JPH04208216A - Preparation of fine liposome - Google Patents
Preparation of fine liposomeInfo
- Publication number
- JPH04208216A JPH04208216A JP33045090A JP33045090A JPH04208216A JP H04208216 A JPH04208216 A JP H04208216A JP 33045090 A JP33045090 A JP 33045090A JP 33045090 A JP33045090 A JP 33045090A JP H04208216 A JPH04208216 A JP H04208216A
- Authority
- JP
- Japan
- Prior art keywords
- lamellar liquid
- aqueous solution
- ribosomes
- liquid crystals
- water
- 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.)
- Pending
Links
- 238000002360 preparation method Methods 0.000 title claims description 18
- 239000002502 liposome Substances 0.000 title abstract 5
- 239000012528 membrane Substances 0.000 claims abstract description 41
- 239000004973 liquid crystal related substance Substances 0.000 claims abstract description 40
- 239000007864 aqueous solution Substances 0.000 claims abstract description 25
- 239000002904 solvent Substances 0.000 claims abstract description 21
- 239000000470 constituent Substances 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000000126 substance Substances 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 150000005846 sugar alcohols Polymers 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 12
- 239000002537 cosmetic Substances 0.000 claims abstract description 7
- 210000003705 ribosome Anatomy 0.000 claims description 49
- 239000000463 material Substances 0.000 claims description 5
- 238000004898 kneading Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 abstract description 22
- 239000003960 organic solvent Substances 0.000 abstract description 15
- 230000014759 maintenance of location Effects 0.000 abstract description 12
- 239000004615 ingredient Substances 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 239000013078 crystal Substances 0.000 abstract 3
- 230000033228 biological regulation Effects 0.000 abstract 1
- 230000008961 swelling Effects 0.000 abstract 1
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 15
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 14
- 239000000203 mixture Substances 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 8
- 150000002632 lipids Chemical class 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
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- 239000004480 active ingredient Substances 0.000 description 6
- 235000011187 glycerol Nutrition 0.000 description 6
- -1 Glyceroglycolipids Chemical class 0.000 description 5
- 229960005003 carbocromen Drugs 0.000 description 5
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
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- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical class CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
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- 235000010323 ascorbic acid Nutrition 0.000 description 4
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- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
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- 150000003432 sterols Chemical class 0.000 description 4
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- 235000000346 sugar Nutrition 0.000 description 4
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 3
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 3
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- 235000013345 egg yolk Nutrition 0.000 description 3
- 210000002969 egg yolk Anatomy 0.000 description 3
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- 229960002139 pilocarpine hydrochloride Drugs 0.000 description 3
- RNAICSBVACLLGM-GNAZCLTHSA-N pilocarpine hydrochloride Chemical compound Cl.C1OC(=O)[C@@H](CC)[C@H]1CC1=CN=CN1C RNAICSBVACLLGM-GNAZCLTHSA-N 0.000 description 3
- QYIXCDOBOSTCEI-QCYZZNICSA-N (5alpha)-cholestan-3beta-ol Chemical compound C([C@@H]1CC2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CCCC(C)C)[C@@]2(C)CC1 QYIXCDOBOSTCEI-QCYZZNICSA-N 0.000 description 2
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
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- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- QYIXCDOBOSTCEI-UHFFFAOYSA-N alpha-cholestanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 QYIXCDOBOSTCEI-UHFFFAOYSA-N 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
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- 239000008280 blood Substances 0.000 description 2
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- 210000004556 brain Anatomy 0.000 description 2
- RNPXCFINMKSQPQ-UHFFFAOYSA-N dicetyl hydrogen phosphate Chemical compound CCCCCCCCCCCCCCCCOP(O)(=O)OCCCCCCCCCCCCCCCC RNPXCFINMKSQPQ-UHFFFAOYSA-N 0.000 description 2
- 229940093541 dicetylphosphate Drugs 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
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- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 2
- FROLUYNBHPUZQU-IIZJPUEISA-N (2R,3R,4S,5R)-2-(hydroxymethyl)-6-[3-[3-[(3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxypropoxy]propoxy]oxane-3,4,5-triol Chemical compound OC[C@H]1OC(OCCCOCCCOC2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@@H](O)[C@H]1O FROLUYNBHPUZQU-IIZJPUEISA-N 0.000 description 1
- JSPNNZKWADNWHI-PNANGNLXSA-N (2r)-2-hydroxy-n-[(2s,3r,4e,8e)-3-hydroxy-9-methyl-1-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoctadeca-4,8-dien-2-yl]heptadecanamide Chemical compound CCCCCCCCCCCCCCC[C@@H](O)C(=O)N[C@H]([C@H](O)\C=C\CC\C=C(/C)CCCCCCCCC)CO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O JSPNNZKWADNWHI-PNANGNLXSA-N 0.000 description 1
- WLRMANUAADYWEA-NWASOUNVSA-N (S)-timolol maleate Chemical compound OC(=O)\C=C/C(O)=O.CC(C)(C)NC[C@H](O)COC1=NSN=C1N1CCOCC1 WLRMANUAADYWEA-NWASOUNVSA-N 0.000 description 1
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 1
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 1
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical group N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
- RGHFKWPGWBFQLN-UHFFFAOYSA-M sodium;5,5-diethylpyrimidin-3-ide-2,4,6-trione Chemical compound [Na+].CCC1(CC)C([O-])=NC(=O)NC1=O RGHFKWPGWBFQLN-UHFFFAOYSA-M 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960001525 thiamylal sodium Drugs 0.000 description 1
- 229960005221 timolol maleate Drugs 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 239000005526 vasoconstrictor agent Substances 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野と本発明の目的〕
本発明は、微細なリボソームの調製法及びその微細なリ
ボソームを含有する医薬用薬剤及び化粧料の調製法に関
するものである。更に詳しくは、本発明は、生体にとっ
て有害な有機溶媒を用いることなく、比較的低温で微細
なリボソームを形成させ、内包物の保持効率が高く、大
量生産に適する微細なリボソームの調製法を提供せんと
するものである。[Detailed Description of the Invention] [Industrial Field of Application and Object of the Invention] The present invention relates to a method for preparing fine ribosomes and a method for preparing pharmaceutical agents and cosmetics containing the fine ribosomes. . More specifically, the present invention provides a method for preparing fine ribosomes that forms fine ribosomes at relatively low temperatures without using organic solvents that are harmful to living organisms, has high inclusion retention efficiency, and is suitable for mass production. This is what I am trying to do.
