EP1082104A1 - Novel poly(methylidene malonate) microspheres, preparation method and pharmaceutical compositions containing them - Google Patents
Novel poly(methylidene malonate) microspheres, preparation method and pharmaceutical compositions containing themInfo
- Publication number
- EP1082104A1 EP1082104A1 EP99915856A EP99915856A EP1082104A1 EP 1082104 A1 EP1082104 A1 EP 1082104A1 EP 99915856 A EP99915856 A EP 99915856A EP 99915856 A EP99915856 A EP 99915856A EP 1082104 A1 EP1082104 A1 EP 1082104A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- microspheres
- preparation
- poly
- solution
- support material
- 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.)
- Ceased
Links
- 239000004005 microsphere Substances 0.000 title claims abstract description 80
- 238000002360 preparation method Methods 0.000 title claims description 29
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 5
- -1 poly(methylidene malonate) Polymers 0.000 title description 14
- 239000000463 material Substances 0.000 claims abstract description 23
- 229920001519 homopolymer Polymers 0.000 claims abstract description 22
- 239000000126 substance Substances 0.000 claims abstract description 21
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 14
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 13
- 239000000839 emulsion Substances 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 31
- 229920000642 polymer Polymers 0.000 claims description 30
- 229920001577 copolymer Polymers 0.000 claims description 28
- 239000003960 organic solvent Substances 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 11
- 239000004094 surface-active agent Substances 0.000 claims description 11
- 230000002209 hydrophobic effect Effects 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- 239000003381 stabilizer Substances 0.000 claims description 9
- 238000001704 evaporation Methods 0.000 claims description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 230000008020 evaporation Effects 0.000 claims description 7
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 6
- 229920001983 poloxamer Polymers 0.000 claims description 5
- 125000006353 oxyethylene group Chemical group 0.000 claims description 4
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 3
- 239000006185 dispersion Substances 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims description 2
- 108010039918 Polylysine Proteins 0.000 claims description 2
- 238000006073 displacement reaction Methods 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 150000004676 glycans Chemical class 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims description 2
- 229920002187 poly[N-2-(hydroxypropyl) methacrylamide] polymer Polymers 0.000 claims description 2
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 claims description 2
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- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 239000005017 polysaccharide Substances 0.000 claims description 2
- 229920000136 polysorbate Polymers 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 235000019422 polyvinyl alcohol Nutrition 0.000 claims 2
- 229920001308 poly(aminoacid) Polymers 0.000 claims 1
- 229940068965 polysorbates Drugs 0.000 claims 1
- 239000013543 active substance Substances 0.000 abstract description 3
- 239000013598 vector Substances 0.000 abstract description 3
- 230000000306 recurrent effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 15
- 238000006116 polymerization reaction Methods 0.000 description 15
- 239000000178 monomer Substances 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 108010058846 Ovalbumin Proteins 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000008346 aqueous phase Substances 0.000 description 12
- 229940092253 ovalbumin Drugs 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- XJDDLMJULQGRLU-UHFFFAOYSA-N 1,3-dioxane-4,6-dione Chemical compound O=C1CC(=O)OCO1 XJDDLMJULQGRLU-UHFFFAOYSA-N 0.000 description 10
- 239000002904 solvent Substances 0.000 description 10
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 8
- 238000005538 encapsulation Methods 0.000 description 8
- 238000010539 anionic addition polymerization reaction Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
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- 239000003999 initiator Substances 0.000 description 5
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- 239000013612 plasmid Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000003760 magnetic stirring Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
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- 238000004626 scanning electron microscopy Methods 0.000 description 3
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- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- MZRVEZGGRBJDDB-UHFFFAOYSA-N N-Butyllithium Chemical compound [Li]CCCC MZRVEZGGRBJDDB-UHFFFAOYSA-N 0.000 description 2
- 125000000129 anionic group Chemical group 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
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- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000010907 mechanical stirring Methods 0.000 description 2
- ZJAOAACCNHFJAH-UHFFFAOYSA-N phosphonoformic acid Chemical compound OC(=O)P(O)(O)=O ZJAOAACCNHFJAH-UHFFFAOYSA-N 0.000 description 2
- 229920001993 poloxamer 188 Polymers 0.000 description 2
- 229920000747 poly(lactic acid) Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000010526 radical polymerization reaction Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 238000005809 transesterification reaction Methods 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
- PSZAEHPBBUYICS-UHFFFAOYSA-N 2-methylidenepropanedioic acid Chemical compound OC(=O)C(=C)C(O)=O PSZAEHPBBUYICS-UHFFFAOYSA-N 0.000 description 1
- ZOLACKDSSUBCNN-UHFFFAOYSA-N 5,6-dimethylcyclohexa-2,4-diene-1-carboxylic acid Chemical compound CC1C(C(O)=O)C=CC=C1C ZOLACKDSSUBCNN-UHFFFAOYSA-N 0.000 description 1
- 238000011735 C3H mouse Methods 0.000 description 1
- 108010078777 Colistin Proteins 0.000 description 1
- BXZVVICBKDXVGW-NKWVEPMBSA-N Didanosine Chemical compound O1[C@H](CO)CC[C@@H]1N1C(NC=NC2=O)=C2N=C1 BXZVVICBKDXVGW-NKWVEPMBSA-N 0.000 description 1
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- OIRDTQYFTABQOQ-UHTZMRCNSA-N Vidarabine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1O OIRDTQYFTABQOQ-UHTZMRCNSA-N 0.000 description 1
- WREGKURFCTUGRC-POYBYMJQSA-N Zalcitabine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)CC1 WREGKURFCTUGRC-POYBYMJQSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 229960004150 aciclovir Drugs 0.000 description 1
- MKUXAQIIEYXACX-UHFFFAOYSA-N aciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
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- 230000002421 anti-septic effect Effects 0.000 description 1
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- 239000003080 antimitotic agent Substances 0.000 description 1
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- 239000003096 antiparasitic agent Substances 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
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- 238000005844 autocatalytic reaction Methods 0.000 description 1
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- 239000002775 capsule Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
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- 229960003346 colistin Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
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- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229960002656 didanosine Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 150000002148 esters Chemical group 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229960005102 foscarnet Drugs 0.000 description 1
- 229960002963 ganciclovir Drugs 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 230000009477 glass transition Effects 0.000 description 1
- 229920000578 graft copolymer Polymers 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000007970 homogeneous dispersion Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
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- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- DSZMPZRQIDKROL-UHFFFAOYSA-N lithium;1-phenylhexylbenzene Chemical compound [Li+].C=1C=CC=CC=1C(CCCC[CH2-])C1=CC=CC=C1 DSZMPZRQIDKROL-UHFFFAOYSA-N 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- VHRYZQNGTZXDNX-UHFFFAOYSA-N methacryloyl chloride Chemical compound CC(=C)C(Cl)=O VHRYZQNGTZXDNX-UHFFFAOYSA-N 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000011206 morphological examination Methods 0.000 description 1
- JORAUNFTUVJTNG-BSTBCYLQSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Chemical compound CC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O.CCC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O JORAUNFTUVJTNG-BSTBCYLQSA-N 0.000 description 1
- IJJSYKQZFFGIEE-UHFFFAOYSA-N naphthalene;potassium Chemical compound [K].C1=CC=CC2=CC=CC=C21 IJJSYKQZFFGIEE-UHFFFAOYSA-N 0.000 description 1
- 125000002524 organometallic group Chemical group 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
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- 229960000502 poloxamer Drugs 0.000 description 1
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000003505 polymerization initiator Substances 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- XDJYMJULXQKGMM-UHFFFAOYSA-N polymyxin E1 Natural products CCC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O XDJYMJULXQKGMM-UHFFFAOYSA-N 0.000 description 1
- KNIWPHSUTGNZST-UHFFFAOYSA-N polymyxin E2 Natural products CC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O KNIWPHSUTGNZST-UHFFFAOYSA-N 0.000 description 1
- 229950008882 polysorbate Drugs 0.000 description 1
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- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- IRAPFUAOCHNONS-UHFFFAOYSA-N potassium;phenylmethylbenzene Chemical compound [K+].C=1C=CC=CC=1[CH-]C1=CC=CC=C1 IRAPFUAOCHNONS-UHFFFAOYSA-N 0.000 description 1
- ZYBHSWXEWOPHBJ-UHFFFAOYSA-N potassium;propan-2-ylbenzene Chemical compound [K+].C[C-](C)C1=CC=CC=C1 ZYBHSWXEWOPHBJ-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- 229960000329 ribavirin Drugs 0.000 description 1
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
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- 238000010254 subcutaneous injection Methods 0.000 description 1
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- 125000001424 substituent group Chemical group 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
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- 239000003826 tablet Substances 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 238000002076 thermal analysis method Methods 0.000 description 1
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- 229960003636 vidarabine Drugs 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 229960000523 zalcitabine Drugs 0.000 description 1
- 229960002555 zidovudine Drugs 0.000 description 1
- HBOMLICNUCNMMY-XLPZGREQSA-N zidovudine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-XLPZGREQSA-N 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/12—Powdering or granulating
- C08J3/14—Powdering or granulating by precipitation from solutions
-
- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
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- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2335/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Derivatives of such polymers
Definitions
- New microspheres based on polyfmethylidene malonate their preparation process and pharmaceutical compositions containing them.
- the present invention relates to new microspheres in particular useful in the pharmaceutical field as particulate vectors intended for the transport of biologically active substances, in particular hydrophilic substances (peptides or proteins), for oral administration.
- biologically active substances in particular hydrophilic substances (peptides or proteins)
- the invention also relates to a process for manufacturing these microspheres and to pharmaceutical compositions containing them.
- microspheres is intended to denote substantially spherical particles, with an average diameter of between 1 ⁇ m and 100 ⁇ m, and preferably between 5 and 100 ⁇ m formed from a continuous network, more or less dense, of a support material.
- microspheres are different from microcapsules which consist of a wall surrounding a cavity. It should however be noted that the microspheres prepared in multiple emulsion can comprise a set of globules dispersed in the continuous network constituting them.
- the total volume of these globules will generally represent a fraction between 1:20 and 1: 2 of the total volume of the microspheres.
- particulate polymer systems can be used to modify the release profile of a therapeutically active substance.
- microspheres based on synthetic polymers such as, for example, poly (lactic acid), poly (lactic-co-glycolic acid), polystyrene, polyepsilonecaprolactone, polymethylmethacrylate, or even methylcellulose or ethylcellulose have been prepared, by various techniques.
- synthetic polymers such as, for example, poly (lactic acid), poly (lactic-co-glycolic acid), polystyrene, polyepsilonecaprolactone, polymethylmethacrylate, or even methylcellulose or ethylcellulose have been prepared, by various techniques.
- microspheres thus obtained are generally non-biodegradable, and when they are, they are characterized by a degradation very delayed in time.
- the degradation is not progressive and occurs all at once after a significant time interval.
- lactic polymers degrade, releasing strongly acidic products which not only cause the autocatalysis of the degradation of the polymer, but are at the origin of the induction of incompatibilities with the encapsulated substances.
