JPH02295917A - Liposome suppressing oxidation of internally capsulated substance and production thereof - Google Patents
Liposome suppressing oxidation of internally capsulated substance and production thereofInfo
- Publication number
- JPH02295917A JPH02295917A JP1118346A JP11834689A JPH02295917A JP H02295917 A JPH02295917 A JP H02295917A JP 1118346 A JP1118346 A JP 1118346A JP 11834689 A JP11834689 A JP 11834689A JP H02295917 A JPH02295917 A JP H02295917A
- Authority
- JP
- Japan
- Prior art keywords
- liposome
- vitamin
- weight
- membrane
- liposomes
- 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
- 239000002502 liposome Substances 0.000 title claims abstract description 72
- 230000003647 oxidation Effects 0.000 title claims abstract description 27
- 238000007254 oxidation reaction Methods 0.000 title claims abstract description 27
- 239000000126 substance Substances 0.000 title claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 title claims description 4
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 claims abstract description 79
- 229930003427 Vitamin E Natural products 0.000 claims abstract description 38
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000011709 vitamin E Substances 0.000 claims abstract description 38
- 235000019165 vitamin E Nutrition 0.000 claims abstract description 38
- 229940046009 vitamin E Drugs 0.000 claims abstract description 38
- 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 claims abstract description 36
- 239000012528 membrane Substances 0.000 claims abstract description 29
- 108010054147 Hemoglobins Proteins 0.000 claims abstract description 19
- 102000001554 Hemoglobins Human genes 0.000 claims abstract description 19
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 18
- 150000003904 phospholipids Chemical class 0.000 claims abstract description 17
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 9
- 239000013543 active substance Substances 0.000 claims description 10
- 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 claims description 8
- WGVKWNUPNGFDFJ-DQCZWYHMSA-N β-tocopherol Chemical compound OC1=CC(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C WGVKWNUPNGFDFJ-DQCZWYHMSA-N 0.000 claims description 6
- 229940087168 alpha tocopherol Drugs 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 229960000984 tocofersolan Drugs 0.000 claims description 5
- 239000002076 α-tocopherol Substances 0.000 claims description 5
- 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 claims description 4
- 150000003611 tocopherol derivatives Chemical class 0.000 claims description 4
- 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 claims description 3
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 claims description 3
- 229940066595 beta tocopherol Drugs 0.000 claims description 3
- 239000000470 constituent Substances 0.000 claims description 3
- RNPXCFINMKSQPQ-UHFFFAOYSA-N dicetyl hydrogen phosphate Chemical compound CCCCCCCCCCCCCCCCOP(O)(=O)OCCCCCCCCCCCCCCCC RNPXCFINMKSQPQ-UHFFFAOYSA-N 0.000 claims description 3
- 229940093541 dicetylphosphate Drugs 0.000 claims description 3
- 235000003441 saturated fatty acids Nutrition 0.000 claims description 3
- 150000004671 saturated fatty acids Chemical class 0.000 claims description 3
- 235000004835 α-tocopherol Nutrition 0.000 claims description 3
- 239000011590 β-tocopherol Substances 0.000 claims description 3