リボソームの研究は１９６４年、１３ ａｎｇ ｌ＋
ａ ｍらが卵黄ホスファチジルコリンの水溶液への懸濁
物を電子顕微鏡、光学顕微鏡で観察し、水溶液のイオン
強度に依存して形態が変化することを見出したことに始
まる。生体膜由来のリン脂質を水溶液中に懸濁すると、
内部に水相を有するスメクチック液晶相の脂質二分子膜
よりなる閉鎖小胞が形成され、この小胞体がリボソーム
と呼ばれるものであるが、リボソームの調製にあたって
は、その脂質組成は適宜選択でき、リボソーム内水相ま
たは膜中に物質を保持できることがら、リボソームは、
生体膜モデルとして脂質の物性や膜の透過性研究、脂質
と膜タンパク貿との相互作用や膜面上で起こる諸反応の
解析などに広く用いられている。また、１９６９年にＷ
ｅｉｓｓｍａｎが遺伝子の酵素欠損にリボソームを用い
て以来、ドラッグデリバリ−としての応用も近年特に注
目されてきている。Research on ribosomes began in 1964, 13 ang l+
A.M. et al. observed a suspension of egg yolk phosphatidylcholine in an aqueous solution using an electron microscope and an optical microscope and found that the form changed depending on the ionic strength of the aqueous solution. When phospholipids derived from biological membranes are suspended in an aqueous solution,
A closed vesicle consisting of a lipid bilayer membrane with a smectic liquid crystal phase and an aqueous phase inside is formed, and this endoplasmic reticulum is called a ribosome.When preparing a ribosome, the lipid composition can be selected as appropriate; Because they can hold substances in their internal aqueous phase or membrane, ribosomes
It is widely used as a biological membrane model to study the physical properties of lipids and membrane permeability, as well as to analyze interactions between lipids and membrane proteins and various reactions that occur on the membrane surface. Also, in 1969, W.
Since Eissman used ribosomes for enzyme deletion in genes, their application as drug delivery has also received particular attention in recent years.
こうしたリボソームの種類には大きく分けて多重層リボ
ソーム（Ｍｕｌｔｉ−１ａｍｅｌｌａｒ ｖｅｓｉｃｌ
ｅ。These types of ribosomes can be broadly divided into multi-lamellar vesicles.
e.
ＭＬＶ、粒子径数μＩＩ）、大きな一枚膜リボソーム（
Ｌａｒｇｅ ｕｎｉ−１ａｍｅｌｌａｒ ｖｅｓｉｃｌ
ｅ、 ＬＵＶ、粒子径０．１〜１．０μｍ）並びに小さ
な一枚膜リボソーム（Ｓｍａｌｌ ｕｎｉ−１ａｍｅｌ
ｌａｒ ｖｅｓｉｃｌｅ、 ＳＵＶ、粒子径２０〜５０
ｎｍ）があり、それぞれ種々の調製法が既に報告されて
いる（Ｓｚｏｋａ、 Ｆ、 Ｊｒ、 ａｎｄ Ｐａｐａ
ｌｌａｄ−ｊｏｐｏｕｌｏｓ Ｄ、、 Ａｎｎ、 Ｒｅ
ｖ、 Ｂｉｏｐｂｙｓ、 Ｂｉｏｅｎｇ、、 ９゜４６
７ （１９８０））。MLV, particle size several μII), large unilamellar ribosome (
Large uni-1amellar vesicl
e, LUV, particle size 0.1-1.0 μm) and small uni-lamellar ribosomes (Small uni-1amel).
lar vesicle, SUV, particle size 20-50
nm), and various preparation methods have been reported (Szoka, F., Jr. and Papa
llad-jopoulos D, Ann, Re
v, Biopbys, Bioeng,, 9°46
7 (1980)).
ところで、これらの従来技術により知られているリボソ
ームの調製は、いずれも実験室スケールの調製にすぎず
、しかも、いずれの方法もリボソーム膜構成物質の溶解
剤としてクロロボルム、エーテル等の人体に望ましくな
い揮発性有機溶媒を使用しているため実用化するには解
決すべき問題が存在している。特にスケール・アップを
した場合の保安作業上の問題、人体への安全性の問題及
び最終的に有機溶媒を除去したとしても、それらの残留
物の生体に及ばず危険性の問題が存在している。近年、
スケール・アップ可能なリボソーム製造装置も市販され
ているが、それらを用いる場合でも、リボソーム膜補強
成分であるコレステロール等のステロール類、セレブロ
シド等の膜への組み込みの為には、あらかじめ均一な膜
を作製する為に、上述のような有機溶媒を使用せねばな
らず、更にはそうした補強成分と組み合わさったリボソ
ームを微細化するにあたっては、たとえ加熱したとして
も、フィルターあるいはノズルをリボソームが通過しに
くく非常に時間がかかり、更なる加熱はスケール・アッ
プ時に保安作業上の問題が生じてくる。特開昭６０−７
９３２号公報記載の多価アルコールを用いる方法では、
前記した如き有機溶媒は用いられていないが、粒子径の
コントロール、特に微細リボソームを得るために撹拌機
、超音波乳化機、高圧乳化機、フィルター等が使用され
ている。これらの処理をほどこさないと粒子径は、はと
んどの場ａＯ１５μｍ以上となるが、これらの処理は内
包有効成分の保持効率を著しく低下させる等の問題があ
る。また、非常に高＠（通常９０°Ｃ以上、ステロール
類添加の際には１６０°Ｃ）にして調製しなければなら
ない為、脂質の分解及び内包有効成分の化学的安定性の
確保に対する問題があり、更には、その生体への安全性
への問題もある。By the way, all of the ribosome preparations known by these conventional techniques are only laboratory-scale preparations, and each method uses chloroborum, ether, and other substances that are undesirable for the human body as dissolving agents for ribosomal membrane constituents. Since a volatile organic solvent is used, there are problems that need to be solved before it can be put into practical use. In particular, there are safety issues when scaling up, safety issues for the human body, and even if the organic solvent is finally removed, the remaining residue may not be dangerous to living organisms. There is. recent years,
Ribosome manufacturing devices that can be scaled up are also commercially available, but even when using them, it is necessary to prepare a uniform membrane in advance in order to incorporate sterols such as cholesterol and cerebroside, which are ribosome membrane reinforcing components, into the membrane. In order to produce these, organic solvents such as those mentioned above must be used, and furthermore, when ribosomes are combined with such reinforcing components, it is difficult for them to pass through a filter or nozzle, even if heated. It is very time consuming and further heating poses safety problems during scale up. Unexamined Japanese Patent Publication 1986-7
In the method using polyhydric alcohol described in Publication No. 932,
Although organic solvents as described above are not used, a stirrer, an ultrasonic emulsifier, a high-pressure emulsifier, a filter, etc. are used to control the particle size, especially to obtain fine ribosomes. If these treatments are not performed, the particle size will be 15 μm or more in most cases, but these treatments have problems such as significantly lowering the retention efficiency of the encapsulated active ingredients. In addition, because it must be prepared at a very high temperature (usually 90°C or higher, 160°C when adding sterols), there are problems with decomposing lipids and ensuring the chemical stability of the active ingredients contained therein. Moreover, there are also problems with its safety to living organisms.