- the microspheres exhibit an extremely low degradation rate, or even zero.
- the persistence time in the body of such particles can limit the repeated application in humans.
- microspheres are characterized, for the most part, by a significant hydrophobicity which promotes strong and often denaturing interactions with the substance to be encapsulated, in particular when the latter is of protein or peptide nature.
- the present application relates to microspheres consisting of a continuous network of a support material in which a substance is optionally dispersed, characterized in that said support material contains at least 70% by weight of a homopolymer consisting of repeating units corresponding to the following general formula (I):
- R represents an alkyl group having from 1 to 6 carbon atoms or a group (CH 2 ) m - COOR 3 in which m is an integer between 1 and 5 and R 3 represents an alkyl group having from 1 to 6 carbon atoms;
- R_ represents an alkyl group having from 1 to 6 carbon atoms
- - n is an integer between 1 and 5.
- microspheres can induce a stimulation of the immune response, when they are associated with an antigen
- This polymeric material is essentially formed of a homopolymer consisting of recurring units of general formula (I).
- Such polymers have the remarkable property of being biocompatible and bioerodible, that is to say that they are capable of degrading chemically or biochemically, by cutting the lateral substituents.
- the erosion rate of the microspheres according to the invention being dependent on the molecular weight of the support material, it can therefore be modulated simply, by using a support material having a molecular weight adapted to the desired erosion rate.
- microspheres according to the present invention therefore exhibit a modulated and progressive bioerosion allowing, for example, the transport of a biologically active substance, dispersed in the support material, to the place in the body where its action will be most effective. .
- the bioerosion of microspheres also prevents their accumulation in the body; their use is therefore no longer limited.
- the above-mentioned homopolymer consists of repeating units corresponding to the general formula (I) in which: R represents an alkyl group having from 1 to 6 carbon atoms; R 2 represents an alkyl group having from 1 to 6 carbon atoms; and n is a number equal to 1; and preferably in which R, and R_ represent a CH 2 -CH 3 group .
- polymers from the poly (methylidene-malonate) family are particularly suitable for the encapsulation of substances hydrophilic, in particular of biological origin, and possibly biologically active.
- biologically active molecule means without limitation any molecule having a prophylactic or curative biological activity, in vitro or in vivo, in particular an anti-infectious agent, in particular an antiseptic, antibiotic, antiviral, antiparasitic or antimitotic agent, especially anticancer.
- Antibiotic or antiseptic agents which can be used can be, for example, rifampicin and colistin.
- antiviral agents non-limiting mention may be made of didanosine, ribavirin, zidovudine, acyclovir, ganciclovir, foscarnet, vidarabine and zalcitabine.
- the support material for the microspheres contains:
- a copolymer comprising at least one block having a hydrophilic character and at least one block having a hydrophobic character, said block of hydrophobic character preferably comprising at least one repeating unit corresponding to the general formula (I).
- the hydrophilic block of the abovementioned copolymer is chosen from a poly (oxyethylene), a poly (vinyl alcohol), a poly (vinylpyrrolidone), a poly (N-2-hydroxypropyl methacrylamide), a poly (hydroxyethylmethacrylate), a poly hydrophilic (amino acid) such as a polylysine, a polysaccharide, and will preferably be a poly (oxyethylene).
- the copolymer may have a block structure, preferably diblock or triblock, or a grafted structure.
- the addition of such copolymers in the support material makes it possible to obtain a homogeneous dispersion of the substance to be encapsulated inside each of the microspheres. It also makes it possible to modulate the hydrophilicity / hydrophobicity ratio of the surface of the microspheres, which makes it possible to avoid or limit the strong and often denaturing interactions with the substance to be encapsulated.
- these copolymers are particularly advantageous for the implementation of the currently preferred process for preparing the microspheres as will be explained in more detail later.
- microspheres in accordance with the present invention can be obtained by implementing a process comprising:
- This multiple emulsion can be obtained in a conventional manner by dispersing a primary emulsion of the water-in-oil type in a second aqueous phase containing a stabilizing agent. This multiple emulsion can also be obtained by a process
- the present invention relates to a process for obtaining microspheres as previously described, comprising: a) the preparation of a first solution of the above-mentioned polymer (s) constituting the support material in a volatile organic solvent optionally containing a surfactant, b) the preparation of a second solution immiscible with the solution obtained in a), optionally containing said substance to be dispersed and optionally a surfactant, c) the preparation of a primary emulsion by dispersion of the second solution in the first solution, the continuous phase consisting of the solution of polymer (s), d) the preparation of a secondary emulsion: - either by dispersing, under stirring, the primary emulsion obtained in c) in a dispersing medium immiscible with said primary emulsion, said dispersing medium optionally containing a stabilizing agent;
- the aforementioned method further comprises: f) isolation of the microspheres by centrifugation g) one or more successive washings of said microspheres h) lyophilization of said microspheres.
- the first step of the process for preparing the microspheres according to the invention therefore comprises the production of an emulsion of the water-in-oil type preferably in the presence of an appropriate surfactant, the oily or organic phase containing the or polymers intended to constitute the support material of said microspheres.
- a solution of the polymer (s) constituting the support material is prepared using an appropriate volatile organic solvent optionally in the presence of a surfactant.
- preformed polymers will be used insofar as the homopolymers essentially constituting the support material for the microspheres can be obtained under conditions allowing good characterization in terms of molar mass and mass dispersity.
- Homopolymers made up of repeating units corresponding to general formula (I) can be prepared from monomers, for example, by following the process described in patent EP 283 346 corresponding to patents US 4,931,584 and US 5,142,098 incorporated here. by reference, said monomers being generally degassed under a vane pump vacuum to constant weight to remove the polymerization inhibitor (SO 2 ).
- These homopolymers will however advantageously be prepared anionically in an aprotic medium, for example by dispersion of the monomer in acetone, followed by the addition of sodium hydroxide with stirring, again followed by evaporation of the acetone and drying of the polymer thus got.
- Other aprotic organic solvents such as acetonitrile, dioxane and tetrahydrofuran can be used in place of acetone.
- the molecular mass of the homopolymer capable of being obtained by the implementation of this process can be perfectly controlled by a judicious choice of the conditions of implementation, and in particular of the concentration of monomer in the organic phase, of the pH and the molarity of the polymerization initiator (sodium hydroxide).
- homopolymers having an average molar mass of 1,000 to 100,000, and preferably 5,000 to 80,000 will be used in the context of the present invention.
- the volatile organic solvent capable of being used for the preparation of the first solution containing the polymer or polymers constituting the support material will generally be chosen so that its boiling point is lower than that of water. This solvent can therefore be easily removed during the final evaporation step, allowing precipitation of the polymer.
- Ethyl acetate is a particularly suitable volatile organic solvent for this purpose.
- the surfactants capable of being used for the stabilization of the primary emulsion can be of various nature and will be added to the organic phase containing the polymer (s) (first solution) and / or to the aqueous phase ( second solution) constituting the dispersed phase.
- This can be for example a poloxamer such as the product sold under the name Pluronic ® F68, or even an alcohol
- (R) poly vinyl such as the product sold under the name Mowiol 40-88, or else a polysorbate, or even a surfactant copolymer whose hydrophobic sequence has a chemical nature identical to that of homopolymer consisting of recurring units corresponding to the general formula (I). It has been shown that such surface-active copolymers and in particular the copolymers of poly (methylidene malonate) and of polyoxyethylene are particularly advantageous insofar as they allow, on the one hand, to obtain a very stable primary emulsion and on the other hand, to obtain a good anchoring of the surfactant in the matrix after evaporation of the solvent.
- surfactant copolymers can be prepared by conventional polymerization techniques well known to those skilled in the art. Among these techniques, use will preferably be made of anionic polymerization, radical polymerization, or else the technique of coupling the precursor sequences of the copolymer, these sequences having previously been functionally functionalized at the end of the chain.
- the anionic polymerization is more particularly suitable for the preparation of block copolymers.
- a block copolymer can thus be obtained:
- the initiating agents capable of being used in the context of these anionic polymerizations will generally be:
- organometallic derivatives such as butyllithium and in particular diphenylhexyllithium;
- alcoholates and in particular macromolecular alcoholates such as a POE alcoholate which can be generated by activation of a hydroxy function using cumylpotassium, diphenylmethylpotassium, naphthalene potassium.
- the anionic polymerization will generally be carried out in a solvent compatible with the various blocks of the copolymer.
- the block copolymers will preferably be prepared by successive anionic polymerization of the ethylene oxide followed by methylidene malonate or by activation of a polyoxyethylenated precursor commercial monohydroxylate and subsequent anionic polymerization of the poly (methylidene malonate) sequence.
- tetrahydrofuran will preferably be used as the polymerization solvent, this product making it possible to work in a homogeneous medium and favorably influencing the polymerization kinetics.
- the monomers used for the preparation of the hydrophilic blocks will generally be commercial products.
- the coupling technique is also more particularly suitable for the preparation of block copolymers.
- This reaction is generally carried out from presynthesized and functionalized homopolymers, in the presence of a coupling agent and optionally an activating agent, in an appropriate solvent.
- the homopolymer of poly (oxyethylene) functionalized with an ⁇ -carboxy group can be obtained for example by transformation with succinic anhydride of a poly (oxyethylene) functionalized with a commercial ⁇ -hydroxy group.
- the homopolymer of poly (methylidene malonate) functionalized with an ⁇ -hydroxy group can be obtained directly by anionic synthesis in an aqueous medium or by anionic synthesis in a solvent using an aqueous sodium hydroxide solution as initiator of the polymerization.
- DCCI dicyclohexylcarbodiimide
- Radical polymerization is more particularly suitable for the preparation of graft copolymers.
- This polymerization is generally carried out from a macromonomer, that is to say from an oligomer carrying at one of its ends an ethylenic group which can be polymerized by radical means and capable of reacting with a monomer to form a copolymer with grafted structure .
- This polymerization will generally be carried out in the presence of an initiator in an appropriate solvent.
- Such a product may be commercial (Aldrich) and will consist, for example, of a poly (oxyethylene) chain with a molar mass of between 308 and 440 g / mol, or will be prepared from a commercial poly (ethylene glycol) monomethyl ether by coupling with methacrylic acid in dichloromethane to form a methoxy terminal function.
- copolymers with grafted structures can also be prepared by transesterification of a poly (oxyethylene) monomethyl ether on side ester chains of a pre-synthesized poly (methylidene malonate). This transesterification will generally be carried out with alcohol in the presence of a catalyst at high temperature.
- Copolymers whose total molecular weight of the hydrophobic blocks is between 1,000 and 80,000 g / mol, and preferably between 1,000 and 50,000 g / mol are particularly suitable in the context of the present invention.
- the primary emulsion used for the preparation of the microspheres according to the invention can be obtained by means of a shearing homogenizer, for example of the Ultraturrax type (13,500 rpm - 5 min).