- 235000007680 β-tocopherol Nutrition 0.000 claims description 3
- 239000008346 aqueous phase Substances 0.000 claims description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 4
- 239000003960 organic solvent Substances 0.000 abstract description 3
- 230000000975 bioactive effect Effects 0.000 abstract 1
- 150000002632 lipids Chemical class 0.000 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 108010061951 Methemoglobin Proteins 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 229930003799 tocopherol Natural products 0.000 description 3
- 239000011732 tocopherol Substances 0.000 description 3
- 235000010384 tocopherol Nutrition 0.000 description 3
- 229960001295 tocopherol Drugs 0.000 description 3
- 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 2
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 description 2
- 235000021360 Myristic acid Nutrition 0.000 description 2
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 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 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- 108010036302 hemoglobin AS Proteins 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940079826 hydrogen sulfite Drugs 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000000936 membranestabilizing effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000021003 saturated fats Nutrition 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- -1 tocopherol ethers Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Dispersion Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Manufacturing Of Micro-Capsules (AREA)
Abstract
Description
（産業上の利用分野）
本発明は、リポソームに関するものである。さらに詳し
くは、内部に封入される薬剤やヘモグロビン等の生理活
性物質の酸化を抑制したリポソームに関するものである
。
（従来の技術）
リポソームを薬剤、あるいはタンパク質、ビタミン、ウ
ィルス、塩類、ホルモンなどの生理活性物質の担体とし
て利用する試みが広く行われている。 また、リポソー
ム内部に生理活性物質としてヘモグロビンを封入し、こ
れを人工赤血球として利用することも広く検討されてい
る。
リポソームを薬物や生理活性物質等の運搬体として利用
する場合、リポソームを生体内に投与しその内水相中に
存在する薬剤や生理活性物質が有効に作用するためには
、生体温度（３６〜３９℃）において長時間その薬効お
よび生理活性機能を維持させなければならない。生体温
度において、酸化によりその活性が失効しやすい薬剤お
よび生理活性物質をリポソーム内に封入する場合には、
その酸化を防止する手段が必要となる。
特に封入される物質がヘモグロビンである場合には、ヘ
モグロビンが酸化されメトヘモグロビンになってしまう
と酸素運搬機能を失うことから、ヘモグロビンの酸化防
止は非常に重要な問題であった。このため、従来のリポ
ソーム型人工赤血球は、酸化を抑制するために、通常、
メト化抑制剤（酸化防止剤もしくは安定化剤）をその製
造過程で添加している。
メト化抑制剤としては、例えば、亜硫酸ナトリウム、亜
硫酸水素す）ＩＪウム、硫酸第一鉄等の無機物質が知ら
れている。これらは、抗酸化作用は確実であるが、生体
に対する毒性も強く、臨床で使用するには好ましくない
。さらに、体内に存在するアスコルビン酸（ビタミンＣ
）、還元型グルタチオン等は、生体認容性の高い酸化抑
制剤として知られているが、抑制効率、および効果の持
続時間または安定性に問題があり、酸化抑制剤としては
不十分なものであった。
一方、リポソームに内包された薬剤、ヘモグロビン等の
酸化は、リポソーム膜を構成する脂質の酸化に起因する
との考えから、膜自体の酸化を抑えるために、膜構成成
分に対して含有量にして１〜２重量％程度のビタミンＥ
を添加して膜の酸化を抑え、これによって内包物の酸化
を抑制する技術が知られているが、この方法によっては
実際に内包物の酸化を抑制することはほとんど不可能で
あった。
（発明が解決しようとする課題）
上述のように酸化抑制剤として使用されるビタミンＥは
、脂溶性物質であるため、不飽和脂質の過酸化反応は効
率よく抑制する。このため不飽和脂質自体の酸化を抑制
するためには上述のような添加率で十分であったが、内
包物の酸化を充分に抑制することはほとんど期待できな
い。また膜成分として飽和脂質を用いた場合には、上述
のような添加量ではビタミンＥを添加する意味はほとん
どなかった。また従来のビタミンＥ添加リポソームは、
生体温度よりかなり低い温度条件下（例えば４℃）にお
いては、内包物の酸化をある程度抑制するものの、生体
温度条件下においては、内包物の酸化を抑制する効果は
ほとんど期待できなかっｔこ 。
本発明者はこれらの技術に鑑み、膜脂質にビタミンＥを
大量に含有させることにより、内包物の酸化を直接に、
しかも生体温度条件下にて抑制することが可能になると
いう事実を新たに知見し、本発明を完成させたものであ
る。
（課題を解決するための手段）
すなわち上記の課題を解決するものは、リポソーム膜中
に、ビタミンＥを含有するリポソームであって、該膜中
のビタミンＥの含有量が２〜４０重量％であるリポソー
ムである。
さらに上記の課題を解決するものは、リポソーム膜中に
リン脂質、コレステロールおよびビタミンＥを含有する
リポソームであって、前記リン脂質に対するコレステロ
ールの含有量が１０〜５０重量％であるリポソームであ
る。
またビタミンＥとしてα−トコフェロール、β−トコフ
ェロール、トコフェロール誘導体ノウチ少なくとも１つ
を含有することが好適である。
リポソーム膜中には、さらに膜電荷物質とじて高級飽和
脂肪ａ、フオスファチジン酸、フオスファチジルグリセ
ロール、ジセチルフオスフエートのうちの少なくとも１
つを含有することが好ましい。
リポソーム内水相中に、酸化を受けやすい薬物または生
理活性物質を含有してなる場合に本発明のリポソームは