従来技術によれば、過激な条件下でないとリボソームの
微細化は困離なことである。その理由としては、他の両
親媒性物質と比べて、レシチンに代表される脂質は、そ
の希薄水溶液でもミセルを形成せず【ＳＤＳのｃｍｃ
１．４５ｘｌＱ−’Ｍ。According to the prior art, it is difficult to miniaturize ribosomes unless under extreme conditions. The reason for this is that, compared to other amphipathic substances, lipids such as lecithin do not form micelles even in dilute aqueous solutions [cmc of SDS].
1.45xlQ-'M.
ＤＰＰＣのｃｍｃ −１０−”Ｍ （Ｓｍｉｔｈ、
Ｒ，ａｎｄ Ｃ。DPPC cmc -10-”M (Smith,
R, and C.
Ｔａｎｆｏｒｄ、 Ｊ、 Ｍｏ１． Ｂｉｏｌ、、 ６
７７５（１９７２））） 、液晶分散系（これは不均一
なＭＬＶ水溶液ともいえる）であり、その分子構造（リ
ボソームを形成しやすい脂質はコーン型、Ｃｕ１ｌｉｓ
、 Ｐ、Ｒ，ａｎｄ ＤｅＸｒｕｉｊｆｆ Ｂ、、
Ｂ、Ｂ−Ａ、、 ５５９．３９９（１９７９）） 、及
び非常に強固な分子間力（これは、水存在下での水との
大きな疎水性効果と強い親木部間のイオン結合（Ｔ、
Ｋａｎｅｋｏ ａｎｄ Ｋ、 ５ｂｉｎｏｄａ、 Ｙｕ
ｋａｇａｋｕ。Tanford, J., Mo1. Biol,, 6
775 (1972))), a liquid crystal dispersion system (which can also be called a non-uniform MLV aqueous solution), and its molecular structure (lipids that tend to form ribosomes are cone-shaped, Culis
, P.R., and DeXruijff B.
B, B-A, 559.399 (1979)), and very strong intermolecular forces (this is due to the large hydrophobic effect with water in the presence of water and the strong ionic bond between parent xylem (T ,
Kaneko and K, 5binoda, Yu
kagaku.
３７７０９（１９８８）、Ｋ、 ５ｈｉｎｏｄａ ａｎ
ｄ Ｔ、 Ｋａｎｅｋｏ、 Ｊ。37709 (1988), K.
dT., Kaneko, J.
Ｄｉｓ、 Ｓｃｉ、 Ｔｅｃ、、 ９５５５（１９８９
））に起因するものである。Dis, Sci, Tec, 9555 (1989
)).
本発明者らは、上記従来技術の問題点を解決するため、
鋭意検討した結果、生体にとって有害な有機溶媒を使用
することなく、比較的低温で微細なリボソームを形成さ
せ、その粒子径を自由に調製することができ、しかも内
包物の保持効率が高く、大量生産に適し、かつ簡便な微
細リボソームの調製法を提供することに成功しｌこ 。In order to solve the problems of the above-mentioned conventional technology, the present inventors
As a result of extensive research, we found that it is possible to form fine ribosomes at relatively low temperatures without using organic solvents that are harmful to living organisms, and that the particle size can be freely adjusted. We have succeeded in providing a method for preparing fine ribosomes that is suitable and simple for production.
本発明方法によれば、大量の薬剤を内包した微細なリボ
ソームを得ることが可能であり、これにより該リボソー
ムを配合した優れたリボソーム製剤を得ることができる
。According to the method of the present invention, it is possible to obtain fine ribosomes containing a large amount of drug, thereby making it possible to obtain excellent ribosome preparations containing the ribosomes.
すなわち、本発明により、リボソーム膜構成物質をその
良溶媒である多価アルコールに溶解し、次いで貧溶媒で
ある別の多価アルコールを添加し、充分に混合すること
によりリボソーム膜構成物質とそれらの多価アルコール
から成るラメラ液晶を形成させ、次に、これに水もしく
はリボソーム膜構成物質に応じて選択された適宜の物質
の水溶液を用いて、これをラメラ液晶が壊れないように
、緩徐に滴下しながら練り込むことにより膨潤したラメ
ラ液晶を形成させ、次いで上記の水又は水溶液と同一も
しくは別の水溶液を用いて、これを連続的に滴下するこ
とを特徴とする微細なリボソームの調製法ならびに、そ
れにより得られた微細なリボソームを利用して医薬およ
び／または化粧料を内包せしめ本発明方法は、生体にと
って無害であり、そのまま最終製品に残留しても投与可
能な水溶性非揮発性有機溶媒と少量の水を用い、それに
よって膨潤したラメラ液晶を形成させ、そのライラ液晶
と水との非常に低い界面張力を利用することにより、そ
の分散エネルギーも非常に低下し、容易にリボソームが
形成されるという界面化学的性質を利用することを特徴
とし、このメカニズム自体全く新規なものである。That is, according to the present invention, the ribosome membrane constituent substances are dissolved in a polyhydric alcohol which is a good solvent, and then another polyhydric alcohol which is a poor solvent is added and thoroughly mixed. Lamellar liquid crystals made of polyhydric alcohol are formed, and then water or an aqueous solution of an appropriate substance selected depending on the ribosomal membrane constituent material is slowly added dropwise to this so as not to break the lamellar liquid crystals. A method for preparing fine ribosomes, which is characterized in that a swollen lamellar liquid crystal is formed by kneading while stirring, and then continuously dripping this using water or an aqueous solution that is the same as or different from the aqueous solution described above; The method of the present invention utilizes the resulting fine ribosomes to encapsulate pharmaceuticals and/or cosmetics.The method of the present invention is a water-soluble non-volatile organic solvent that is harmless to living organisms and can be administered even if it remains in the final product. By using a small amount of water to form a swollen lamellar liquid crystal, and by taking advantage of the extremely low interfacial tension between the Lyra liquid crystal and water, the dispersion energy is also extremely low, making it easy to form ribosomes. This mechanism itself is completely new, as it utilizes the surface chemical properties of oxidation.