- the substance to be encapsulated is generally added to the dispersed aqueous phase of the primary emulsion. 11 5309 PCT / FR99 / 01005
- the second step of the process for preparing the microspheres in accordance with the invention comprises the preparation of a secondary emulsion:
- the dispersing medium which is immiscible with the primary emulsion is an aqueous phase into which the primary emulsion is preferably introduced drop by drop and the emulsion is also produced for example using a homogenizer of the Ultraturrax type ( 8,000 rpm; 5 mins).
- Polyvinyl alcohol constitutes a stabilizing agent which is particularly suitable for the preparation of the secondary emulsion.
- this second step can be followed by an additional step of displacement of the organic solvent.
- the third essential step of the process for preparing the microspheres in accordance with the invention consists in evaporating the volatile organic solvent which was used for the preparation of the solution of the polymer (s). In the particular case where this solvent is ethyl acetate, this evaporation takes place for a period of approximately 12 hours at room temperature, with mechanical stirring (1,400 rpm).
- these microspheres will have an average diameter of between 1 ⁇ m and 100 ⁇ m, preferably between 5 ⁇ m and 50 ⁇ m for their application as vectors in the pharmaceutical field.
- the microspheres obtained at the end of the third step will be isolated by centrifugation, washed and possibly lyophilized.
- the present invention also relates to pharmaceutical compositions containing the microspheres which have just been described. These compositions will generally be suitable for a 12 5309 PCT / FR99 / 01005
- oral administration and will be presented, for example, in the form of tablets, capsules, powders or granules.
- PMM 2.1.2 polymer consisting of recurring monomer units corresponding to the formula
- microspheres thus obtained have an average diameter of 6 microns and 14.2% of the ovalbumin used in the preparation are encapsulated in the microspheres of PMM 2.1.2, which corresponds to an encapsulation of 2.5% (w / w).
- This preparation is administered orally to C3H mice at a dose of 100 micrograms of encapsulated ovalbumin (per mouse and per day) for 5 consecutive days. The last force-feeding takes place 7 days before the 14 55309 PCT / FR99 / 01005
- a POE-PMM 2.1.2 block copolymer was used. This copolymer was obtained by successive polymerization of the two monomers, starting with the preparation of the POE block, by the implementation of the following experimental protocol.
- the reactor in which the polymerization is carried out 250 ml is connected to a vacuum ramp allowing to work under high vacuum and to get rid of protic impurities.
- the solvent (THF, 150 ml) purified of any trace of moisture is cryodistilled in the reactor at -70 ° C.
- the initiator (potassium Terbutanolate (0.1N / THF); 10 ml) is then added using a syringe through a septum.
- the ethylene oxide (5 g) is then introduced by cryodistillation.
- the polymerization is carried out at room temperature for 48 hours. After this period, a sample makes it possible to control, by gel permeation chromatography, the molar mass (4,000 g / mol) and the polymolecularity index (1.13) of the first sequence.
- the MM 2.1.2 (0.5 ml) freshly degassed under vacuum to remove the SO 2 used as a polymerization inhibitor, is then added quickly and all at once at room temperature. 15 5309 PCT / FR99 / 01005
- the copolymer is deactivated by adding methanol and precipitated in diethyl ether.
- the procedure described in example 1 is followed, but the ovalbumin (60 mg) is replaced by 2 mg of the peptide V3 28 of the V3 BRU loop of HIV gp 120 (sequence NNTRKSIHI GPGRAFYATGDIIGDIRQA).
- the microspheres obtained have an average size of 5.8 microns and 70% of the V3 28 peptide used is encapsulated in the microspheres, which corresponds to an encapsulation of 0.48% w / w.
- the study carried out in scanning electron microscopy reveals smooth and spherical particles.
- the procedure is as in Example 4, but Pluronic F 68 is added to the aqueous phase containing the peptide at a concentration of 2%.
- the microspheres obtained have a size of 7.0 microns and 70% of the V3 28 peptide used is encapsulated in the microspheres.
- Example 2 The procedure is as in Example 1, but the internal aqueous phase consists of 1 ml of 0.5M acetic acid containing 3 mg of type IL collagen.
- the microspheres thus obtained have an average diameter of 6 microns and 66.6% of the collagen put in in the preparation are encapsulated in the microspheres of PMM2.1.2, which corresponds to an encapsulation of 0.7% (w / w).
- Example 2 The procedure is as in Example 1, but the internal aqueous phase consists of 1 ml of distilled water.
- the microspheres thus obtained do not contain a biologically active substance. Their average diameter is 7.0 microns.
- Example 2 The procedure described in Example 1 is followed, but the ovalbumin (60 mg) is replaced by the plasmid pCDNA3 (5 mg).
- the microspheres obtained have an average size of 7 ⁇ m and 9.8% of plasmid used are encapsulated in the microspheres which corresponds to an encapsulation of 0.17% (w / w).
- microspheres thus obtained have an average diameter of 7 ⁇ m and 12% of the plasmid used are encapsulated in the microspheres which corresponds to an encapsulation of 0.22% (w / w).
- Example 2 The procedure described in Example 1 is followed, but the ovalbumin (60 mg) is replaced by the oligonucleotide (pdT16) (2 mg).
- the microspheres obtained have an average size of 4.8 ⁇ m and 20.6% of oligonucleotide used are encapsulated in the microspheres which corresponds to an encapsulation of 0.19% (w / w).
- microspheres obtained have an average diameter of 5.7 ⁇ m and 23% of oligonucleotide used are encapsulated in the microspheres which corresponds to an encapsulation of 0.21% (w / w).
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Abstract
Description
Nouvelles microsphères à base de polyfméthylidène malonate). leur procédé de préparation et compositions pharmaceutiques les contenant.New microspheres based on polyfmethylidene malonate). their preparation process and pharmaceutical compositions containing them.
La présente invention a pour objet de nouvelles microspheres notamment utiles dans le domaine pharmaceutique comme vecteurs particulaires destinés au transport de substances biologiquement actives, en particulier de substances hydrophiles (peptides ou protéines), en vue d'une administration orale.The present invention relates to new microspheres in particular useful in the pharmaceutical field as particulate vectors intended for the transport of biologically active substances, in particular hydrophilic substances (peptides or proteins), for oral administration.
L'invention a également pour objet un procédé de fabrication de ces microsphères et des compositions pharmaceutiques les contenant.The invention also relates to a process for manufacturing these microspheres and to pharmaceutical compositions containing them.
Dans le cadre de la présente description, on entend désigner par le terme "microsphères" des particules sensiblement sphériques, d'un diamètre moyen compris entre 1 μm et 100 μm, et de préférence entre 5 et 100 μm formées d'un réseau continu, plus ou moins dense, d'un matériau support.In the context of the present description, the term "microspheres" is intended to denote substantially spherical particles, with an average diameter of between 1 μm and 100 μm, and preferably between 5 and 100 μm formed from a continuous network, more or less dense, of a support material.
Ces microsphères se différencient des microcapsules qui sont constituées d'une paroi entourant une cavité. Il est cependant à noter que les microsphères préparées en émulsion multiple peuvent comporter un ensemble de globules dispersés dans le réseau continu les constituant.These microspheres are different from microcapsules which consist of a wall surrounding a cavity. It should however be noted that the microspheres prepared in multiple emulsion can comprise a set of globules dispersed in the continuous network constituting them.
Dans ce dernier cas, le volume total de ces globules représentera généralement une fraction comprise entre 1 : 20 et 1 : 2 du volume total des microsphères. Au cours des dernières années, de nombreuses études ont permis de montrer que des systèmes particulaires à base de polymères peuvent être utilisés pour modifier le profil de libération d'une substance thérapeutiquement active.In the latter case, the total volume of these globules will generally represent a fraction between 1:20 and 1: 2 of the total volume of the microspheres. In recent years, numerous studies have shown that particulate polymer systems can be used to modify the release profile of a therapeutically active substance.
C'est ainsi que des microsphères à base de polymères synthétiques, comme par exemple, de poly(acide lactique), de poly(acide lactique-co- glycolique), de polystyrène, de polyepsilonecaprolactone, de polyméthylméthacrylate, ou encore à base de méthylcellulose ou d'éthylcellulose ont été préparées, par des techniques variées.This is how microspheres based on synthetic polymers, such as, for example, poly (lactic acid), poly (lactic-co-glycolic acid), polystyrene, polyepsilonecaprolactone, polymethylmethacrylate, or even methylcellulose or ethylcellulose have been prepared, by various techniques.
Cependant, les microspheres ainsi obtenues sont généralement non biodégradables, et lorsqu'elles le sont, elles se caractérisent par une dégradation très retardée dans le temps.However, the microspheres thus obtained are generally non-biodegradable, and when they are, they are characterized by a degradation very delayed in time.
Ainsi, dans le cas des microsphères à base d'acide poly(lactique), par exemple, la dégradation n'est pas progressive et se produit en une seule fois après un intervalle de temps important.Thus, in the case of microspheres based on poly (lactic acid), for example, the degradation is not progressive and occurs all at once after a significant time interval.
En outre, les polymères lactiques se dégradent en libérant des produits fortement acides qui, non seulement entraînent l'autocatalyse de la dégradation du polymère, mais sont à l'origine de l'induction d'incompatibilités avec les substances encapsulées.In addition, lactic polymers degrade, releasing strongly acidic products which not only cause the autocatalysis of the degradation of the polymer, but are at the origin of the induction of incompatibilities with the encapsulated substances.
Dans le cas des autres polymères utilisés, les microsphères présentent une vitesse de dégradation extrêmement faible, voire nulle. Le temps de rémanence dans l'organisme de telles particules peut en limiter l'application répétée chez l'homme.In the case of the other polymers used, the microspheres exhibit an extremely low degradation rate, or even zero. The persistence time in the body of such particles can limit the repeated application in humans.
Enfin, les microsphères connues se caractérisent, pour la plupart, par une hydrophobicité importante qui favorise des interactions fortes et souvent dénaturantes avec la substance à encapsuler, en particulier lorsque cette dernière est de nature protéinique ou peptidique.Finally, the known microspheres are characterized, for the most part, by a significant hydrophobicity which promotes strong and often denaturing interactions with the substance to be encapsulated, in particular when the latter is of protein or peptide nature.
Il a été découvert, et ceci constitue le fondement de la présente invention, qu'il était possible de réaliser de nouvelles microsphères qui permettent de remédier aux inconvénients des microsphères de l'état de la technique.It has been discovered, and this constitutes the basis of the present invention, that it was possible to produce new microspheres which make it possible to remedy the drawbacks of the microspheres of the prior art.
Ainsi, selon un premier aspect, la présente demande a pour objet des microsphères constituées d'un réseau continu d'un matériau support dans lequel est éventuellement dispersée une substance, caractérisées en ce que ledit matériau support contient au moins 70 % en poids d'un homopolymère constitué d'unités récurrentes répondant à la formule générale (I) suivante :Thus, according to a first aspect, the present application relates to microspheres consisting of a continuous network of a support material in which a substance is optionally dispersed, characterized in that said support material contains at least 70% by weight of a homopolymer consisting of repeating units corresponding to the following general formula (I):
COOR1 COOR 1
— CH.- CH.