その効果を特に発揮する。 例えば、生理活性物質はヘ
モグロビンである。 さらに上記課題を解決するリポソ
ームは、リポソーム膜構成成分にビタミンＥを含有量に
して５〜２００重量％混合し、得られた混合物を用いて
リポソームを形成することによって得られる。
さらに上記課題を解決するリポソームは、リン脂質を含
有するリポソーム膜構成成分に、含有量にして５〜２０
０重量％のビタミンＥと、前記リン脂質に対する含有量
にして１０〜５０重量％のコレステロールを混合し、得
られた混合物を用いてリポソームを形成することによっ
て得られる。
さらに本発明に係るリポソームは、リポソーム膜中にリ
ン脂質、コレステロールおよびビタミンＥを含有するリ
ポソームであって、前記リン脂質に対するコレステロー
ルの含有量が１０〜５０重量％であるリポソームである
。
本発明におけるリポソーム膜形成脂質は特に制限はなく
、リポソームを形成するものであれば天然または合成の
脂質が使用可能であるが、特に飽和のリン脂質が好適に
使用される。その例としては、レシチン（ホスファチジ
ルコリン）、ホスファチジルエタノールアミン
スファチジルセリン、ホスファチジルイシノトール、ホ
スファチジルグリセロール、スフィンゴミエリンあるい
はこれらを常法に従って水素添加したものが挙げられ、
これらを組み合わせて用いることもできる。
膜中には、内包物の酸化を抑制するためにビタミンＥが
添加される。ビタミンＥとしては、α−トコフェロール
、β−トコフェロール、マタハトコフェロール誘導体（
トコフェロールエステル、トコフェロールエーテル）が
使用できるが、特にＱ−ａ−トコフェロール、ｄ４−σ
ートコフェロール、Ｑ−σートコフェロールｉ［、ｄ１
２−α−トコフェロール酢酸が好ましい。ビタミンＥの
添加量としては、２〜４０重量％が好適である。添加量
が４０重量％より大きいと、それ以上含有しても効果が
上がらなくなり、一方２重量％より小さいと膜脂質の酸
化は十分抑制できるものの、内包物の酸化を抑制するに
は不十分になる。
本発明において膜安定物質として添加されるコレステロ
ールの添加量は、リン脂質に対する含有量が１０〜５０
重量％が好ましく、特に好ましいのは１０〜３０重量％
である。含有量で５０重量％を越える場合．には、内包
物の酸化を直接、および生体温度条件下にて抑制するＩ
；めに必要とされるビタミンＥの含有量を保持すること
が難しくなる。一方ｌＯ重量％を下回る場合には、十分
な膜安定作用が得られないこととなる。
また膜構成成分には、所望によって高級飽和脂肪酸、フ
ォスファチジン酸、７オスフアチジルグリセロール、ジ
セチル７オス７エート等の膜荷電物質を添加してもよい
。
リポソーム内に取り込まれるヘモグロビンは、赤血球を
常法にしたがって溶血し、分−分子量５万の膜を使用し
た限外ろ過により濃縮したものが使用される。
本発明のリポソームは、リン脂質を含有するリポソーム
膜構成成分にビタミンＥの含有量が５〜２００重量％、
リン脂質の含有量が１０〜５０重量％のコレステロール
を添加し、クロロホルム、エタノール等の有機溶媒に溶
解し、得られた溶液から溶媒をエバポレーション、凍結
乾燥、スプレードライ法等の方法により留去し、残留物
に上記２のヘモグロビン含有水溶液を加え、該水溶液を
振とうまたは超音波処理により均一に懸濁し、この懸濁
液を用いてリポソームを形成させることによって得られ
る。ビタミンＥが５重量％以下であると、内包物の酸化
を抑制する効果が不十分となり、２００重量％以上添加
しても含有率は°上昇しないからである。懸濁液をリポ
ソームに調整するには、フレンチプレス法、超音波法、
逆相法等種々公知の方法がとられ得る。
得られたリポソームの脂質中にはビタミンＥが含有され
ているが、その含有量は必ずしも最初に添加した仕込み
量と同一ではない。脂質中に含有されなかったビタミン
Ｅはリポソーム洗浄工程において除去される。得られた
リポソーム中のビタミンＥの含有率は、リポソーム溶液
中より有機溶媒にてビタミンＥを抽出し、紫外吸収する
ことにより算出できる。
次に実施例および比較例を示して本発明をさらに詳細に
説明する。(Industrial Application Field) The present invention relates to liposomes. More specifically, the present invention relates to liposomes in which oxidation of physiologically active substances such as drugs and hemoglobin encapsulated therein is suppressed. (Prior Art) Attempts have been made widely to utilize liposomes as carriers of drugs or physiologically active substances such as proteins, vitamins, viruses, salts, and hormones. Furthermore, encapsulating hemoglobin as a physiologically active substance inside liposomes and using this as an artificial red blood cell is also widely considered. When liposomes are used as carriers for drugs, physiologically active substances, etc., the biological temperature (36 to It is necessary to maintain its medicinal efficacy and physiologically active function for a long time at 39°C. When encapsulating in liposomes drugs and physiologically active substances whose activity is likely to be lost due to oxidation at biological temperatures,
A means to prevent the oxidation is required. In particular, when the substance to be encapsulated is hemoglobin, preventing hemoglobin from oxidizing has been a very important issue because if hemoglobin is oxidized to methemoglobin, it loses its oxygen transport function. For this reason, conventional liposome-type artificial red blood cells usually require
Memethization inhibitors (antioxidants or stabilizers) are added during the manufacturing process. Inorganic substances such as sodium sulfite, hydrogen sulfite, ferrous sulfate, and the like are known as memethization inhibitors. Although these have certain antioxidant effects, they are also highly toxic to living organisms and are not preferred for clinical use. Furthermore, ascorbic acid (vitamin C
