual moisture content of the lyophilisates was ⁇ 3%. There is no significant difference in the residual moisture between the individual batches. It is known that the residual moisture has a significant influence on the stability of the product.
- Example 8 Lyophilization of homogeneous lipoplexes consisting of DOPE / DAC-Chol 70/30 (DAC30) and nucleic acid
- the primary packaging corresponded to that in example 5.
- the water content of lipoplexes is an important factor for the long-term stability of the product.
- the lyophilization program used has no destabilizing influence on the lipoplex size.
- FIG. 7 shows the results using the example of the commercially available lipid Lipofectin (Invitrogen life technologies, Carlsbad, USA).
- both the lipoplex size and the transfection efficiency varied. The latter is very much influenced by the way the two solutions are brought together and mixed. Lipoplexes produced by LzD showed twice as compared to the DzL lipoplexes high transfection efficiency.
- the particle size also varied ( Figure 7) and depended on the mixing process. Similar experiments were also carried out with the lipid DAC30, and in summary it can be said that the DzL lipoplexes transfect somewhat better than LzD lipoplexes. With lipofectin, the reverse mix order led to better results.
- Pretreatment of the liposomes for the Lipoplex transfection properties The pretreatment of the liposomes, extruded versus non-extruded, has an influence on the transfection efficiency and the particle size of the lipoplexes produced.
- Table 24 shows the transfection efficiency (expressed in% of transfected cells) of DAC30 / pAH7-EGFP 4: 1 (w / w) lipoplexes, which was carried out by complexing the plasmid with liposomes that were either non-extruded or 1x through an 800 nm extrusion membrane extruded and mixed over the Y-piece.
- the transfection efficiency for lipoplexes which were produced with extruded liposomes was twice as high as that for lipoplexes from non-extruded liposomes.
- HA SMC human aorta smooth muscle cell
- A-10 SMC rat smooth muscle cell
- the storage stability of the lipoplexes DAC30 / pMCP-1 5: 1 (w / w) (as lyophilisate) was determined at different temperatures (see FIG. 8). The following parameters were determined: size of the lipoplexes, homogeneity (polydispersity index Pl), DNA content, DNA integrity of the ccc form, residual moisture (given as quotient: measured value / zero value) and bioactivity (expressed as transfection efficiency of the test batch based on an internal standard) , Storage of these lipoplexes at 2-8 ° C for 8 months (Table 27) shows that the size of the lipoplexes is in the range of 300 - 330 nm with Pl ⁇ 0.3. The DNA content and the integrity of the DNA (expressed as% ccc form) hardly changed within the scope of the measurement accuracy. The residual moisture did not change either.
- FIG. 8 shows the results for the bioactivity of the lipoplexes (stored at 4 ° C.) over a period of 8 months. It can be seen that the quotient of the bioactivity of the batch to be tested assumes values of around 2 based on an internal standard.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Dispersion Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Manufacturing Of Micro-Capsules (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP04764641A EP1663152A2 (de) | 2003-09-05 | 2004-08-31 | Verfahren zur herstellung von homogenen liposomen und lipoplexen |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US50073503P | 2003-09-05 | 2003-09-05 | |
EP03019663A EP1512393A1 (de) | 2003-09-08 | 2003-09-08 | Verfahren zur Herstellung von homogenen Liposomen und Lipoplexen |
EP04764641A EP1663152A2 (de) | 2003-09-05 | 2004-08-31 | Verfahren zur herstellung von homogenen liposomen und lipoplexen |