しかも本発明の方法によれば、ラメラ液晶中に有効成分
を封じ込めるので、その保持効率も高く、またこのラメ
ラ液晶は高温にしなくとも例えば３０°Ｃでも形成され
、更には、膜構成物質に対する良溶媒と貧溶媒の比率あ
るいはそれらの溶媒に対する膜構成物質の濃度を調製す
ることにより任意の粒子径のリボソームが得られる。Moreover, according to the method of the present invention, since the active ingredient is confined in the lamellar liquid crystal, its retention efficiency is high, and the lamellar liquid crystal can be formed even at, for example, 30°C without being heated to a high temperature, and furthermore, it has good properties against film-constituting substances. Ribosomes of any particle size can be obtained by adjusting the ratio of the solvent to the poor solvent or the concentration of membrane constituents relative to the solvent.
従って、先に挙げたリボソームの工業的生産性を困難な
らしめている要因及びリボソーム膜構成物質、内包有効
成分の分解、更には有毒性有機溶媒の他、蛇面活性剤な
どの最終製品中への残留性及びスケール・アップに伴う
設備上・操作上の問題等の諸問題は、本発明の方法によ
って解消されたといえる。Therefore, the above-mentioned factors that make the industrial productivity of ribosomes difficult, as well as the decomposition of ribosome membrane constituents and the active ingredients contained therein, as well as toxic organic solvents and surfactants in the final product. It can be said that various problems such as persistence and equipment and operational problems associated with scale-up have been solved by the method of the present invention.
以下、本発明を詳細に述べる。The present invention will be described in detail below.
本発明において使用されるリボソーム膜構成物質きは、
主要膜構成成分の他必要に応じて膜補強成分、荷電成分
、酸化防止剤、防腐・殺菌剤などが用いられる。The ribosomal membrane constituent materials used in the present invention are:
In addition to the main membrane components, membrane reinforcing components, charged components, antioxidants, preservatives/sterilizers, etc. are used as necessary.
主要膜構成成分は例えば卵黄レンチン、大豆レシチン、
水添した卵黄レシチン・大豆レシチン、及びＤＭＰＣ，
ＤＰＰＣ％ＤＳＰＣに代表される合成レシチン、卵黄ス
フィンゴミエリン、牛脳スフィンゴミエリン等に代表さ
れるリン脂質の他、ジガラクトシルジグリセリドのよう
な糖脂質、ジアルキル型合成界面活性剤等の一種又は二
種以上の混合物である。The main membrane constituents are, for example, egg yolk lentin, soybean lecithin,
Hydrogenated egg yolk lecithin, soybean lecithin, and DMPC,
In addition to synthetic lecithin represented by DPPC%DSPC, phospholipids represented by egg yolk sphingomyelin, bovine brain sphingomyelin, etc., one or more types of glycolipids such as digalactosyl diglyceride, dialkyl type synthetic surfactants, etc. It is a mixture of
また、膜補強成分としては、コレステロール、コレスタ
ノール等のステロール類、セレブロシーｑ−へヒ
ド、ガングリオシド等のスフィンゴ糖脂質、セラミド、
グリセロ糖脂質及び配糖体等が用いられ、荷電成分とし
ては、ホスファチジルエタノールアミン、ホスファチジ
ルイノシＩ・−ル、ボス７アチジルセリン、ホスファチ
ジルグリセロール、ホスファチジン酸、ジセチルホスフ
ェート、ステアリルアミン等が用いられ、更に酸化防止
剤としては、α−トコフェロール、防腐・殺菌剤として
は、パラベン類、塩化ベンザルコニウム等を加えてリボ
ソーム膜を形成させてもよい。これらリボソームの膜構
成物質の比率は特定されないが、好ましくは主要膜構成
成分１重量部に対して、膜補強成分を０〜１．０重量部
程度、荷電成分をθ〜０．２重量部程度加えるのが望ま
しい。In addition, membrane reinforcing components include cholesterol, sterols such as cholestanol, glycosphingolipids such as cerebrocytium q-hehyde and ganglioside, ceramide,
Glyceroglycolipids, glycosides, etc. are used, and as charged components, phosphatidylethanolamine, phosphatidylinosyl, bos 7 atidylserine, phosphatidylglycerol, phosphatidic acid, dicetyl phosphate, stearylamine, etc. are used. As an antioxidant, α-tocopherol, as a preservative/sterilizer, parabens, benzalkonium chloride, etc. may be added to form a ribosome membrane. The ratio of these ribosome membrane constituents is not specified, but preferably the membrane reinforcing component is about 0 to 1.0 parts by weight and the charged component is about θ to 0.2 parts by weight to 1 part by weight of the main membrane constituents. It is desirable to add.
本発明方法で使用されるリボソーム膜構成物質に対する
良溶媒としての水溶性非揮発性有機溶媒の例としてはプ
ロピレングリコール％Ｉ、３−ブタンジオール等の多価
アルコール類の単独又は混合物、貧溶媒としての水溶性
非揮発性有−１〇−
機溶媒の例としてはグリセリン、ポリエチレングリコー
ル４００、Ｄ−ソルビト−ル等の多価アルコール類の単
独又は混合物が挙げられる。これら有機溶媒の混合比率
及び使用量は所望の粒子径に依存するが、好ましくは良
溶媒：貧溶媒＝１：０〜０．２ ： ０．８、使用量は
リボソーム膜構成物質１重量部に対し約０〜２重量部程
度となるようにするのが望ましい。Examples of water-soluble non-volatile organic solvents as good solvents for the ribosomal membrane constituents used in the method of the present invention include propylene glycol%I, polyhydric alcohols such as 3-butanediol alone or in mixtures, and as poor solvents. Examples of water-soluble non-volatile organic solvents include polyhydric alcohols such as glycerin, polyethylene glycol 400, and D-sorbitol, alone or in mixtures. The mixing ratio and usage amount of these organic solvents depend on the desired particle size, but preferably good solvent: poor solvent = 1:0 to 0.2: 0.8, and the usage amount is based on 1 part by weight of the ribosome membrane constituent material. It is desirable that the amount is about 0 to 2 parts by weight.