), reduced glutathione, etc. are known as oxidation inhibitors with high biotolerance, but they have problems with inhibition efficiency, duration of effect, and stability, and are insufficient as oxidation inhibitors. Ta. On the other hand, it is believed that the oxidation of drugs, hemoglobin, etc. encapsulated in liposomes is caused by the oxidation of the lipids that make up the liposome membrane. ~2% by weight of vitamin E
Although there is a known technique for suppressing the oxidation of the film by adding , thereby suppressing the oxidation of the inclusions, it has been almost impossible to actually suppress the oxidation of the inclusions using this method. (Problems to be Solved by the Invention) As mentioned above, vitamin E, which is used as an oxidation inhibitor, is a fat-soluble substance and therefore efficiently suppresses the peroxidation reaction of unsaturated lipids. Therefore, although the above-mentioned addition rate was sufficient to suppress the oxidation of the unsaturated lipid itself, it is hardly expected to sufficiently suppress the oxidation of the inclusions. Furthermore, when saturated lipids were used as membrane components, there was almost no point in adding vitamin E at the above-mentioned addition amount. In addition, conventional vitamin E-added liposomes
Although the oxidation of inclusions is suppressed to some extent under temperature conditions considerably lower than the biological temperature (for example, 4°C), little effect in suppressing the oxidation of inclusions can be expected under biological temperature conditions. In view of these techniques, the present inventor has made it possible to directly prevent the oxidation of inclusions by incorporating a large amount of vitamin E into membrane lipids.
Moreover, the present invention was completed based on the new finding that it is possible to suppress the effect under biological temperature conditions. (Means for solving the problem) That is, what solves the above problem is a liposome containing vitamin E in the liposome membrane, and the content of vitamin E in the membrane is 2 to 40% by weight. It is a liposome. Furthermore, what solves the above-mentioned problem is a liposome containing phospholipid, cholesterol, and vitamin E in the liposome membrane, and the content of cholesterol relative to the phospholipid is 10 to 50% by weight. Further, it is preferable to contain at least one of α-tocopherol, β-tocopherol, and tocopherol derivative Nouchi as vitamin E. The liposome membrane further contains at least one of higher saturated fat a, phosphatidic acid, phosphatidylglycerol, and dicetyl phosphate as a membrane-charged substance.
It is preferable to contain one. The liposome of the present invention particularly exhibits its effects when the aqueous phase within the liposome contains a drug or physiologically active substance that is susceptible to oxidation. For example, the physiologically active substance is hemoglobin. Furthermore, a liposome that solves the above problems can be obtained by mixing 5 to 200% by weight of vitamin E with liposome membrane constituents and forming a liposome using the resulting mixture. Furthermore, the liposome that solves the above problems has a content of 5 to 20% in the liposome membrane component containing phospholipids.