COO — (CH2)n— COOR2 COO - (CH 2 ) n - COOR 2
dans laquelle :in which :
- R, représente un groupe alkyle ayant de 1 à 6 atomes de carbone ou un groupe (CH2)m - COOR3 dans lequel m est un nombre entier compris entre 1 et 5 et R3 représente un groupe alkyle ayant de 1 à 6 atomes de carbone ;- R, represents an alkyl group having from 1 to 6 carbon atoms or a group (CH 2 ) m - COOR 3 in which m is an integer between 1 and 5 and R 3 represents an alkyl group having from 1 to 6 carbon atoms;
- R_ représente un groupe alkyle ayant de 1 à 6 atomes de carbone ; et- R_ represents an alkyl group having from 1 to 6 carbon atoms; and
- n est un nombre entier compris entre 1 et 5.- n is an integer between 1 and 5.
Il a été montré qu'en raison de la nature chimique des polymères formant leur matrice, ces nouvelles microsphères :It has been shown that due to the chemical nature of the polymers forming their matrix, these new microspheres:
- présentent une cinétique de dégradation progressive et modulée ; - permettent d'encapsuler avec une grande efficacité des substances hydrophiles, notamment d'origine biologique ;- exhibit progressive and modulated degradation kinetics; - allow hydrophilic substances, especially of biological origin, to be encapsulated with great efficiency;
Il a en outre été observé, d'une façon tout à fait surprenante et inattendue, que ces microsphères : - peuvent induire une stimulation de la réponse immunitaire, lorsqu'elles sont associées à un antigène ;It has also been observed, in a completely surprising and unexpected manner, that these microspheres: - can induce a stimulation of the immune response, when they are associated with an antigen;
- permettent, dans certains cas, la suppression de réactions pathologiques d'hypersensibilité (induction d'une tolérance), lorsqu'elles sont administrées par voie orale. C'est donc la nature du matériau polymérique formant la matrice des microsphères qui constitue l'originalité de la présente invention.- allow, in some cases, the suppression of pathological hypersensitivity reactions (induction of tolerance), when administered orally. It is therefore the nature of the polymeric material forming the matrix of the microspheres which constitutes the originality of the present invention.
Ce matériau polymérique est essentiellement formé d'un homopolymère constitué d'unités récurrentes de formule générale (I).This polymeric material is essentially formed of a homopolymer consisting of recurring units of general formula (I).
De tels polymères présentent la propriété remarquable d'être biocompatibles et bioérodables, c'est à dire qu'ils sont susceptibles de se dégrader par voie chimique ou biochimique, par coupure des substituants latéraux.Such polymers have the remarkable property of being biocompatible and bioerodible, that is to say that they are capable of degrading chemically or biochemically, by cutting the lateral substituents.
La vitesse d'érosion des microsphères conformes à l'invention étant dépendante du poids moléculaire du matériau support, elle peut donc être modulée simplement, en utilisant un matériau support ayant un poids moléculaire adapté à la vitesse d'érosion désirée.The erosion rate of the microspheres according to the invention being dependent on the molecular weight of the support material, it can therefore be modulated simply, by using a support material having a molecular weight adapted to the desired erosion rate.
Les microsphères selon la présente invention présentent donc une bioérosion modulée et progressive permettant, par exemple, le transport d'une substance biologiquement active, dispersée dans le matériau support, jusqu'à l'endroit de l'organisme où son action sera la plus efficace. La bioérosion des microspheres évite également leur accumulation dans l'organisme ; leur utilisation n'est donc plus limitée.The microspheres according to the present invention therefore exhibit a modulated and progressive bioerosion allowing, for example, the transport of a biologically active substance, dispersed in the support material, to the place in the body where its action will be most effective. . The bioerosion of microspheres also prevents their accumulation in the body; their use is therefore no longer limited.
Selon une caractéristique particulière, l'homopolymère précité est constitué d'unités récurrentes répondant à la formule générale (I) dans laquelle : R, représente un groupe alkyle ayant de 1 à 6 atomes de carbone ; R2 représente un groupe alkyle ayant de 1 à 6 atomes de carbone ; et n est un nombre égal à 1 ; et de préférence dans laquelle R, et R_ représentent un groupement CH2-CH3.According to a particular characteristic, the above-mentioned homopolymer consists of repeating units corresponding to the general formula (I) in which: R represents an alkyl group having from 1 to 6 carbon atoms; R 2 represents an alkyl group having from 1 to 6 carbon atoms; and n is a number equal to 1; and preferably in which R, and R_ represent a CH 2 -CH 3 group .
Ces différents types de polymères de la famille des poly(méthylidène- malonate) conviennent particulièrement à l'encapsulation de substances hydrophiles, notamment d'origine biologique, et éventuellement biologiquement actives.These different types of polymers from the poly (methylidene-malonate) family are particularly suitable for the encapsulation of substances hydrophilic, in particular of biological origin, and possibly biologically active.
Par "molécule biologiquement active", on entend de manière non limitative toute molécule ayant une activité biologique prophylactique ou curative, in vitro ou in vivo, notamment un agent anti-infectieux, en particulier un agent antiseptique, antibiotique, antiviral, antiparasitaire ou antimitotique, notamment anticancéreux.The term “biologically active molecule” means without limitation any molecule having a prophylactic or curative biological activity, in vitro or in vivo, in particular an anti-infectious agent, in particular an antiseptic, antibiotic, antiviral, antiparasitic or antimitotic agent, especially anticancer.
Des agents antibiotiques ou antiseptiques utilisables peuvent être par exemple la rifampicine et la colistine. A titre d'exemples d'agents antiviraux, on peut citer de manière non limitative la didanosine, la ribavirine, la zidovudine, l'acyclovir, le ganciclovir, le foscarnet, la vidarabine et la zalcitabine.Antibiotic or antiseptic agents which can be used can be, for example, rifampicin and colistin. As examples of antiviral agents, non-limiting mention may be made of didanosine, ribavirin, zidovudine, acyclovir, ganciclovir, foscarnet, vidarabine and zalcitabine.
Le cis-plastine et le taxol peuvent par exemple être utilisés en tant qu'agents anticancéreux. Selon un mode de réalisation actuellement préféré de l'invention, le matériau support des microsphères contient :Cis-plastin and taxol can for example be used as anticancer agents. According to a currently preferred embodiment of the invention, the support material for the microspheres contains:
- de 90 % à 99,5 % en poids d'un homopolymère tel que défini précédemment ; et- From 90% to 99.5% by weight of a homopolymer as defined above; and
- de 0,5 % à 10 % en poids d'un copolymère comportant au moins une séquence présentant un caractère hydrophile et au moins une séquence présentant un caractère hydrophobe, ladite séquence à caractère hydrophobe comprenant de préférence au moins une unité récurrente répondant à la formule générale (I).from 0.5% to 10% by weight of a copolymer comprising at least one block having a hydrophilic character and at least one block having a hydrophobic character, said block of hydrophobic character preferably comprising at least one repeating unit corresponding to the general formula (I).
Avantageusement, la séquence à caractère hydrophile du copolymère précité est choisie parmi un poly(oxyéthylène), un poly(alcoolvinylique), une poly(vinylpyrrolidone), un poly(N-2-hydroxypropyl méthacrylamide), un poly(hydroxyéthylméthacrylate), un poly(amino acide) hydrophile tel qu'une polylysine, un polysaccharide, et sera de préférence un poly(oxyéthylène).Advantageously, the hydrophilic block of the abovementioned copolymer is chosen from a poly (oxyethylene), a poly (vinyl alcohol), a poly (vinylpyrrolidone), a poly (N-2-hydroxypropyl methacrylamide), a poly (hydroxyethylmethacrylate), a poly hydrophilic (amino acid) such as a polylysine, a polysaccharide, and will preferably be a poly (oxyethylene).
Le copolymère peut avoir une structure à blocs, de préférence di-blocs ou tri-blocs, ou une structure greffée. L'addition de tels copolymères dans le matériau support permet d'obtenir une dispersion homogène de la substance à encapsuler à l'intérieur de chacune des microsphères. Elle permet également de moduler le rapport hydrophilie/hydrophobie de la surface des microsphères, ce qui permet d'éviter ou de limiter les interactions fortes et souvent dénaturantes avec la substance à encapsuler.The copolymer may have a block structure, preferably diblock or triblock, or a grafted structure. The addition of such copolymers in the support material makes it possible to obtain a homogeneous dispersion of the substance to be encapsulated inside each of the microspheres. It also makes it possible to modulate the hydrophilicity / hydrophobicity ratio of the surface of the microspheres, which makes it possible to avoid or limit the strong and often denaturing interactions with the substance to be encapsulated.
En outre, ces copolymères, dont la nature chimique de la séquence hydrophobe est identique à celle de l'homopolymère constituant pour l'essentiel les microsphères, sont particulièrement avantageux pour la mise en oeuvre du procédé actuellement préféré de préparation des microsphères comme cela sera expliqué plus en détail par la suite.In addition, these copolymers, the chemical nature of the hydrophobic block of which is identical to that of the homopolymer essentially constituting the microspheres, are particularly advantageous for the implementation of the currently preferred process for preparing the microspheres as will be explained in more detail later.
D'une façon générale, les microsphères conformes à la présente invention peuvent être obtenues par la mise en oeuvre d'un procédé comportant :In general, the microspheres in accordance with the present invention can be obtained by implementing a process comprising:
- la préparation d'une émulsion multiple à trois phases, dont la phase intermédiaire est constituée d'une solution du ou des polymères constituant le matériau support dans un solvant organique volatil, etthe preparation of a three-phase multiple emulsion, the intermediate phase of which consists of a solution of the polymer or polymers constituting the support material in a volatile organic solvent, and
- l'évaporation dudit solvant organique, dans des conditions permettant de provoquer la précipitation du polymère autour des gouttelettes constituant la phase interne.- Evaporation of said organic solvent, under conditions allowing the precipitation of the polymer around the droplets constituting the internal phase.