また、リボソーム膜構成物質と上記多価アルコールの組
み合わせにより形成されたラメラ液晶相を膨潤させた、
いわゆる膨潤ラメラ液晶を形成させる為の水性溶液とし
ては、水、緩衝液、生理食塩水、糖水溶液及びこれらの
混合液などが用いられるが、分子間力が大きく、水分散
性が非常に悪いスフィンゴミエリン等を用いる場合、主
要膜構成成分との相互作用の強い内包有効成分を用いる
場合、あるいは膜構造を強固にする膜補強成分を多量に
用いる場合（主要膜構成成分１重量部に対し当該膜補強
成分を約０．３重量部以上用いる場合等）には、膨潤ラ
メラ液晶を形成させる為の水性溶液としては約３０％以
上の高濃度糖水溶液を用いるのが望ましい。この際に用
いる糖の例としては、ゲルコール、マンノース、ソルビ
トール、シュクロース、マルｉ・−ス、ｌ−レバロース
等単糖、二糖類等の単独又は混合物が挙げられる。膨潤
ラメラ液晶を形成させる為の水性溶液の使用量は、良溶
媒／貧溶媒の比率によるが、リボソーム膜構成物質−多
価アルコール液晶１重量部に対して約０〜２重量部が望
ましい。また、この膨潤ラメラ液晶を更に希釈する為の
水性溶液としては、水、ｐＨ緩衝液、生理食塩水、糖水
溶液及びこれらの混合いずれでも良い。In addition, the lamellar liquid crystal phase formed by the combination of the ribosomal membrane constituent material and the polyhydric alcohol is swollen.
Water, buffer solutions, physiological saline, aqueous sugar solutions, and mixtures of these are used as aqueous solutions to form so-called swollen lamellar liquid crystals, but sphingols have large intermolecular forces and have very poor water dispersibility. When using myelin, etc., when using an encapsulated active ingredient that has a strong interaction with the main membrane constituents, or when using a large amount of membrane reinforcing ingredients that strengthen the membrane structure (per 1 part by weight of the main membrane constituents) When the reinforcing component is used in an amount of about 0.3 parts by weight or more), it is desirable to use a high concentration sugar aqueous solution of about 30% or more as the aqueous solution for forming the swollen lamellar liquid crystal. Examples of sugars used in this case include monosaccharides and disaccharides such as gelcole, mannose, sorbitol, sucrose, malice, l-levalose, etc., singly or in mixtures. The amount of the aqueous solution used to form the swollen lamellar liquid crystal depends on the ratio of good solvent/poor solvent, but is preferably about 0 to 2 parts by weight per 1 part by weight of the ribosome membrane constituent material-polyhydric alcohol liquid crystal. The aqueous solution for further diluting the swollen lamellar liquid crystal may be water, pH buffer, physiological saline, aqueous sugar solution, or a mixture thereof.
ここで、主要膜構成成分と良溶媒の混合系にステロール
類等の膜補強成分を添加して均一溶液とする為には、約
８０°Ｃまで加温、撹拌することにより、容易に混合せ
しめることが可能であり、そこにグリセリン等の貧溶媒
を加えて、いったんラメラ液晶を形成させてしまえば、
もはやそのように加温して用いる必要はない。Here, in order to add membrane reinforcing components such as sterols to the mixed system of the main membrane components and a good solvent and make a homogeneous solution, it is possible to easily mix them by heating to about 80°C and stirring. Once a lamellar liquid crystal is formed by adding a poor solvent such as glycerin,
It is no longer necessary to heat the product in this way.
本発明に係る微細リボソーム調製法における温度は、一
般に脂質の相転移温度（Ｔｃ）Ｕ上で行った方が効率は
良く、従って、有効成分の薬剤を内包化する場合におい
ても、薬剤を含有する水性溶液はあらかじめＴｃ以上に
しておくことが望ましい。また、本発明にかかるリボソ
ーム調製法において、リボソームへの薬剤の保持効率を
アップさせるには、保持させる薬剤をできるだけ少量の
水性溶液に溶かし込み、これをラメラ液晶相内に封じ込
める（または薬剤を水性溶液にとかさず、直接ラメラ液
晶相内に（練り込められれば、より良い）ことが重要で
ある。The temperature in the microribosome preparation method according to the present invention is generally more efficient when carried out above the phase transition temperature (Tc) U of lipids. Therefore, even when encapsulating a drug as an active ingredient, It is desirable to prepare the aqueous solution at a temperature higher than Tc in advance. In addition, in the ribosome preparation method according to the present invention, in order to increase the efficiency of drug retention in ribosomes, the drug to be retained is dissolved in as small an aqueous solution as possible, and this is sealed within the lamellar liquid crystal phase (or the drug is dissolved in an aqueous solution). It is important to not dissolve it in a solution, but to directly incorporate it into the lamellar liquid crystal phase (it is better if it is incorporated).
本発明の微細なリボソーム製剤に保持させる医薬品用薬
剤としては、用いる水溶性非揮発性有機溶媒、水性溶液
に溶解するものなら特に制限はないが、塩酸エチレフリ
ン、アミノフィリンに代表される強心剤、シチコリン、
塩酸グロプラノロール等の循環器増強剤、カルボクロメ
ン等の血管拡張剤、デキストラン硫酸等の動脈硬化剤、
塩酸フェニレフリン等の血管収縮剤、チアミラールナト
リウム、ペンＩ・バルビタ〜ルナトリウム、塩酸ケタミ
ン等の全身麻酔剤、塩酸ヒロカルビン、塩酸カルテオロ
ール、マレイン酸チモロール等の緑内障剤等が用いられ
る。The pharmaceutical agent to be retained in the fine ribosomal preparation of the present invention is not particularly limited as long as it dissolves in the water-soluble non-volatile organic solvent used and the aqueous solution.
Cardiovascular enhancers such as glopranolol hydrochloride, vasodilators such as carbochromene, arteriosclerotic agents such as dextran sulfate,
Vasoconstrictors such as phenylephrine hydrochloride, general anesthetics such as thiamylal sodium, pen I barbital sodium, ketamine hydrochloride, and glaucoma agents such as hylocarbin hydrochloride, carteolol hydrochloride, and timolol maleate are used.
本発明に係る微細なリボソームの調製法において製剤に
保持される化粧料用薬剤としては、用いる水溶性非揮発
性有機溶媒、水性溶液に溶解するものなら特に制限はな
いが、アスコルビン酸フォスフエイト、インフェルラ酸
すトリウムなどの美白剤、グリチルリチン酸カリウムな
どの抗炎症剤、ヒアルロン酸ナトリウム、コンドロイチ
ン硫酸などの保湿剤等が用いられる。In the method for preparing fine ribosomes according to the present invention, there are no particular restrictions on the cosmetic agents retained in the formulation as long as they are soluble in the water-soluble non-volatile organic solvent used and the aqueous solution, but include ascorbic acid phosphate, infer Whitening agents such as thorium lactate, anti-inflammatory agents such as potassium glycyrrhizinate, and moisturizing agents such as sodium hyaluronate and chondroitin sulfate are used.