It is obtained by mixing 0% by weight of vitamin E and 10 to 50% by weight of cholesterol based on the phospholipid, and forming a liposome using the resulting mixture. Furthermore, the liposome according to the present invention is a liposome containing phospholipid, cholesterol, and vitamin E in the liposome membrane, and the content of cholesterol relative to the phospholipid is 10 to 50% by weight. The liposome membrane-forming lipid in the present invention is not particularly limited, and natural or synthetic lipids can be used as long as they form liposomes, but saturated phospholipids are particularly preferably used. Examples include lecithin (phosphatidylcholine), phosphatidylethanolamine sphatidylserine, phosphatidylisinotole, phosphatidylglycerol, sphingomyelin, or hydrogenated products of these according to conventional methods.
These can also be used in combination. Vitamin E is added to the membrane to suppress oxidation of inclusions. Vitamin E includes α-tocopherol, β-tocopherol, and Matacha tocopherol derivatives (
tocopherol esters, tocopherol ethers) can be used, in particular Q-a-tocopherol, d4-σ
tocopherol, Q-σ tocopherol i[, d1
2-α-tocopherol acetic acid is preferred. The amount of vitamin E added is preferably 2 to 40% by weight. If the amount added is more than 40% by weight, the effect will not increase even if it is added more than that, while if it is less than 2% by weight, the oxidation of membrane lipids can be sufficiently suppressed, but it will not be enough to suppress the oxidation of inclusions. Become. In the present invention, the amount of cholesterol added as a membrane stabilizing substance is 10 to 50% relative to phospholipids.
% by weight is preferred, particularly preferably 10-30% by weight.
It is. If the content exceeds 50% by weight. contains I which suppresses the oxidation of inclusions directly and under biological temperature conditions.
; it becomes difficult to maintain the vitamin E content required for the body. On the other hand, if it is less than 10% by weight, a sufficient film stabilizing effect cannot be obtained. Further, membrane-charged substances such as higher saturated fatty acids, phosphatidic acid, 7-osphatidylglycerol, and dicetyl 7-os 7-ate may be added to the membrane constituents, if desired. The hemoglobin to be incorporated into the liposome is obtained by hemolyzing red blood cells in a conventional manner and concentrating by ultrafiltration using a membrane with a molecular weight of 50,000 min. The liposome of the present invention has a vitamin E content of 5 to 200% by weight in the liposome membrane component containing phospholipid,
Cholesterol with a phospholipid content of 10 to 50% by weight is added, dissolved in an organic solvent such as chloroform or ethanol, and the solvent is distilled off from the resulting solution by methods such as evaporation, freeze drying, and spray drying. Then, the hemoglobin-containing aqueous solution described in 2 above is added to the residue, the aqueous solution is uniformly suspended by shaking or ultrasonication, and this suspension is used to form liposomes. This is because if the content of vitamin E is 5% by weight or less, the effect of suppressing the oxidation of the inclusions will be insufficient, and the content will not increase even if 200% by weight or more is added. To prepare the suspension into liposomes, use the French press method, ultrasound method,
Various known methods such as a reverse phase method may be used. Although vitamin E is contained in the lipid of the obtained liposome, the content is not necessarily the same as the amount initially added. Vitamin E not contained in the lipids is removed in the liposome washing step. The content of vitamin E in the obtained liposome can be calculated by extracting vitamin E from the liposome solution with an organic solvent and absorbing it under ultraviolet light. Next, the present invention will be explained in further detail by showing Examples and Comparative Examples.