Cette émulsion multiple peut être obtenue de façon classique en dispersant une émulsion primaire du type eau-dans-huile dans une seconde phase aqueuse contenant un agent stabilisant. Cette émulsion multiple peut également être obtenue par un procédéThis multiple emulsion can be obtained in a conventional manner by dispersing a primary emulsion of the water-in-oil type in a second aqueous phase containing a stabilizing agent. This multiple emulsion can also be obtained by a process
"inverse" consistant à verser une solution aqueuse dans une émulsion primaire de type eau-dans-huile. D'une façon tout à fait inattendue, ce procédé "inverse" a permis d'obtenir des résultats tout à fait remarquables parfois même meilleurs que ceux obtenus par la technique classique précitée. Ainsi, selon un deuxième aspect, la présente invention concerne un procédé d'obtention de microsphères telles que précédemment décrites, comprenant : a) la préparation d'une première solution du ou des polymère(s) précité(s) constituant le matériau support dans un solvant organique volatil contenant éventuellement un agent tensio-actif, b) la préparation d'une seconde solution non miscible avec la solution obtenue en a), contenant éventuellement ladite substance à disperser et éventuellement un agent tensio-actif, c) la préparation d'une émulsion primaire par dispersion de la seconde solution dans la première solution, la phase continue étant constituée par la solution de polymère(s), d) la préparation d'une émulsion secondaire : - soit en dispersant, sous agitation, l'émulsion primaire obtenue en c) dans un milieu dispersant non miscible avec ladite émulsion primaire, ledit milieu dispersant contenant éventuellement un agent stabilisant ;"reverse" consisting in pouring an aqueous solution into a primary emulsion of the water-in-oil type. Quite unexpectedly, this "reverse" process has made it possible to obtain quite remarkable results, sometimes even better than those obtained by the aforementioned conventional technique. Thus, according to a second aspect, the present invention relates to a process for obtaining microspheres as previously described, comprising: a) the preparation of a first solution of the above-mentioned polymer (s) constituting the support material in a volatile organic solvent optionally containing a surfactant, b) the preparation of a second solution immiscible with the solution obtained in a), optionally containing said substance to be dispersed and optionally a surfactant, c) the preparation of a primary emulsion by dispersion of the second solution in the first solution, the continuous phase consisting of the solution of polymer (s), d) the preparation of a secondary emulsion: - either by dispersing, under stirring, the primary emulsion obtained in c) in a dispersing medium immiscible with said primary emulsion, said dispersing medium optionally containing a stabilizing agent;
- soit en versant sous agitation, dans ladite émulsion primaire, une solution constituée d'un milieu non miscible avec ladite émulsion primaire, ledit milieu contenant éventuellement un agent stabilisant, e) l'évaporation dudit solvant organique sous agitation.- Or by pouring, with stirring, into said primary emulsion, a solution consisting of a medium immiscible with said primary emulsion, said medium optionally containing a stabilizing agent, e) evaporation of said organic solvent with stirring.
Selon une caractéristique particulière de l'invention, le procédé précité comprend en outre : f) l'isolation des microsphères par centrifugation g) un ou plusieurs lavages successifs desdites microsphères h) la lyophilisation desdites microsphères.According to a particular characteristic of the invention, the aforementioned method further comprises: f) isolation of the microspheres by centrifugation g) one or more successive washings of said microspheres h) lyophilization of said microspheres.
La première étape du procédé de préparation des microsphères conforme à l'invention comporte donc la réalisation d'une émulsion de type eau-dans-huile de préférence en présence d'un agent tensio-actif approprié, la phase huileuse ou organique contenant le ou les polymères destinés à constituer le matériau support desdites microsphères.The first step of the process for preparing the microspheres according to the invention therefore comprises the production of an emulsion of the water-in-oil type preferably in the presence of an appropriate surfactant, the oily or organic phase containing the or polymers intended to constitute the support material of said microspheres.
Dans un premier temps, une solution du ou des polymère(s) constituant le matériau support est préparée à l'aide d'un solvant organique volatil approprié éventuellement en présence d'un agent tensio-actif. Avantageusement, on utilisera dans cette étape, des polymères préformés dans la mesure où les homopolymères constituant pour l'essentiel le matériau support des microsphères peuvent être obtenus dans des conditions permettant une bonne caractérisation en terme de masse molaire et de dispersité en masse.Firstly, a solution of the polymer (s) constituting the support material is prepared using an appropriate volatile organic solvent optionally in the presence of a surfactant. Advantageously, in this step, preformed polymers will be used insofar as the homopolymers essentially constituting the support material for the microspheres can be obtained under conditions allowing good characterization in terms of molar mass and mass dispersity.
Les homopolymères constitués d'unités récurrentes répondant à la formule générale (I) peuvent être préparés à partir de monomères, par exemple, en suivant le procédé décrit dans le brevet EP 283346 correspondant aux brevets US 4 931 584 et US 5 142 098 incorporés ici par référence, lesdits monomères étant généralement dégazés sous vide de pompe à palettes jusqu'à poids constant pour éliminer l'inhibiteur de polymérisation (SO2). Ces homopolymères seront cependant avantageusement préparés par voie anionique en milieu aprotique, par exemple par dispersion du monomère dans l'acétone, suivie de l'ajout de soude sous agitation, encore suivie de l'évaporation de l'acétone et du séchage du polymère ainsi obtenu. D'autres solvants organiques aprotiques tels que l'acétonitrile, le dioxane et le tétrahydrofurane peuvent être utilisés en lieu et place de l'acétone.Homopolymers made up of repeating units corresponding to general formula (I) can be prepared from monomers, for example, by following the process described in patent EP 283 346 corresponding to patents US 4,931,584 and US 5,142,098 incorporated here. by reference, said monomers being generally degassed under a vane pump vacuum to constant weight to remove the polymerization inhibitor (SO 2 ). These homopolymers will however advantageously be prepared anionically in an aprotic medium, for example by dispersion of the monomer in acetone, followed by the addition of sodium hydroxide with stirring, again followed by evaporation of the acetone and drying of the polymer thus got. Other aprotic organic solvents such as acetonitrile, dioxane and tetrahydrofuran can be used in place of acetone.
La masse moléculaire de l'homopolymère susceptible d'être obtenu par la mise en oeuvre de ce procédé peut être parfaitement maîtrisée par un choix judicieux des conditions de mise en oeuvre, et en particulier de la concentration en monomère dans la phase organique, du pH et de la molarité de l'amorceur de polymérisation (soude).The molecular mass of the homopolymer capable of being obtained by the implementation of this process can be perfectly controlled by a judicious choice of the conditions of implementation, and in particular of the concentration of monomer in the organic phase, of the pH and the molarity of the polymerization initiator (sodium hydroxide).
D'une façon générale, on utilisera dans le cadre de la présente invention des homopolymères présentant une masse molaire moyenne de 1.000 à 100.000, et de préférence de 5.000 à 80.000. Le solvant organique volatil susceptible d'être utilisé pour la préparation de la première solution contenant le ou les polymères constituant le matériau support sera généralement choisi de telle sorte que son point d'ébullition soit inférieur à celui de l'eau. Ce solvant pourra donc être éliminé facilement lors de l'étape finale d'évaporation en permettant la précipitation du polymère. L'acétate d'éthyle constitue un solvant organique volatil particulièrement approprié à cet effet.In general, homopolymers having an average molar mass of 1,000 to 100,000, and preferably 5,000 to 80,000, will be used in the context of the present invention. The volatile organic solvent capable of being used for the preparation of the first solution containing the polymer or polymers constituting the support material will generally be chosen so that its boiling point is lower than that of water. This solvent can therefore be easily removed during the final evaporation step, allowing precipitation of the polymer. Ethyl acetate is a particularly suitable volatile organic solvent for this purpose.
Les tensio-actifs susceptibles d'être utilisés pour la stabilisation de l'émulsion primaire peuvent être de nature variée et seront ajoutés à la phase organique contenant le(s) polymère(s) (première solution) et/ou à la phase aqueuse (seconde solution) constituant la phase dispersée.The surfactants capable of being used for the stabilization of the primary emulsion can be of various nature and will be added to the organic phase containing the polymer (s) (first solution) and / or to the aqueous phase ( second solution) constituting the dispersed phase.
Il peut s'agir par exemple d'un poloxamer tel que le produit commercialisé sous la dénomination Pluronic® F68, ou bien encore d'un alcoolThis can be for example a poloxamer such as the product sold under the name Pluronic ® F68, or even an alcohol
(R) poly vinylique tel que le produit commercialisé sous la dénomination Mowiol 40- 88, ou bien encore d'un polysorbate, ou bien encore d'un copolymère tensio-actif dont la séquence hydrophobe présente une nature chimique identique à celle de l'homopolymère constitué d'unités récurrentes répondant à la formule générale (I). Il a été montré que de tels copolymères tensio-actifs et en particulier les copolymères de poly(méthylidène malonate) et de polyoxyéthylène sont particulièrement avantageux dans la mesure où ils permettent d'une part, d'obtenir une émulsion primaire très stable et d'autre part, d'obtenir un bon ancrage du tensio-actif dans la matrice après évaporation du solvant.(R) poly vinyl such as the product sold under the name Mowiol 40-88, or else a polysorbate, or even a surfactant copolymer whose hydrophobic sequence has a chemical nature identical to that of homopolymer consisting of recurring units corresponding to the general formula (I). It has been shown that such surface-active copolymers and in particular the copolymers of poly (methylidene malonate) and of polyoxyethylene are particularly advantageous insofar as they allow, on the one hand, to obtain a very stable primary emulsion and on the other hand, to obtain a good anchoring of the surfactant in the matrix after evaporation of the solvent.
Les copolymères tensio-actifs précités peuvent être préparés par des techniques de polymérisation classiques bien connues de l'homme de métier. Parmi ces techniques, on utilisera de préférence la polymérisation par voie anionique, la polymérisation par voie radicalaire, ou encore la technique de couplage des séquences précurseurs du copolymère, ces séquences ayant été au préalable fonctionnalisées en bout de chaîne de façon adéquate.The above-mentioned surfactant copolymers can be prepared by conventional polymerization techniques well known to those skilled in the art. Among these techniques, use will preferably be made of anionic polymerization, radical polymerization, or else the technique of coupling the precursor sequences of the copolymer, these sequences having previously been functionally functionalized at the end of the chain.
La polymérisation par voie anionique convient plus particulièrement à la préparation de copolymères à blocs.The anionic polymerization is more particularly suitable for the preparation of block copolymers.
Elle comporte l'addition séquentielle des monomères et permet d'obtenir des copolymères de structure parfaitement définie, les quantités d'amorceurs et de monomères engagés permettant de contrôler le degré de polymérisation de chacune des séquences. Un copolymère à blocs peut être ainsi obtenu :It involves the sequential addition of the monomers and makes it possible to obtain copolymers of perfectly defined structure, the quantities of initiators and of monomers involved making it possible to control the degree of polymerization of each of the blocks. A block copolymer can thus be obtained:
- soit par polymérisation anionique d'un premier monomère et réaction sur la chaîne en croissance d'un second monomère ;- Either by anionic polymerization of a first monomer and reaction on the growing chain of a second monomer;
- soit par activation d'un polymère précurseur qui servira d'amorceur à la polymérisation d'un second monomère. Les agents d'amorçage susceptibles d'être utilisés dans le cadre de ces polymérisations par voie anionique seront généralement :- Or by activation of a precursor polymer which will serve as an initiator for the polymerization of a second monomer. The initiating agents capable of being used in the context of these anionic polymerizations will generally be:
- d'une part, les dérivés organométalliques comme le butyllithium et en particulier le diphénylhexyllithium ;- on the one hand, organometallic derivatives such as butyllithium and in particular diphenylhexyllithium;
- d'autre part, des alcoolates et en particulier les alcoolates macromoléculaires tels qu'un alcoolate de POE qui peuvent être générés par activation d'une fonction hydroxy à l'aide de cumylpotassium, de diphénylméthylpotassium, de naphtalène potassium.- on the other hand, alcoholates and in particular macromolecular alcoholates such as a POE alcoholate which can be generated by activation of a hydroxy function using cumylpotassium, diphenylmethylpotassium, naphthalene potassium.