このようにして薬剤を保持した均一粒径のリボソーム製
剤が再現性良くしかも大量に得られるが、このリボソー
ム製剤はこのまま使用しても良く、また透析、ゲルろ過
、遠心分離等の手段によりリボソームに保持されなかっ
た薬剤を分離除去して使用しても良い。In this way, a drug-retaining ribosome preparation with a uniform particle size can be obtained with good reproducibility and in large quantities.This ribosome preparation can be used as is, or it can be separated into ribosomes by means such as dialysis, gel filtration, or centrifugation. The unretained drug may be separated and removed for use.
次に実施例により本発明の具体化例を示すが、これらの
実施例は何ら本発明を限定するものではない。Next, specific examples of the present invention will be shown through Examples, but these Examples are not intended to limit the present invention in any way.
実施例１
微細リボソームの調製
プロピレングリコール４．５ｇを７５℃に加温し、これ
に卵黄レシチン（純度９５％） ０．６ｇ、コレステロ
ール０．３ｇ、ホスファチジルセリンＯ，１ｇを加え撹
拌し、均一な溶液とした。次に同一温度条件でグリセリ
ン４．５ｇを加えて液晶ゲルを調製した。これにｌｏｇ
の５０％グルコース／２０ｍＭトリス塩酸緩衝液（ｐＨ
７，４）を撹拌しながら１滴ずつ滴下して、膨潤ラメラ
液晶を形成させた。更に９０ｇの２０ｍＭ トリス塩酸
緩衝液（ｐＨ７，４）を撹拌しながら滴下していき、透
明なリボソーム溶液を得た。このリボソームの粒子径を
コールタ−・カウンター（モデ、ルＮ４ＭＤ）により測
定したところ、１１０２ｎの均一粒子分布のリボソーム
であることがわかった。一方、膨潤ラメラ液晶を形成さ
せず、−気にｂｕｆｆｅｒを加えて、撹拌した場合には
、３１Ｏｎｍと６０Ｑｎｍにピークを持つ、不均一なリ
ボソームとなった。Example 1 Preparation of fine ribosomes 4.5 g of propylene glycol was heated to 75°C, and 0.6 g of egg yolk lecithin (95% purity), 0.3 g of cholesterol, and 1 g of phosphatidylserine O were added and stirred to form a homogeneous It was made into a solution. Next, 4.5 g of glycerin was added under the same temperature conditions to prepare a liquid crystal gel. log to this
of 50% glucose/20mM Tris-HCl buffer (pH
7, 4) was added drop by drop with stirring to form a swollen lamellar liquid crystal. Furthermore, 90 g of 20 mM Tris-HCl buffer (pH 7.4) was added dropwise with stirring to obtain a transparent ribosome solution. When the particle diameter of this ribosome was measured using a Coulter counter (Model N4MD), it was found that the ribosome had a uniform particle distribution of 1102 nm. On the other hand, when a swollen lamellar liquid crystal was not formed and a buffer was added and stirred, non-uniform ribosomes with peaks at 31 Onm and 60 Q nm were obtained.
実施例２
目薬用微細リボソームの調製
プロピレングリコール６ｇを７５°Ｃに加温し、これに
卵黄レシチン０．６ｇ、コレステロール０．３ｇ、ステ
アリルアミン０．ｈを加え、撹拌し、均一溶液とした。Example 2 Preparation of fine ribosomes for eye drops 6 g of propylene glycol was heated to 75°C, and to this was added 0.6 g of egg yolk lecithin, 0.3 g of cholesterol, and 0.0 g of stearylamine. h was added and stirred to obtain a homogeneous solution.
次に同一温度条件でグリセリン３ｇを加えて、ｌＯ％上
記膜成分の３３．３％グリセリン・プロピレングリコー
ルの液晶ゲルを調製した。この液晶ゲル１ｇをとり、緑
内障側塩酸ピロカルピン０．１ｇを添加し、７５°Ｃに
てよく混合し、これに２０ｍＭ トリス塩酸緩衝液（ｐ
ｌ’１７．４） １ ｇを撹拌しながら１滴ずつ滴下し
、更に同一のｂｕｆｆｅｒ７．９ｇを撹拌しながら滴下
して、透明なリボソーム溶液を得た。このリボソーム溶
液をセファデックスＧ−５０ゲルカラム（ファルマシア
製）で外相の塩酸ピロカルピンを除去した。次いで、リ
ボソーム内に保持された塩酸ピロカルピンを高速液体ク
ロマトグラフィーにより測定したところ、保持効率は２
０．４％であった。このリボソームの粒子径は１３２ｎ
ｍであった。一方、同一の系＝１５−
をイクストルーダー（Ｅｘｔｒｕｄｅｒ）にてリボソー
ムを調製したところ、粒子径はＩ １２ｎｍで、保持効
率は５．３％にすぎなかった。Next, 3 g of glycerin was added under the same temperature conditions to prepare a liquid crystal gel of 33.3% glycerin/propylene glycol in 10% of the above membrane components. Take 1 g of this liquid crystal gel, add 0.1 g of pilocarpine hydrochloride on the glaucoma side, mix well at 75°C, and add 20 mM Tris-HCl buffer (p
l'17.4) 1 g was added dropwise one by one while stirring, and 7.9 g of the same buffer was further added dropwise while stirring to obtain a transparent ribosome solution. Pilocarpine hydrochloride in the external phase was removed from this ribosome solution using a Sephadex G-50 gel column (manufactured by Pharmacia). Next, when pilocarpine hydrochloride retained in ribosomes was measured by high performance liquid chromatography, the retention efficiency was 2.
It was 0.4%. The particle size of this ribosome is 132n
It was m. On the other hand, when ribosomes were prepared from the same system = 15- using an extruder, the particle diameter was 12 nm and the retention efficiency was only 5.3%.
実施例３
医療用微細リボソームの調製
１．３−ブチレングリコール３ｇを８０°Ｃに加温し、
これにＤＰＰＣ（純度９８％） Ｏ，ｈｔ、コレスタノ
ール０．３ｇを加え、撹拌し、均一溶液とした。次に同
一温度条件でポリエチレングリコール４００ ４ｇを加
えて、１２，５％上記膜成分の５７．１％ポリエヂレン
グリコール４００・１．３− フチレンゲリコールの液
晶ゲルを調製した。この液晶ゲル１ｇをとり、血管拡張
剤カルボクロメン０．１ｇを添加し、８０ ’Ｏにてよ
く混合し、これに２０＋ｎＭｌ−リス塩酸５ａｌｉｎｅ
ｂｕｆｆｅｒ（ｐＨ７，４） ｌ ９を撹拌しながら
１滴ずつ滴下し、更に同一のｂｕｆｆｅｒ ７．ｈを撹
拌しながら滴下して、澄明なりポソーム溶液を得た。Example 3 Preparation of medical microribosomes 1. 3 g of 3-butylene glycol was heated to 80°C,
To this, DPPC (purity 98%) O,ht and 0.3 g of cholestanol were added and stirred to form a homogeneous solution. Next, 4 g of polyethylene glycol 400 was added under the same temperature conditions to prepare a liquid crystal gel of 57.1% polyethylene glycol 400.1.3-phthylene gelicol, which was 12.5% of the above film component. Take 1 g of this liquid crystal gel, add 0.1 g of the vasodilator carbochromene, mix well at 80'O, and add 20+nMl-lis-hydrochloric acid 5aline.