【実施例１】
水素添加卵黄レシチン２ｇ、コレステロール１ｇ１ミリ
スチン酸０．１７ｇ１ｄＱ−α−トコフェロール酢酸２
．４６ｇをジクロロメタン５０ｍｆｆに溶解し、エバポ
レーションによりジクロロメタンを除去しｔ；。得られ
た混合脂質に５０重量％ヘモグロビン水溶液３０ｍｆｆ
を加え、振とう混合後フレンチプレス細胞破砕機（ＯＨ
ＴＡＫＥ社製）を用いて、５００ ｋｇｆ／ ｃｒｓ２
の圧力でフレンチプレス処理を１０回繰り返した。得ら
れたフレンチプレス処理液を遠心分離処理（１７，ＯＯ
Ｏｒｐｍ Ｘ ３０ｍ１ｎ ａｔ ４℃）し、生
理食塩水で３回洗浄し、リポソームに取り込まれなかっ
たヘモグロビン、ビタミンＥをデカンテーションで除去
し、沈澱物としてヘモグロビン内包リポソーム（ヘモグ
ロビン濃度５重量％、１０ｍ１２）を得た。このヘモグ
ロビン内包リポソームにクロロホルムを添加して、ビタ
ミンＥを抽出し含有量を測定したところリン脂質に対し
含有量の８．７重量％のビタミンＥが含有されていた。
このヘモグロビン内包リポソームを３７℃でインキユベ
ートシ、経時的なメトヘモグロビン濃度を測定したとこ
ろ、２４時間で１６重量％、４８時間で１９重量％であ
った。メトヘモグロビン濃度はＣＯオキシメーター（Ｉ
ｎｓＬｒｕｍｅｎｔａｔｉｏｎ Ｌａｂｏｒａｔｏｒｙ
社製）を用いて測定した。[Example 1] 2 g of hydrogenated egg yolk lecithin, 1 g of cholesterol, 0.17 g of myristic acid, 2 g of dQ-α-tocopherol acetic acid
．． 46 g was dissolved in 50 mff of dichloromethane, and the dichloromethane was removed by evaporation. 30mff of 50% by weight hemoglobin aqueous solution was added to the obtained mixed lipid.
After shaking and mixing, use a French press cell disrupter (OH
(manufactured by TAKE), 500 kgf/ crs2
The French press process was repeated 10 times at a pressure of . The obtained French press treatment liquid was centrifuged (17, OO
Hemoglobin and vitamin E not incorporated into the liposomes were removed by decantation, and hemoglobin-containing liposomes (hemoglobin concentration 5% by weight, 10ml) were obtained as a precipitate. I got it. When chloroform was added to this hemoglobin-containing liposome to extract vitamin E and its content was measured, it was found that the content of vitamin E was 8.7% by weight based on the phospholipid. When this hemoglobin-containing liposome was incubated at 37° C. and the methemoglobin concentration was measured over time, it was 16% by weight at 24 hours and 19% by weight at 48 hours. Methemoglobin concentration was measured using a CO oximeter (I
nsLrumentation Laboratory
(manufactured by S.A.).
ｄａ−ａ−トコフェロールを添加しない以外は実施例１
と同様の混合比でヘモグロビン内包リポソームを調製し
、同様の方法でメトヘモグロビン濃度を測定したところ
２４時間で３５重量％、４８時間で５０重量％であった
。
また、この試料を１力月（４℃）保存した後に、同様の
方法でメトヘモグロビン濃度を測定したところメトヘモ
グロビン濃度に大きな変化は認められなかった。Example 1 except that da-a-tocopherol was not added.
When a hemoglobin-containing liposome was prepared at the same mixing ratio as above and the methemoglobin concentration was measured in the same manner, it was 35% by weight at 24 hours and 50% by weight at 48 hours. Further, when this sample was stored for one month (4° C.) and the methemoglobin concentration was measured in the same manner, no significant change was observed in the methemoglobin concentration.
【実施例２】
水素添加卵黄レシチン２ｇｓ ミリスチン酸０゜１７
ｇ、 ｄＱ−ａ−トコフェロール酢酸１．２３ ｇに対
しコレステロールの添加量をＯｇ、０．２５ｇ。
０．５ｇ、０．７５ｇ、Ｉｇとした５種類の脂質混合物
を作成し、実施例１と同様の方法で５種類のヘモグロビ
ン内包リポソームを得た。実施例１と同様の方法にてそ
れぞれのビタミンＥ含有量を測定したところ、それぞれ
９．１重量％、８．５重量％、４．３重量％、３．６重
量％、２．１重量％であった。それぞれについて２４時
間（３７℃）保存した後、メトヘモグロビン濃度を測定
した。第１図はその結果を示すグラフである。図に示す
ように、コレステロール無添加の場合に、メトヘモグロ
ビン濃度が最も低くなる。これはコレステロールの添加
量減少に伴い、コレステロール減少分に代えてビタミン
Ｅが膜中に取り込まれ、その含有量が増大し、抗酸化作
用が強くなったためである。また実施例においては、膜
電荷物質としてミリスチン酸を添加したが、その他の高
級飽和脂肪酸、フォスファチジン酸、フォス７アチジル
グリセロール、ジセチルフォスフェートのうちの少なく
とも１つを単独にまたは組み合わせて使用してもほぼ同
様の効果が得られる。
（発明の効果）
以上、詳述したように、本発明に係るリポソームは、ビ
タミンＥをその膜中に大量に含有するため、内包した薬
物やヘモグロビン等の生理活性物質の酸化による劣化を
直接抑制することができ、また生体温度条件下において
も内包物の酸化を効果的に抑制することができる。
また本発明によれば、リポソーム膜中のビタミンＥ量を
増大させることによりリポソーム膜中のコレステロール
量を減少させたリポソームを提供することができる。[Example 2] Hydrogenated egg yolk lecithin 2gs myristic acid 0°17