La polymérisation par voie anionique sera généralement réalisée dans un solvant compatible avec les diverses séquences du copolymère. Dans le cas où la séquence à caractère hydrophile est constituée d'un poly(oxyéthylène) et la séquence à caractère hydrophobe est constituée d'un poly(méthylidène malonate), les copolymères à blocs seront préparés de préférence par polymérisation anionique successive de l'oxyde d'éthylène puis du méthylidène malonate ou par activation d'un précurseur polyoxyéthyléné monohydroxylé commercial et polymérisation anionique subséquente de la séquence poly(méthylidène malonate).The anionic polymerization will generally be carried out in a solvent compatible with the various blocks of the copolymer. In the case where the hydrophilic block consists of a poly (oxyethylene) and the hydrophobic block consists of a poly (methylidene malonate), the block copolymers will preferably be prepared by successive anionic polymerization of the ethylene oxide followed by methylidene malonate or by activation of a polyoxyethylenated precursor commercial monohydroxylate and subsequent anionic polymerization of the poly (methylidene malonate) sequence.
D'une façon générale, on utilisera de préférence le tétrahydrofurane comme solvant de polymérisation, ce produit permettant de travailler en milieu homogène et influençant favorablement la cinétique de polymérisation.In general, tetrahydrofuran will preferably be used as the polymerization solvent, this product making it possible to work in a homogeneous medium and favorably influencing the polymerization kinetics.
Les monomères utilisés pour la préparation des séquences hydrophiles seront généralement des produits commerciaux.The monomers used for the preparation of the hydrophilic blocks will generally be commercial products.
La technique de couplage convient également plus particulièrement à la préparation de copolymères à blocs. Cette réaction est généralement réalisée à partir d'homopolymères présynthétisés et fonctionnalisés, en présence d'un agent de couplage et éventuellement d'un agent d'activation, dans un solvant approprié.The coupling technique is also more particularly suitable for the preparation of block copolymers. This reaction is generally carried out from presynthesized and functionalized homopolymers, in the presence of a coupling agent and optionally an activating agent, in an appropriate solvent.
Dans le cas de la préparation des copolymères préférés selon l'invention, dont la séquence hydrophile est constituée d'un poly(oxyéthylène) et la séquence hydrophobe est constituée d'un poly(méthylidène malonate), on utilisera avantageusement un homopolymère du poly(oxyéthylène) fonctionnalisé par un groupement α-carboxy et un homopolymère du poly(méthylidène malonate) fonctionnalisé par un groupement α-hydroxy.In the case of the preparation of the preferred copolymers according to the invention, the hydrophilic block of which consists of a poly (oxyethylene) and the hydrophobic block of which consists of a poly (methylidene malonate), a homopolymer of the poly ( oxyethylene) functionalized by an α-carboxy group and a homopolymer of poly (methylidene malonate) functionalized by an α-hydroxy group.
L'homopolymère du poly(oxyéthylène) fonctionnalisé par un groupement α-carboxy peut être obtenu par exemple par transformation par l'anhydride succinique d'un poly(oxyéthylène) fonctionnalisé par un groupement α-hydroxy commercial.The homopolymer of poly (oxyethylene) functionalized with an α-carboxy group can be obtained for example by transformation with succinic anhydride of a poly (oxyethylene) functionalized with a commercial α-hydroxy group.
L'homopolymère du poly(méthylidène malonate) fonctionnalisé par un groupement α-hydroxy peut être obtenu directement par synthèse anionique en milieu aqueux ou par synthèse anionique dans un solvant en utilisant une solution aqueuse de soude comme amorceur de la polymérisation.The homopolymer of poly (methylidene malonate) functionalized with an α-hydroxy group can be obtained directly by anionic synthesis in an aqueous medium or by anionic synthesis in a solvent using an aqueous sodium hydroxide solution as initiator of the polymerization.
En tant qu'agent de couplage particulièrement adapté à cette polymérisation, on utilisera avantageusement le dicyclohexylcarbodiimide (DCCI). La réaction de couplage peut être éventuellement activée par catalyse basique et se déroulera généralement dans un solvant compatible avec les homopolymères, comme en particulier le dichlorométhane dans le cas particulier des copolymères préférés de l'invention. 10 5309 PCT/FR99/01005As a coupling agent which is particularly suitable for this polymerization, dicyclohexylcarbodiimide (DCCI) will advantageously be used. The coupling reaction can optionally be activated by basic catalysis and will generally take place in a solvent compatible with homopolymers, such as in particular dichloromethane in the particular case of the preferred copolymers of the invention. 10 5309 PCT / FR99 / 01005
La polymérisation par voie radicalaire convient plus particulièrement à la préparation de copolymères greffés.Radical polymerization is more particularly suitable for the preparation of graft copolymers.
Cette polymérisation est généralement réalisée à partir d'un macromonomère, c'est à dire d'un oligomère portant à l'une de ses extrémités un groupement éthylénique polymérisable par voie radicalaire et susceptible de réagir avec un monomère pour former un copolymère à structure greffée.This polymerization is generally carried out from a macromonomer, that is to say from an oligomer carrying at one of its ends an ethylenic group which can be polymerized by radical means and capable of reacting with a monomer to form a copolymer with grafted structure .
Cette polymérisation sera généralement réalisée en présence d'un amorceur dans un solvant approprié.This polymerization will generally be carried out in the presence of an initiator in an appropriate solvent.
Dans le cas de la préparation des copolymères dont la séquence hydrophile est constituée d'un poly(oxyéthylène) divers macromomeres fonctionnalisés pourront être utilisés.In the case of the preparation of the copolymers whose hydrophilic block consists of a poly (oxyethylene) various functionalized macromomers may be used.
On préférera plus particulièrement utiliser un macromonomère de poly(oxyéthylène) fonctionnalisé par un groupement méthacryloyle.It is more particularly preferred to use a poly (oxyethylene) macromonomer functionalized with a methacryloyl group.
Un tel produit peut être commercial (Aldrich) et sera constitué par exemple d'une chaîne de poly(oxyéthylène) de masse molaire comprise entre 308 et 440 g/mol, ou sera préparé à partir d'un poly(éthylèneglycol)monométhyléther commercial par couplage avec l'acide méthacrylique dans le dichlorométhane pour former une fonction terminale méthoxy.Such a product may be commercial (Aldrich) and will consist, for example, of a poly (oxyethylene) chain with a molar mass of between 308 and 440 g / mol, or will be prepared from a commercial poly (ethylene glycol) monomethyl ether by coupling with methacrylic acid in dichloromethane to form a methoxy terminal function.
On peut encore préparer un tel macromonomère par activation d'un poly(oxyéthylène) et réaction subséquente sur le chlorure de méthacryloyle.One can also prepare such a macromonomer by activation of a poly (oxyethylene) and subsequent reaction on methacryloyl chloride.
Les copolymères à structures greffées peuvent également être préparés par transestérification d'un poly(oxyéthylène) monométhyléther sur des chaînes latérales esters d'un poly(méthylidène malonate) pré-synthétisé. Cette transestérification sera généralement réalisée avec de l'alcool en présence d'un catalyseur à température élevée.The copolymers with grafted structures can also be prepared by transesterification of a poly (oxyethylene) monomethyl ether on side ester chains of a pre-synthesized poly (methylidene malonate). This transesterification will generally be carried out with alcohol in the presence of a catalyst at high temperature.
Des copolymères dont la masse molaire totale des séquences à caractère hydrophobe est comprise entre 1.000 et 80.000 g/mol, et de préférence entre 1.000 et 50.000 g/mol conviennent particulièrement dans le cadre de la présente invention. D'une façon générale, l'émulsion primaire servant à la préparation des microsphères selon l'invention pourra être obtenue au moyen d'un homogénéisateur cisaillant par exemple de type Ultraturrax (13.500 tr/mn - 5 mn).Copolymers whose total molecular weight of the hydrophobic blocks is between 1,000 and 80,000 g / mol, and preferably between 1,000 and 50,000 g / mol are particularly suitable in the context of the present invention. In general, the primary emulsion used for the preparation of the microspheres according to the invention can be obtained by means of a shearing homogenizer, for example of the Ultraturrax type (13,500 rpm - 5 min).
La substance à encapsuler est généralement additionnée à la phase aqueuse dispersée de l'émulsion primaire. 11 5309 PCT/FR99/01005The substance to be encapsulated is generally added to the dispersed aqueous phase of the primary emulsion. 11 5309 PCT / FR99 / 01005
La deuxième étape du procédé de préparation des microsphères conformes à l'invention comprend la préparation d'une émulsion secondaire :The second step of the process for preparing the microspheres in accordance with the invention comprises the preparation of a secondary emulsion:
- soit en dispersant, sous agitation, l'émulsion primaire obtenue à la première étape dans un milieu dispersant non miscible avec ladite émulsion primaire, ledit milieu dispersant contenant éventuellement un agent stabilisant ;- Either by dispersing, with stirring, the primary emulsion obtained in the first step in a dispersing medium immiscible with said primary emulsion, said dispersing medium optionally containing a stabilizing agent;
- soit en versant sous agitation dans ladite émulsion primaire, une solution constituée d'un milieu non miscible avec ladite émulsion primaire, ledit milieu contenant éventuellement un agent stabilisant.- Either by pouring with stirring into said primary emulsion, a solution consisting of a medium immiscible with said primary emulsion, said medium optionally containing a stabilizing agent.
Généralement, le milieu dispersant non miscible avec l'émulsion primaire est une phase aqueuse dans laquelle l'émulsion primaire est introduite de préférence goutte à goutte et l'émulsion est également réalisée par exemple à l'aide d'un homogénéisateur du type Ultraturrax (8.000 tr/mn ; 5mn).Generally, the dispersing medium which is immiscible with the primary emulsion is an aqueous phase into which the primary emulsion is preferably introduced drop by drop and the emulsion is also produced for example using a homogenizer of the Ultraturrax type ( 8,000 rpm; 5 mins).
L'alcool polyvinylique constitue un agent stabilisant particulièrement approprié pour la préparation de l'émulsion secondaire. Eventuellement, cette deuxième étape peut être suivie d'une étape supplémentaire de déplacement du solvant organique.Polyvinyl alcohol constitutes a stabilizing agent which is particularly suitable for the preparation of the secondary emulsion. Optionally, this second step can be followed by an additional step of displacement of the organic solvent.