PCT/EP2004/009675 WO2005023219A2 (de) | 2003-09-05 | 2004-08-31 | Verfahren zur herstellung von homogenen liposomen und lipoplexen |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1663152A2 true EP1663152A2 (de) | 2006-06-07 |
Family
ID=34130097
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP03019663A Withdrawn EP1512393A1 (de) | 2003-09-05 | 2003-09-08 | Verfahren zur Herstellung von homogenen Liposomen und Lipoplexen |
EP04764641A Withdrawn EP1663152A2 (de) | 2003-09-05 | 2004-08-31 | Verfahren zur herstellung von homogenen liposomen und lipoplexen |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP03019663A Withdrawn EP1512393A1 (de) | 2003-09-05 | 2003-09-08 | Verfahren zur Herstellung von homogenen Liposomen und Lipoplexen |
Country Status (5)
Country | Link |
---|---|
US (1) | US20050064026A1 (de) |
EP (2) | EP1512393A1 (de) |
CA (1) | CA2537772A1 (de) |
TW (1) | TW200524636A (de) |
WO (1) | WO2005023219A2 (de) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112138144A (zh) * | 2019-06-28 | 2020-12-29 | 杭州生物医药创新研究中心 | 一种包含活性生物因子的脂质体及其制备方法 |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1674081A1 (de) * | 2004-12-23 | 2006-06-28 | KTB Tumorforschungsgesellschaft mbH | Herstellung von lipidbasierten Nanopartikeln unter Einsatz einer dualen asymmetrischen Zentrifuge |
CN101378777A (zh) * | 2006-02-07 | 2009-03-04 | 日本生物学的制剂有限公司 | 新型疫苗载体 |
WO2010031877A1 (en) * | 2008-09-22 | 2010-03-25 | Nanologica Ab | Hybrid silica -polycarbonate porous membranes and porous polycarbonate replicas obtained thereof |
EP2902013A1 (de) * | 2008-10-16 | 2015-08-05 | Marina Biotech, Inc. | Verfahren und Zusammensetzungen zur liposomalen und effizienten Abgabe von Gen-Silencing-Therapeutika |
EP2386647A1 (de) * | 2010-05-10 | 2011-11-16 | Qiagen GmbH | Verfahren zur Transfektion einer eurkaryotischen Zelle |
PL2591114T3 (pl) | 2010-07-06 | 2017-08-31 | Glaxosmithkline Biologicals Sa | Immunizacja dużych ssaków małymi dawkami rna |
WO2012006377A2 (en) | 2010-07-06 | 2012-01-12 | Novartis Ag | Delivery of rna to trigger multiple immune pathways |
AU2011276234B2 (en) | 2010-07-06 | 2016-02-25 | Glaxosmithkline Biologicals S.A. | Liposomes with lipids having an advantageous pKa- value for RNA delivery |
EP3970742B1 (de) * | 2010-08-31 | 2022-05-25 | GlaxoSmithKline Biologicals S.A. | Pegylierte liposomen zur auslieferung von immunogen-kodierender rns |
EP3520813B1 (de) | 2010-10-11 | 2023-04-19 | GlaxoSmithKline Biologicals S.A. | Antigenfreisetzungsplattformen |
CA2840989A1 (en) | 2011-07-06 | 2013-01-10 | Novartis Ag | Immunogenic combination compositions and uses thereof |
GB201204632D0 (en) * | 2012-03-16 | 2012-05-02 | Univ Belfast | Delivery system |
CN111246845A (zh) * | 2017-10-20 | 2020-06-05 | 生物技术Rna制药有限公司 | 适用于治疗的脂质体rna制剂的制备和储存 |
CN113817589B (zh) * | 2021-09-03 | 2022-12-30 | 南昌大学 | 一种细胞转染装置、细胞转染方法及微流道制作方法 |
US20230346977A1 (en) | 2022-04-13 | 2023-11-02 | Universitat Autònoma De Barcelona | Treatment of neuromuscular diseases via gene therapy that expresses klotho protein |
WO2024102954A1 (en) | 2022-11-10 | 2024-05-16 | Massachusetts Institute Of Technology | Activation induced clipping system (aics) |
CN115957351B (zh) * | 2022-11-15 | 2023-06-27 | 中国人民解放军海军第九七一医院 | 一种碘海醇注射液及其制备方法 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5094785A (en) * | 1986-12-10 | 1992-03-10 | Ciba Corning Diagnostics Corp. | Process for stabilizing liposomes |
US6623671B2 (en) * | 1990-10-05 | 2003-09-23 | Royden M. Coe | Liposome extrusion process |
DE19944262A1 (de) * | 1999-09-15 | 2001-03-29 | Cardiogene Gentherapeutische S | Pharmazeutische Zusammensetzung in Form eines Nukleinsäure-Lipid-Komplexes, ihre Herstellung und Verwendung in der Gentherapie |
US20030157113A1 (en) * | 1999-12-28 | 2003-08-21 | Terman David S. | Compositions and methods for treatment of neoplastic disease |
GB0106041D0 (en) * | 2001-03-12 | 2001-05-02 | Cancer Res Ventures Ltd | Lipids and liposomes |