Buffer (pH 7,4) l 9 was added drop by drop with stirring, and then added to the same buffer 7. h was added dropwise with stirring to obtain a clear posome solution.
このリボソーム溶液をセファデックスＧ−５０ゲルカラ
ムで外相のカルボクロメンを除去し、リボソーム内のカ
ルボクロメンを高速液体クロマトグラフイーにより測定
したところ、保持効率は、１１．１％であった（粒子径
１５２旧ｎ）。一方、同一の系を旧ｃｒｏｆ １ｕｉｄ
ｉｚｅｒにてリボソームを調製したところ、粒子径はｌ
３０ ｎ ｍで保持効率は２．２％にすぎず、同様に
、ラメラ液晶を用いず、後でカルボクロメンｂｕｆｆｅ
ｒ溶液を添加した場合には、１．８％にすぎなかった。The carbochromene in the external phase of this ribosome solution was removed using a Sephadex G-50 gel column, and the carbochromene in the ribosome was measured by high performance liquid chromatography, and the retention efficiency was 11.1% (particle size 152 Old n). On the other hand, the same system is the old crof 1uid
When ribosomes were prepared using an iser, the particle size was 1
At 30 nm, the retention efficiency was only 2.2%, and similarly, without using lamellar liquid crystals and later with carbochromene buffe.
When r solution was added, it was only 1.8%.
実施例４
医薬用微細リボソームの調製
プロピレングリコール６ｇを７５°Ｃに加温し、これに
牛脳スフィンゴミエリン０．（３ｇ、コレステロール０
．３ｇ、ホスファチジルグリセロール０，１ｇを加え、
撹拌し、均一溶液とした。次に同一温度条件で、グリセ
リン６ｇを加えて、７．７％上記膜成分の５０％グリセ
リン・プロピレングリコールの液晶ゲルを調製した。こ
の液晶ゲルＩｇをとり、動脈硬化剤デキストラン硫酸帆
１ｇを添加し、７５℃にてよく混合し、これに１ｇの５
０％シュクロース／２０ｍＭトリス塩酸緩衝液（ｐＨ７
，４）を撹拌しながら１滴ずつ滴下して、更に５．６９
の２０ｍＭｌ・リス塩酸緩衝液を撹拌しながら滴下して
、澄明なりボソーム溶液を得た。このリボソームを実施
例３と同様に分離処理して、リボソーム内のデキストラ
ン硫酸を高速液体クロマトグラフィーにより測定したと
ころ、保持効率は９．９％であった（粒子ザイズｌ１８
ｎｍ）。Example 4 Preparation of fine ribosomes for pharmaceutical use 6 g of propylene glycol was heated to 75°C, and 0.5 g of bovine brain sphingomyelin was added to it. (3g, cholesterol 0
．． Add 3g and 0.1g of phosphatidylglycerol,
Stir to obtain a homogeneous solution. Next, under the same temperature conditions, 6 g of glycerin was added to prepare a liquid crystal gel of 7.7% glycerin/propylene glycol containing 50% of the above membrane components. Take this liquid crystal gel Ig, add 1 g of arteriosclerotic agent dextran sulfate, mix well at 75°C, and add 1 g of 5
0% sucrose/20mM Tris-HCl buffer (pH 7)
, 4) one drop at a time while stirring, and then add 5.69
20 mM of Lis-HCl buffer was added dropwise with stirring to obtain a clear bosome solution. This ribosome was separated in the same manner as in Example 3, and dextran sulfate in the ribosome was measured by high performance liquid chromatography, and the retention efficiency was 9.9% (particle size 18
nm).
一方、同一の系をラメラ液晶を形成させずにリボソーム
を調製したところ、その粒子径は１１０００ｎ以上でし
かも不均一であり、数時間後には沈澱を生じてしまった
。On the other hand, when ribosomes were prepared from the same system without forming lamellar liquid crystals, the particle size was more than 11,000 nm and non-uniform, and precipitation occurred after several hours.
実施例５
化粧料用微細リボソームの調製
アスコルビン酸ホスフェイト含有美自リボソーム化粧料
プロピレングリコール４．５ｇを７５℃に加温し、これ
に卵黄レシチン０．（ｉｇ、コレステロール０．３ｇ、
ジセチルリン酸帆１ｇを加え、撹拌し、均一溶液とした
。次に同一温度条件で、グリセリン４．５ｇを加えて、
ｌＯ％上記膜成分の５０％グリセリン・プロピレングリ
コールの液晶ゲルを調製した。Example 5 Preparation of fine ribosomes for cosmetics 4.5g of ascorbic acid phosphate-containing beauty ribosome cosmetics propylene glycol was heated to 75°C, and 0.5g of egg yolk lecithin was added to it. (ig, cholesterol 0.3g,
1 g of dicetyl phosphate was added and stirred to form a homogeneous solution. Next, under the same temperature conditions, add 4.5 g of glycerin.
A liquid crystal gel containing 50% glycerin/propylene glycol of the above membrane components was prepared.
この液晶ゲル１９をとり、０．３ｇのアスコルビン酸ホ
スフェイトを添加し、４０℃にてよく混合し、混合しな
がら、これに０．５ｇの５０％グルコース水溶液を添加
し、更に８．２ｇの水を滴下して、澄明なりポソーム溶
液を得た。このリボソームを実施例３と同様に分離処理
して、アスコルビン酸ホスフェイトを高速液体クロマト
グラフィーにて測定したところ、保持効率は１８．７％
（粒子ザイズ１４１ｎｍ）であった。Take this liquid crystal gel 19, add 0.3 g of ascorbic acid phosphate, mix well at 40°C, add 0.5 g of 50% glucose aqueous solution to this while mixing, and add 8.2 g of water. was added dropwise to obtain a clear posome solution. When this ribosome was separated in the same manner as in Example 3 and ascorbic acid phosphate was measured by high performance liquid chromatography, the retention efficiency was 18.7%.