g, the amount of cholesterol added to 1.23 g of dQ-a-tocopherol acetic acid was Og, 0.25 g. Five types of lipid mixtures of 0.5 g, 0.75 g, and Ig were prepared, and five types of hemoglobin-containing liposomes were obtained in the same manner as in Example 1. When the respective vitamin E contents were measured in the same manner as in Example 1, they were 9.1% by weight, 8.5% by weight, 4.3% by weight, 3.6% by weight, and 2.1% by weight, respectively. Met. After each sample was stored for 24 hours (37°C), the methemoglobin concentration was measured. FIG. 1 is a graph showing the results. As shown in the figure, methemoglobin concentration is lowest when no cholesterol is added. This is because, as the amount of added cholesterol decreases, vitamin E is incorporated into the membrane to replace the decreased amount of cholesterol, its content increases, and its antioxidant effect becomes stronger. In the examples, myristic acid was added as a membrane charge substance, but at least one of other higher saturated fatty acids, phosphatidic acid, phos-7 atidylglycerol, and dicetyl phosphate was added alone or in combination. Almost the same effect can be obtained by using it. (Effects of the Invention) As detailed above, since the liposome according to the present invention contains a large amount of vitamin E in its membrane, it directly suppresses deterioration due to oxidation of the encapsulated drug and physiologically active substances such as hemoglobin. Furthermore, oxidation of inclusions can be effectively suppressed even under biological temperature conditions. Further, according to the present invention, it is possible to provide a liposome in which the amount of cholesterol in the liposome membrane is reduced by increasing the amount of vitamin E in the liposome membrane.
第１図は、コレステロール重量比と、２４時間（３７℃
）保存後におけるメトヘモグロビン濃度との関係を表し
たグラフである。Figure 1 shows the cholesterol weight ratio and 24 hours (37°C
) is a graph showing the relationship with methemoglobin concentration after storage.
Claims (8)
ームであって、該膜中のビタミンＥの含有量が２〜４０
重量％であるリポソーム。(1) A liposome containing vitamin E in the liposome membrane, the content of vitamin E in the membrane being 2 to 40%.
% by weight of liposomes.
びビタミンＥを含有するリポソームであって、前記リン
脂質に対するコレステロールの含有量が１０〜５０重量
％であるリポソーム。(2) A liposome containing phospholipids, cholesterol, and vitamin E in the liposome membrane, wherein the content of cholesterol relative to the phospholipids is 10 to 50% by weight.
フェロール、トコフェロール誘導体のうち少なくとも１
つを含有する請求項１または２に記載のリポソーム。(3) At least one of α-tocopherol, β-tocopherol, and tocopherol derivatives as vitamin E
The liposome according to claim 1 or 2, comprising:
飽和脂肪酸、フォスファチジン酸、フォスファチジルグ
リセロール、ジセチルフォスフェートのうちの少なくと
も１つを含有する請求項１ないし３のいずれかに記載の
リポソーム。(4) The liposome membrane further contains at least one of higher saturated fatty acids, phosphatidic acid, phosphatidylglycerol, and dicetyl phosphate as a membrane-charged substance. liposomes.
または生理活性物質を含有してなる請求項１ないし４の
いずれかに記載のリポソーム。(5) The liposome according to any one of claims 1 to 4, wherein the liposome's internal aqueous phase contains a drug or physiologically active substance that is susceptible to oxidation.
のリポソーム。(6) The liposome according to claim 5, wherein the physiologically active substance is hemoglobin.
重量％混合し、得られた混合物を用いてリポソームを形
成することを特徴とするリポソームの製法。(7) Add 5-200% of vitamin E to the liposome membrane constituents.
A method for producing liposomes, which comprises mixing % by weight and forming liposomes using the resulting mixture.