La troisième étape essentielle du procédé de préparation des microsphères conformes à l'invention, consiste à évaporer le solvant organique volatil ayant servi à la préparation de la solution du ou des polymères. Dans le cas particulier où ce solvant est l'acétate d'éthyle, cette évaporation s'effectue pendant une durée d'environ 12 heures à température ambiante, sous agitation mécanique (1.400 tr/mn).The third essential step of the process for preparing the microspheres in accordance with the invention consists in evaporating the volatile organic solvent which was used for the preparation of the solution of the polymer (s). In the particular case where this solvent is ethyl acetate, this evaporation takes place for a period of approximately 12 hours at room temperature, with mechanical stirring (1,400 rpm).
L'homme de métier choisira de façon appropriée les différentes conditions de mise en oeuvre de ces trois premières étapes essentielles du procédé conforme à la présente invention en fonction des caractéristiques physicochimiques et morphologiques des microsphères recherchées.Those skilled in the art will appropriately choose the different conditions for implementing these first three essential steps of the process according to the present invention as a function of the physicochemical and morphological characteristics of the microspheres sought.
D'une façon générale, ces microsphères présenteront un diamètre moyen compris entre 1 μm et 100 μm, de préférence entre 5 μm et 50 μm pour leur application en tant que vecteurs dans le domaine pharmaceutique. D'une façon générale, les microsphères obtenues à l'issue de la troisième étape seront isolées par centrifugation , lavées et éventuellement lyophilisées.In general, these microspheres will have an average diameter of between 1 μm and 100 μm, preferably between 5 μm and 50 μm for their application as vectors in the pharmaceutical field. Generally, the microspheres obtained at the end of the third step will be isolated by centrifugation, washed and possibly lyophilized.
Selon un troisième aspect, la présente invention concerne également des compositions pharmaceutiques contenant les microsphères qui viennent d'être décrites. Ces compositions seront généralement appropriées pour une 12 5309 PCT/FR99/01005According to a third aspect, the present invention also relates to pharmaceutical compositions containing the microspheres which have just been described. These compositions will generally be suitable for a 12 5309 PCT / FR99 / 01005
administration par voie orale et se présenteront par exemple sous la forme de comprimés, de gélules, de poudres ou de granules.oral administration and will be presented, for example, in the form of tablets, capsules, powders or granules.
La présente invention va maintenant être illustrée par les exemples non limitatifs suivants : Dans ces exemples, les abréviations suivantes ont été utilisées :The present invention will now be illustrated by the following nonlimiting examples: In these examples, the following abbreviations have been used:
OE : oxyde d'éthylèneOE: ethylene oxide
POE : poly(oxyéthylène)POE: poly (oxyethylene)
MM 2.1.2 : méthylidène malonate répondant à la formule :MM 2.1.2: methylidene malonate corresponding to the formula:
OO
.C— OCH2CH3 .C— OCH 2 CH 3
H2C=C OH 2 C = CO
C— O— CH,- ^C— O— CH, - ^
I II I
O OCH2CH3 O OCH 2 CH 3
encore dénommé : 1-éthoxycarbonyl-l-éthoxycarbonylméthylène- oxycarbonyléthènealso known as: 1-ethoxycarbonyl-l-ethoxycarbonylmethylene- oxycarbonylethene
PMM 2.1.2 : polymère constitué d'unités monomères récurrentes répondant à la formulePMM 2.1.2: polymer consisting of recurring monomer units corresponding to the formula
COOCH2CH3 COOCH 2 CH 3
-CH2 Ç-CH 2 Ç
CO— O— CH2 COOCH2CH3 CO— O— CH 2 COOCH 2 CH 3
Par ailleurs, dans ces exemples : - la taille des microsphères a été mesurée par la technique du compteur Coulter et l'examen morphologique réalisé en microscopie électronique à balayage, soit sur les microsphères brutes de fabrication, soit après cryofracture ; 13 5309 PCT/FR99/01005Furthermore, in these examples: - the size of the microspheres was measured by the Coulter counter technique and the morphological examination carried out by scanning electron microscopy, either on the raw microspheres of manufacture, or after cryofracture; 13 5309 PCT / FR99 / 01005
- la masse moléculaire des polymères a été déterminée par chromatographie de perméation de gel (CPG).- The molecular weight of the polymers was determined by gel permeation chromatography (CPG).
EXEMPLE 1EXAMPLE 1
100 mg de méthylidène malonate 2.1.2 sont dissous dans 10 ml d'acétone sous agitation magnétique. 100 micro litres de soude 0,1 N sont ajoutés progressivement sous agitation magnétique. La polymérisation est maintenue pendant 5 minutes puis 100 microlitres d'HCl 0,1 N sont ajoutés toujours sous agitation magnétique. L'acétone est entièrement évaporé sous vide. Le polymère obtenu est ensuite lavé à l'aide d'environ 100 ml d'eau distillée puis séché sous vide. La masse moléculaire de ce polymère est de 30.000.100 mg of methylidene malonate 2.1.2 are dissolved in 10 ml of acetone with magnetic stirring. 100 micro liters of 0.1 N sodium hydroxide are gradually added with magnetic stirring. The polymerization is maintained for 5 minutes then 100 microliters of 0.1 N HCl are added, still with magnetic stirring. The acetone is completely evaporated in vacuo. The polymer obtained is then washed using approximately 100 ml of distilled water and then dried under vacuum. The molecular mass of this polymer is 30,000.
280 mg de polyméthylidène malonate sont dissous dans 10 ml d'acétate d'éthyle. 1 ml de phase aqueuse contenant 60 mg d'ovalbumine est émulsionné dans la phase organique sous agitation à l'aide d'un Ultraturrax à une vitesse de280 mg of polymethylidene malonate are dissolved in 10 ml of ethyl acetate. 1 ml of aqueous phase containing 60 mg of ovalbumin is emulsified in the organic phase with stirring using an Ultraturrax at a speed of
13.500 rpm pendant 5 minutes. Cette émulsion est ensuite ajoutée à 100 ml d'une solution aqueuse d'alcool polyvinylique à 2 %, l'agitation étant réalisée à l'aide d'un Ultraturrax à une vitesse de 8.000 rpm pendant 5 minutes. L'évaporation de l'acétate d'éthyle est réalisée à température ambiante pendant la nuit, sous agitation mécanique (pale tournante) à une vitesse de 1.400 rpm. Les microsphères sont recueillies après centrifugation à 4.000 rpm pendant 10 minutes puis lavées 6 fois avec de l'eau distillée et chaque fois soumises à une nouvelle centrifugation. Après la dernière centrifugation, les microsphères sont remises en suspension dans un volume de 3 ml d'eau distillée puis lyophilisées.13,500 rpm for 5 minutes. This emulsion is then added to 100 ml of a 2% aqueous polyvinyl alcohol solution, the stirring being carried out using an Ultraturrax at a speed of 8,000 rpm for 5 minutes. The ethyl acetate is evaporated at room temperature overnight, with mechanical stirring (rotating blade) at a speed of 1,400 rpm. The microspheres are collected after centrifugation at 4,000 rpm for 10 minutes then washed 6 times with distilled water and each time subjected to a new centrifugation. After the last centrifugation, the microspheres are resuspended in a volume of 3 ml of distilled water and then lyophilized.
Les microsphères ainsi obtenues ont un diamètre moyen de 6 microns et 14,2 % de l'ovalbumine mise en oeuvre dans la préparation sont encapsulés dans les microsphères de PMM 2.1.2, ce qui correspond à une encapsulation de 2,5 % (w/w).The microspheres thus obtained have an average diameter of 6 microns and 14.2% of the ovalbumin used in the preparation are encapsulated in the microspheres of PMM 2.1.2, which corresponds to an encapsulation of 2.5% (w / w).
Cette préparation est administrée par voie orale à des souris C3H à la dose de 100 microgrammes d'ovalbumine encapsulée (par souris et par jour) pendant 5 jours consécutifs. Le dernier gavage a lieu 7 jours avant la 14 55309 PCT/FR99/01005This preparation is administered orally to C3H mice at a dose of 100 micrograms of encapsulated ovalbumin (per mouse and per day) for 5 consecutive days. The last force-feeding takes place 7 days before the 14 55309 PCT / FR99 / 01005
sensibilisation des animaux à l'ovalbumine qui est effectuée par injection sous- cutanée d'ovalbumine libre (100 microgrammes par souris) aux jours JO et J14. 90 % des souris survivent à la deuxième injection d'ovalbumine alors que moins de 30 % des souris gavées par les microsphères sans ovalbumine ou par la même dose d'ovalbumine non encapsulée survivent.sensitization of animals to ovalbumin which is carried out by subcutaneous injection of free ovalbumin (100 micrograms per mouse) on days OJ and D14. 90% of the mice survive the second injection of ovalbumin while less than 30% of the mice force-fed by microspheres without ovalbumin or by the same dose of unencapsulated ovalbumin survive.
EXEMPLE 2EXAMPLE 2
On procède suivant l'exemple 1 mais du Pluronic F 68 est ajouté dans la phase aqueuse contenant l'ovalbumine à la concentration de 2 %.The procedure is as in Example 1, but Pluronic F 68 is added to the aqueous phase containing ovalbumin at a concentration of 2%.
EXEMPLE 3EXAMPLE 3
On procède suivant l'exemple 1 mais 20 mg de copolymère de POE- PMM sont ajoutés dans la phase organique contenant le polymère.The procedure is as in Example 1, but 20 mg of POE-PMM copolymer are added to the organic phase containing the polymer.
Dans cet exemple, un copolymère à blocs POE-PMM 2.1.2 a été utilisé. Ce copolymère a été obtenu par polymérisation successive des deux monomères en commençant par la préparation du bloc POE, par la mise en oeuvre du protocole expérimental suivant. Le réacteur dans lequel est effectué la polymérisation (250 ml) est raccordé à une rampe à vide permettant de travailler sous vide poussé et de s'affranchir des impuretés protiques.In this example, a POE-PMM 2.1.2 block copolymer was used. This copolymer was obtained by successive polymerization of the two monomers, starting with the preparation of the POE block, by the implementation of the following experimental protocol. The reactor in which the polymerization is carried out (250 ml) is connected to a vacuum ramp allowing to work under high vacuum and to get rid of protic impurities.
Le solvant (THF, 150 ml) purifié de toute trace d'humidité est cryodistillé dans le réacteur à - 70°C. L'amorceur (Terbutanolate de potassium (0.1N/THF) ; 10 ml) est ensuite ajouté à l'aide d'une seringue au travers d'un septum.The solvent (THF, 150 ml) purified of any trace of moisture is cryodistilled in the reactor at -70 ° C. The initiator (potassium Terbutanolate (0.1N / THF); 10 ml) is then added using a syringe through a septum.
L'oxyde d'éthylène (5 g) est alors introduit par cryodistillation. La polymérisation s'effectue à température ambiante pendant 48 heures. Après cette durée, un prélèvement permet de contrôler, par chromatographie par perméation de gel, la masse molaire (4.000 g/mol) et l'indice de polymolécularité (1.13) de la première séquence.The ethylene oxide (5 g) is then introduced by cryodistillation. The polymerization is carried out at room temperature for 48 hours. After this period, a sample makes it possible to control, by gel permeation chromatography, the molar mass (4,000 g / mol) and the polymolecularity index (1.13) of the first sequence.