(Particle size: 141 nm).
既知のリボソーム調製法に比べて本発明法が優れている
点としては以下のことが挙げられる。The advantages of the method of the present invention over known ribosome preparation methods include the following.
■、良溶媒と貧溶媒の比率を変えることにより任意のほ
ぼ均一な粒子径のリボソームを得ることができる。(2) By changing the ratio of good and poor solvents, ribosomes with any substantially uniform particle size can be obtained.
２、膜補強成分を添加しても、貧溶媒の添加により室温
付近でもラメラ液晶を形成させることができるので、高
温に加熱する必要がない。2. Even if a film reinforcing component is added, lamellar liquid crystals can be formed even at room temperature by adding a poor solvent, so there is no need to heat the film to high temperatures.
３、 ラメラ液晶相に大量の薬剤を封入できるので、他
の方法に比べて保持効率が高い。3. Since a large amount of drug can be encapsulated in the lamellar liquid crystal phase, the retention efficiency is higher than other methods.
４、 スフィンゴミエリン等の分子間力の大きい脂質で
も、１１００ｎ前後のリボソームを得ることができる。4. Even with lipids with large intermolecular forces such as sphingomyelin, ribosomes of around 1100n can be obtained.
５、 ピークの均質な粒子径のリボソームが再現良く得
られる。5. Ribosomes with uniform particle size peaks can be obtained with good reproducibility.
６、大量生産が容易である。6. Easy to mass produce.
本発明方法により調製された微細リボソームは安定であ
り、例えば窒素ガス置換、冷蔵庫放置（５℃）で状態は
変化しない（調製直後の粒子径１１０２ｎが１年６ケ月
後１０１０５ｎ。The fine ribosomes prepared by the method of the present invention are stable, and their state does not change even when the ribosomes are replaced with nitrogen gas or left in the refrigerator (5°C) (the particle diameter immediately after preparation is 1102n, but after 1 year and 6 months it is 10105n).
また、抗酸化剤を添加した系では、室温でも安定であっ
た。Furthermore, the system containing an antioxidant was stable even at room temperature.
また、本発明方法により得られる微細リボソームを用い
ることにより、薬剤の経皮（あるいは経角膜）等の透過
性及び薬剤の血中安定性も非常に優れていた。例えば、
スフィンゴミエリン／コレステロール／ホスファチジル
グリセロール（６０／３０／１０）の系を用いて、カル
ボキシ７ルオロセインを内包させ、その血中動態を日本
内色種ウサギを用いて調べたところ、半減期は５時間以
上であり、リボソーム化しない場合に比べてのＡＵＧ（
ニアリア・アンダー・カーブ）も約２０倍であった。Furthermore, by using the fine ribosomes obtained by the method of the present invention, the transdermal (or transcorneal) permeability of the drug and the blood stability of the drug were also very excellent. for example,
Carboxy7-fluorosein was encapsulated using the sphingomyelin/cholesterol/phosphatidylglycerol (60/30/10) system, and its blood dynamics were investigated using Japanese colored rabbits, and the half-life was over 5 hours. , and the AUG (
(nearly under curve) was also approximately 20 times larger.
特許出願人 ポーラ化成工業株式会社Patent applicant: POLA CHEMICAL INDUSTRIES, INC.
Claims (1)
コールに溶解し、次いで貧溶媒である別の多価アルコー
ルを添加し、充分に混合することによりリボソーム膜構
成物質とそれらの多価アルコールから成るラメラ液晶を
形成させ、次に、これに水もしくはリボソーム膜構成物
質に応じて選択された適宜の物質の水溶液を用いて、こ
れをラメラ液晶が壊れないように、緩徐に滴下しながら
練り込むことにより膨潤したラメラ液晶を形成させ、次
いで上記の水又は水溶液と同一もしくは別の水溶液を用
いて、これを連続的に滴下することを特徴とする微細な
リボソームの調製法。 ２）医薬および／または化粧料を内包せしめた請求項１
）に記載の微細なリボソームの調製法。[Claims] 1) A ribosome membrane constituent substance is dissolved in a polyhydric alcohol which is a good solvent, and then another polyhydric alcohol which is a poor solvent is added and mixed thoroughly to dissolve the ribosome membrane constituent substance. Lamellar liquid crystals made of these polyhydric alcohols are formed, and then water or an aqueous solution of an appropriate substance selected depending on the ribosomal membrane constituent material is used to slowly dissolve the lamellar liquid crystals so that the lamellar liquid crystals do not break. A method for preparing fine ribosomes, which is characterized by forming a swollen lamellar liquid crystal by kneading it while dropping it, and then continuously dropping this using the same or different aqueous solution as the above water or aqueous solution. . 2) Claim 1 containing a medicine and/or cosmetics
) Preparation method for fine ribosomes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33045090A JPH04208216A (en) | 1990-11-30 | 1990-11-30 | Preparation of fine liposome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33045090A JPH04208216A (en) | 1990-11-30 | 1990-11-30 | Preparation of fine liposome |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04208216A true JPH04208216A (en) | 1992-07-29 |
Family
ID=18232757
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33045090A Pending JPH04208216A (en) | 1990-11-30 | 1990-11-30 | Preparation of fine liposome |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04208216A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000169327A (en) * | 1998-12-03 | 2000-06-20 | Pias Arise Kk | Low irritant preparation for external use for skin and bathing agent |
| KR100429384B1 (en) * | 2001-02-15 | 2004-04-29 | 한국콜마 주식회사 | Cosmetic product and its manufacturing method using water in oil in gel network(W/O/G) system |
| WO2011081136A1 (en) | 2009-12-28 | 2011-07-07 | 株式会社資生堂 | Cosmetic preparation |
| JP2014101294A (en) * | 2012-11-19 | 2014-06-05 | Pola Chem Ind Inc | O/w/o emulsifier-type skin external preparation |
-
1990
- 1990-11-30 JP JP33045090A patent/JPH04208216A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000169327A (en) * | 1998-12-03 | 2000-06-20 | Pias Arise Kk | Low irritant preparation for external use for skin and bathing agent |
| KR100429384B1 (en) * | 2001-02-15 | 2004-04-29 | 한국콜마 주식회사 | Cosmetic product and its manufacturing method using water in oil in gel network(W/O/G) system |
| WO2011081136A1 (en) | 2009-12-28 | 2011-07-07 | 株式会社資生堂 | Cosmetic preparation |
| JP2014101294A (en) * | 2012-11-19 | 2014-06-05 | Pola Chem Ind Inc | O/w/o emulsifier-type skin external preparation |
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