リポソーム膜構成成分に対する含有量が５〜２００重量
％のビタミンＥと、前記リン脂質に対する含有量が１０
〜５０重量％のコレステロールを混合し、得られた混合
物を用いてリポソームを形成することを特徴とするリポ
ソームの製法。(8) In the liposome membrane component containing phospholipid, vitamin E is added in an amount of 5 to 200% by weight relative to the liposome membrane component, and vitamin E is added in an amount of 10% by weight relative to the phospholipid.
A method for producing liposomes, which comprises mixing up to 50% by weight of cholesterol and forming liposomes using the resulting mixture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1118346A JPH02295917A (en) | 1989-05-10 | 1989-05-10 | Liposome suppressing oxidation of internally capsulated substance and production thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1118346A JPH02295917A (en) | 1989-05-10 | 1989-05-10 | Liposome suppressing oxidation of internally capsulated substance and production thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02295917A true JPH02295917A (en) | 1990-12-06 |
Family
ID=14734421
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1118346A Pending JPH02295917A (en) | 1989-05-10 | 1989-05-10 | Liposome suppressing oxidation of internally capsulated substance and production thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02295917A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5569464A (en) * | 1993-04-02 | 1996-10-29 | Wakamoto Pharmaceutical Co., Ltd. | Stable aqueous dispersions containing liposomes |
| JP2000502087A (en) * | 1995-12-15 | 2000-02-22 | ナショナル リサーチ カウンシル オブ カナダ | Archeosomes, arceosomes containing coenzyme Q, and other types of liposomes containing coenzyme Q, as adjuvants and delivery vehicles |
| WO2010061880A1 (en) * | 2008-11-26 | 2010-06-03 | 中外製薬株式会社 | Vesicle preparation |
| JPWO2012102364A1 (en) * | 2011-01-27 | 2014-06-30 | サンスター株式会社 | Polyunsaturated fatty acid-containing composition |
| JP2015193580A (en) * | 2014-03-31 | 2015-11-05 | テルモ株式会社 | Methods for producing amphotericin b liposomes |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60188329A (en) * | 1984-03-07 | 1985-09-25 | Ajinomoto Co Inc | Transfer accelerator for liposome inclusion substance |
| JPS6295134A (en) * | 1985-10-21 | 1987-05-01 | Nippon Saafuakutanto Kogyo Kk | Production of liposome |
| JPS62152531A (en) * | 1985-12-26 | 1987-07-07 | Dai Ichi Seiyaku Co Ltd | Preparation of liposome |
-
1989
- 1989-05-10 JP JP1118346A patent/JPH02295917A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60188329A (en) * | 1984-03-07 | 1985-09-25 | Ajinomoto Co Inc | Transfer accelerator for liposome inclusion substance |
| JPS6295134A (en) * | 1985-10-21 | 1987-05-01 | Nippon Saafuakutanto Kogyo Kk | Production of liposome |
| JPS62152531A (en) * | 1985-12-26 | 1987-07-07 | Dai Ichi Seiyaku Co Ltd | Preparation of liposome |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5569464A (en) * | 1993-04-02 | 1996-10-29 | Wakamoto Pharmaceutical Co., Ltd. | Stable aqueous dispersions containing liposomes |
| JP2000502087A (en) * | 1995-12-15 | 2000-02-22 | ナショナル リサーチ カウンシル オブ カナダ | Archeosomes, arceosomes containing coenzyme Q, and other types of liposomes containing coenzyme Q, as adjuvants and delivery vehicles |
| WO2010061880A1 (en) * | 2008-11-26 | 2010-06-03 | 中外製薬株式会社 | Vesicle preparation |
| CN102292069A (en) * | 2008-11-26 | 2011-12-21 | 中外制药株式会社 | Vesicle preparation |
| JPWO2010061880A1 (en) * | 2008-11-26 | 2012-04-26 | 中外製薬株式会社 | Vesicle formulation |
| JP5600299B2 (en) * | 2008-11-26 | 2014-10-01 | 中外製薬株式会社 | Vesicle formulation |
| JPWO2012102364A1 (en) * | 2011-01-27 | 2014-06-30 | サンスター株式会社 | Polyunsaturated fatty acid-containing composition |
| JP2015193580A (en) * | 2014-03-31 | 2015-11-05 | テルモ株式会社 | Methods for producing amphotericin b liposomes |
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