Le MM 2.1.2 (0,5 ml) fraîchement dégazé sous vide pour enlever le SO2 utilisé comme inhibiteur de polymérisation, est alors rajouté rapidement et en une seule fois à température ambiante. 15 5309 PCT/FR99/01005The MM 2.1.2 (0.5 ml) freshly degassed under vacuum to remove the SO 2 used as a polymerization inhibitor, is then added quickly and all at once at room temperature. 15 5309 PCT / FR99 / 01005
Après 5 heures, le copolymère est désactivé par ajout de méthanol et précipité dans l'éther diéthylique.After 5 hours, the copolymer is deactivated by adding methanol and precipitated in diethyl ether.
5 motifs dérivés de MM 2.1.2 sont fixés au POE, ce qui correspond à une masse molaire pour le PMM 2.1.2 de 1.150 g/mol. L'analyse thermique du copolymère révèle une température de transition vitreuse de - 16°C ainsi qu'un pic de fusion de 45°C (ΔH = 117 J/g).5 motifs derived from MM 2.1.2 are attached to POE, which corresponds to a molar mass for PMM 2.1.2 of 1,150 g / mol. The thermal analysis of the copolymer reveals a glass transition temperature of - 16 ° C as well as a melting peak of 45 ° C (ΔH = 117 J / g).
EXEMPLE 4EXAMPLE 4
On procède suivant la technique décrite dans l'exemple 1, mais l'ovalbumine (60 mg) est remplacée par 2 mg du peptide V3 28 de la boucle V3 BRU de la gp 120 du VIH (séquence NNTRKSIHI GPGRAFYATGDIIGDIRQA). Les microsphères obtenues ont une taille moyenne de 5.8 microns et 70 % du peptide V3 28 mis en oeuvre sont encapsulés dans les microsphères ce qui correspond à une encapsulation de 0.48 % w/w. L'étude réalisée en microscopie électronique à balayage révèle des particules lisses et de forme sphérique.The procedure described in example 1 is followed, but the ovalbumin (60 mg) is replaced by 2 mg of the peptide V3 28 of the V3 BRU loop of HIV gp 120 (sequence NNTRKSIHI GPGRAFYATGDIIGDIRQA). The microspheres obtained have an average size of 5.8 microns and 70% of the V3 28 peptide used is encapsulated in the microspheres, which corresponds to an encapsulation of 0.48% w / w. The study carried out in scanning electron microscopy reveals smooth and spherical particles.
EXEMPLE 5EXAMPLE 5
On procède suivant l'exemple 4 mais du Pluronic F 68 est ajouté dans la phase aqueuse contenant le peptide à la concentration de 2 %. Les microsphères obtenues ont une taille de 7.0 microns et 70 % du peptide V3 28 mis en oeuvre sont encapsulés dans les microsphères.The procedure is as in Example 4, but Pluronic F 68 is added to the aqueous phase containing the peptide at a concentration of 2%. The microspheres obtained have a size of 7.0 microns and 70% of the V3 28 peptide used is encapsulated in the microspheres.
EXEMPLE 6EXAMPLE 6
On procède suivant l'exemple 4 mais 20 mg de copolymère de POE- PMM sont ajoutés dans la phase aqueuse contenant le peptide. 16 5309 PCT/FR99/01005The procedure is as in Example 4, but 20 mg of POE-PMM copolymer are added to the aqueous phase containing the peptide. 16 5309 PCT / FR99 / 01005
EXEMPLE 7EXAMPLE 7
On procède suivant l'exemple 4 mais 20 mg de copolymère de POE- PMM sont ajoutés dans la phase organique contenant le polymère.The procedure is as in Example 4, but 20 mg of POE-PMM copolymer are added to the organic phase containing the polymer.
EXEMPLE 8EXAMPLE 8
On procède suivant l'exemple 1 mais la phase aqueuse interne est constituée de 1 ml d'acide acétique 0,5M contenant 3 mg de collagène de type IL Les microsphères ainsi obtenues ont un diamètre moyen de 6 microns et 66.6 % du collagène mis en oeuvre dans la préparation sont encapsulés dans les microsphères de PMM2.1.2, ce qui correspond à une encapsulation de 0.7 % (w/w).The procedure is as in Example 1, but the internal aqueous phase consists of 1 ml of 0.5M acetic acid containing 3 mg of type IL collagen. The microspheres thus obtained have an average diameter of 6 microns and 66.6% of the collagen put in in the preparation are encapsulated in the microspheres of PMM2.1.2, which corresponds to an encapsulation of 0.7% (w / w).
EXEMPLE 9EXAMPLE 9
On procède suivant l'exemple 1 mais la phase aqueuse interne est constituée de 1 ml d'eau distillée. Les microsphères ainsi obtenues ne contiennent pas de substance biologiquement active. Leur diamètre moyen est de 7.0 microns.The procedure is as in Example 1, but the internal aqueous phase consists of 1 ml of distilled water. The microspheres thus obtained do not contain a biologically active substance. Their average diameter is 7.0 microns.
EXEMPLE 10EXAMPLE 10
On procède suivant la technique décrite dans l'exemple 1 mais l'ovalbumine (60 mg) est remplacée par le plasmide pCDNA3 (5 mg). Les microsphères obtenues ont une taille moyenne de 7 μm et 9,8 % de plasmide mis en oeuvre sont encapsulés dans les microsphères ce qui correspond à une encapsulation de 0,17 % (w/w).The procedure described in Example 1 is followed, but the ovalbumin (60 mg) is replaced by the plasmid pCDNA3 (5 mg). The microspheres obtained have an average size of 7 μm and 9.8% of plasmid used are encapsulated in the microspheres which corresponds to an encapsulation of 0.17% (w / w).
L'étude réalisée en microscopie électronique à balayage révèle des particules lisses et de forme sphérique.The study carried out in scanning electron microscopy reveals smooth and spherical particles.
EXEMPLE 11EXAMPLE 11
On procède suivant l'exemple 10 mais du Pluronic® est ajouté dans la phase aqueuse contenant le plasmide, à la concentration de 2 %. 17 5309 PCT/FR99/01005The procedure according to Example 10 but Pluronic ® is added to the aqueous phase containing plasmid, at a concentration of 2%. 17 5309 PCT / FR99 / 01005
Les microsphères ainsi obtenues ont un diamètre moyen de 7 μm et 12 % du plasmide mis en oeuvre sont encapsulés dans les microsphères ce qui correspond à une encapsulation de 0,22 % (w/w).The microspheres thus obtained have an average diameter of 7 μm and 12% of the plasmid used are encapsulated in the microspheres which corresponds to an encapsulation of 0.22% (w / w).
EXEMPLE 12EXAMPLE 12
On procède suivant la technique décrite dans l'exemple 1 mais l'ovalbumine (60 mg) est remplacée par l'oligonucléotide (pdT16) (2 mg). Les microsphères obtenues ont une taille moyenne de 4,8 μm et 20,6 % d'oligonucléotide mis en oeuvre sont encapsulés dans les microsphères ce qui correspond à une encapsulation de 0,19 % (w/w).The procedure described in Example 1 is followed, but the ovalbumin (60 mg) is replaced by the oligonucleotide (pdT16) (2 mg). The microspheres obtained have an average size of 4.8 μm and 20.6% of oligonucleotide used are encapsulated in the microspheres which corresponds to an encapsulation of 0.19% (w / w).
EXEMPLE 13EXAMPLE 13
On procède suivant l'exemple 12 mais du Pluronic® à la concentration de 2 % est ajouté dans la phase aqueuse contenant l'oligonucléotide.One proceeds according to Example 12 but Pluronic ® at a concentration of 2% is added to the aqueous phase containing the oligonucleotide.
Les microsphères obtenues ont un diamètre moyen de 5,7 μm et 23 % d'oligonucléotide mis en oeuvre sont encapsulés dans les microsphères ce qui correspond à une encapsulation de 0.21 % (w/w). The microspheres obtained have an average diameter of 5.7 μm and 23% of oligonucleotide used are encapsulated in the microspheres which corresponds to an encapsulation of 0.21% (w / w).
Claims
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| FR9805424 | 1998-04-29 | ||
| FR9805424A FR2778100B1 (en) | 1998-04-29 | 1998-04-29 | NOVEL POLY (METHYLIDENE MALONATE) MICROSPHERES, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| PCT/FR1999/001005 WO1999055309A1 (en) | 1998-04-29 | 1999-04-28 | Novel poly(methylidene malonate) microspheres, preparation method and pharmaceutical compositions containing them |
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| MX2020010192A (en) * | 2018-07-25 | 2020-10-20 | Firmenich & Cie | Process for preparing microcapsules. |
| WO2020123579A1 (en) * | 2018-12-11 | 2020-06-18 | Sirrus, Inc. | Polymerization of 1,1-dicarbonyl 1-alkenes |
| US20220411561A1 (en) | 2019-07-19 | 2022-12-29 | Arkema France | Curable compositions useful for obtaining non-sensitizing cured products |
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| KR940003548U (en) * | 1992-08-14 | 1994-02-21 | 김형술 | Laundry dryer |
| DE19508049C2 (en) | 1995-02-23 | 1997-02-06 | Schering Ag | Use of methylene malon diester derivatives for the production of gas-containing microparticles |
| FR2755136B1 (en) * | 1996-10-25 | 1999-01-22 | Virsol | PROCESS FOR THE PREPARATION OF METHYLIDENE MALONATE NANOPARTICLES, NANOPARTICLES CONTAINING ONE OR MORE BIOLOGICALLY ACTIVE MOLECULES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
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- 1999-04-28 EP EP99915856A patent/EP1082104A1/en not_active Ceased
- 1999-04-28 SK SK1631-2000A patent/SK16312000A3/en unknown
- 1999-04-28 CA CA002330481A patent/CA2330481A1/en not_active Abandoned
- 1999-04-28 BR BR9910016-9A patent/BR9910016A/en not_active IP Right Cessation
- 1999-04-28 HU HU0101649A patent/HUP0101649A3/en unknown
- 1999-04-28 US US09/673,255 patent/US6440461B1/en not_active Expired - Lifetime
- 1999-04-28 PL PL99343687A patent/PL343687A1/en unknown
- 1999-04-28 AU AU34288/99A patent/AU752644B2/en not_active Ceased
- 1999-04-28 RU RU2000129800/15A patent/RU2227018C2/en not_active IP Right Cessation
- 1999-04-28 AR ARP990101963A patent/AR019114A1/en unknown
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- 1999-04-28 JP JP2000545509A patent/JP2002513042A/en active Pending
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| AU752644B2 (en) | 2002-09-26 |
| FR2778100B1 (en) | 2001-05-04 |
| PL343687A1 (en) | 2001-08-27 |
| HUP0101649A3 (en) | 2002-10-28 |
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