-
2003
- 2003-09-08 EP EP03019663A patent/EP1512393A1/de not_active Withdrawn
-
2004
- 2004-08-24 US US10/925,324 patent/US20050064026A1/en not_active Abandoned
- 2004-08-31 CA CA002537772A patent/CA2537772A1/en not_active Abandoned
- 2004-08-31 EP EP04764641A patent/EP1663152A2/de not_active Withdrawn
- 2004-08-31 WO PCT/EP2004/009675 patent/WO2005023219A2/de active Application Filing
- 2004-09-03 TW TW093126751A patent/TW200524636A/zh unknown
Non-Patent Citations (1)
Title |
---|
See references of WO2005023219A2 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112138144A (zh) * | 2019-06-28 | 2020-12-29 | 杭州生物医药创新研究中心 | 一种包含活性生物因子的脂质体及其制备方法 |
CN112138144B (zh) * | 2019-06-28 | 2023-02-24 | 杭州生物医药创新研究中心 | 一种包含活性生物因子的脂质体及其制备方法 |
Also Published As
Publication number | Publication date |
---|---|
TW200524636A (en) | 2005-08-01 |
EP1512393A1 (de) | 2005-03-09 |
WO2005023219A2 (de) | 2005-03-17 |
US20050064026A1 (en) | 2005-03-24 |
WO2005023219A3 (de) | 2007-02-01 |
CA2537772A1 (en) | 2005-03-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1663152A2 (de) | Verfahren zur herstellung von homogenen liposomen und lipoplexen | |
DE69837339T2 (de) | Veränderung der Wirkstoffladung in multivesikulären Liposomen | |
EP1838286B1 (de) | Herstellung von lipidbasierten nanopartikeln unter einsatz einer dualen asymmetrischen zentrifuge | |
DE60036861T2 (de) | Verkapselung biologisch aktiver komplexe in liposomen | |
DE69631149T2 (de) | Formulierungen in form von emulsionen zur verabreichung von nukleinsäuren an zellen | |
EP0056781B1 (de) | Verfahren zur Herstellung von liposomalen Arzneimitteln | |
DE60123583T2 (de) | Dehydratisierungs-/rehydratisierungsverfahren zur herstellung von liposome | |
DE69837274T2 (de) | Verfahren zur verkapselung von nukleinsäuren in lipiddoppelschichten | |
DE69535297T2 (de) | Zubereitung multivesikulärer liposomen zur gesteuerten freisetzung von wirkstoffen | |
DE69923315T2 (de) | Verfahren zur herstellung von liposomen | |
DE69826488T2 (de) | Stabile partikuläre komplexe lamellarer struktur mit neutraler oder negativer gesamtladung | |
DE69825137T2 (de) | Liposomale Erythropoietin-Dispersion | |
EP1814843B1 (de) | (2r)-enantiomer von dotap-chlorid | |
DE69819338T2 (de) | Lipid haltige zusammenstellungen und deren verwendungen | |
DE60019134T2 (de) | Verfahren und zusammensetzungen für die abgabe von pharmazeutischen wirkstoffen | |
DE69837819T2 (de) | Kondensierte plasmid-liposome komplex zur transfektion | |
EP1734928A2 (de) | Serumstabile amphotere liposomen | |
DE4216644B4 (de) | Liposomen enthaltende Arzneimittel | |
DE69820861T2 (de) | Teilchenförmige arzneimittelträger | |
DE60025494T2 (de) | Epothilon zusammensetzungen | |
WO2004100928A1 (de) | Injizierbare liposomale depots zum wirkstoffdelivery | |
EP0488142B1 (de) | Verfahren zur Verkapselung fester oder flüssiger, lipophiler Wirkstoffe zu diesen Wirkstoff enthaltenden Phospholipid-Liposomen sowie Arzneimittel diese Liposomen enthaltend | |
EP0470437B1 (de) | Wässriges Liposomensystem | |
CN108309930B (zh) | 一种氧氟沙星液晶凝胶纳米粒滴眼液及其制备方法 | |
JP2024505154A (ja) | ウチデロンリポソーム組成物、及びその製造方法並びに使用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR |
|
AX | Request for extension of the european patent |
Extension state: AL HR LT LV MK |
|
DAX | Request for extension of the european patent (deleted) | ||
PUAK | Availability of information related to the publication of the international search report |
Free format text: ORIGINAL CODE: 0009015 |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: SCHUBERT, ROLF Inventor name: PESCHKA-SUESS, REGINE Inventor name: HOERMANN, HANS Inventor name: DENKINGER, NICOLE Inventor name: GARIDEL, PATRIK |
|
17P | Request for examination filed |
Effective date: 20070801 |
|
RBV | Designated contracting states (corrected) |
Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20100302 |