JPS62284000A - Physiologically active protein bonded lipid microsphere - Google Patents
Physiologically active protein bonded lipid microsphereInfo
- Publication number
- JPS62284000A JPS62284000A JP61126697A JP12669786A JPS62284000A JP S62284000 A JPS62284000 A JP S62284000A JP 61126697 A JP61126697 A JP 61126697A JP 12669786 A JP12669786 A JP 12669786A JP S62284000 A JPS62284000 A JP S62284000A
- Authority
- JP
- Japan
- Prior art keywords
- lms
- physiologically active
- drug
- active protein
- microsphere
- 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
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 28
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 28
- 239000004005 microsphere Substances 0.000 title claims abstract description 9
- 150000002632 lipids Chemical class 0.000 title claims abstract description 6
- 229940079593 drug Drugs 0.000 claims abstract description 21
- 239000003814 drug Substances 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 abstract description 15
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 abstract description 6
- 229960004857 mitomycin Drugs 0.000 abstract description 5
- 235000012424 soybean oil Nutrition 0.000 abstract description 5
- 239000003549 soybean oil Substances 0.000 abstract description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 206010006187 Breast cancer Diseases 0.000 abstract description 2
- 208000026310 Breast neoplasm Diseases 0.000 abstract description 2
- 239000008280 blood Substances 0.000 abstract description 2
- 210000004369 blood Anatomy 0.000 abstract description 2
- 235000011187 glycerol Nutrition 0.000 abstract description 2
- 230000003327 cancerostatic effect Effects 0.000 abstract 3
- 239000003795 chemical substances by application Substances 0.000 abstract 3
- QMXCRMQIVATQMR-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-pyridin-2-ylsulfanylpropanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSC1=CC=CC=N1 QMXCRMQIVATQMR-UHFFFAOYSA-N 0.000 abstract 1
- 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 abstract 1
- 239000007795 chemical reaction product Substances 0.000 abstract 1
- 230000009849 deactivation Effects 0.000 abstract 1
- 229940083466 soybean lecithin Drugs 0.000 abstract 1
- 238000000322 laser mass spectrometry Methods 0.000 description 32
- 229940079322 interferon Drugs 0.000 description 9
- 102000014150 Interferons Human genes 0.000 description 7
- 108010050904 Interferons Proteins 0.000 description 7
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 6
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 6
- -1 emulsification aids Substances 0.000 description 6
- 229960005356 urokinase Drugs 0.000 description 6
- SLKDGVPOSSLUAI-PGUFJCEWSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine zwitterion Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCCCC SLKDGVPOSSLUAI-PGUFJCEWSA-N 0.000 description 4
- 102100035695 Gamma-aminobutyric acid receptor-associated protein Human genes 0.000 description 3
- 101001001372 Homo sapiens Gamma-aminobutyric acid receptor-associated protein Proteins 0.000 description 3
- 102000015696 Interleukins Human genes 0.000 description 3
- 108010063738 Interleukins Proteins 0.000 description 3
- 238000012377 drug delivery Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- CGIGDMFJXJATDK-UHFFFAOYSA-N Indomethacin Natural products CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 description 1
- 108010002350 Interleukin-2 Chemical class 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- NPPQSCRMBWNHMW-UHFFFAOYSA-N Meprobamate Chemical compound NC(=O)OCC(C)(CCC)COC(N)=O NPPQSCRMBWNHMW-UHFFFAOYSA-N 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- VGQOVCHZGQWAOI-UHFFFAOYSA-N UNPD55612 Natural products N1C(O)C2CC(C=CC(N)=O)=CN2C(=O)C2=CC=C(C)C(O)=C12 VGQOVCHZGQWAOI-UHFFFAOYSA-N 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- VGQOVCHZGQWAOI-HYUHUPJXSA-N anthramycin Chemical compound N1[C@@H](O)[C@@H]2CC(\C=C\C(N)=O)=CN2C(=O)C2=CC=C(C)C(O)=C12 VGQOVCHZGQWAOI-HYUHUPJXSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229950000812 dexamethasone palmitate Drugs 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- LPEPZBJOKDYZAD-UHFFFAOYSA-N flufenamic acid Chemical compound OC(=O)C1=CC=CC=C1NC1=CC=CC(C(F)(F)F)=C1 LPEPZBJOKDYZAD-UHFFFAOYSA-N 0.000 description 1
- 229960004369 flufenamic acid Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229960000905 indomethacin Drugs 0.000 description 1
- 239000002799 interferon inducing agent Substances 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 235000004213 low-fat Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229960001428 mercaptopurine Drugs 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 229960005205 prednisolone Drugs 0.000 description 1
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
３、発明の詳細な説明
〔産業上の利用分野〕
本発明は、生理活性蛋白をその表面に共有結合したりピ
ッドマイクロスフェア−（以下、ＬＭＳとする）に関す
る。Detailed Description of the Invention 3. Detailed Description of the Invention [Field of Industrial Application] The present invention relates to pid microspheres (hereinafter referred to as LMS) having physiologically active proteins covalently bonded to their surfaces.
最近、薬剤の生体内動態を研究する一分野として、生物
薬剤学が脚光を浴びてきておυ、薬剤を効率良く目的部
位に運ぶために、種々の工夫をこらしたドラッグ・デリ
バリ−・システム（医薬品送達システム） （ＤＤＳ）
の開発に力が注がれてきている。ＤＤＳの中に、リボゾ
ームや高分子化合物をキャリアーとして用いる方法があ
るが、キャリアーとしてＬＭＳを用いる方法がある。＝
Ｓは、現在の薬剤キャリアーの中では、安全性、生物学
的崩壊性、応用範冊の広さ、ターゲツティング効果、工
業化など何れの面からみても、実用価値が高いものと評
価できる。Recently, biopharmaceutical science has been in the spotlight as a field that studies the in vivo dynamics of drugs.Drug delivery systems (Drug delivery systems) have been developed to efficiently deliver drugs to the target site. drug delivery system) (DDS)
Efforts are being focused on the development of Among DDS, there is a method using ribosomes or polymer compounds as a carrier, and there is a method using LMS as a carrier. =
Among current drug carriers, S can be evaluated as having high practical value in terms of safety, biological degradability, wide range of applications, targeting effect, and industrialization.
しかし、ＬＭＳは、製剤上の特徴でもめ、るが、脂肪相
を界面活性剤が取り囲むという構造のために、水溶性の
薬物に応用しにくいという欠点がある。そのため、脂溶
性の低い薬物には、その薬物に脂溶性官能基を導入する
か、或いは工を構成する大豆油にエイコサペンタエン散
又はエイシンなどの薬物溶解性の高い物質を混入するこ
とが行われ、それによ、Ｑ ＬＭＳ内部に薬物を成る程
度封入させることができる。However, although LMS has some formulation characteristics, it has the disadvantage that it is difficult to apply to water-soluble drugs because of its structure in which a surfactant surrounds a fatty phase. Therefore, for drugs with low fat solubility, either a fat-soluble functional group is introduced into the drug, or a substance with high drug solubility, such as eicosapentaene powder or eisin, is mixed into the soybean oil that makes up the drug. , thereby allowing the drug to be encapsulated to a certain extent inside the Q LMS.
このような方法によっても、抗体、インターフェロン、
インターロイキン、組織型プラスミノーゲン活性体、ク
ロキナーゼなどの生理活性を有する蛋白を、ＬＭＳ内部
に封入させることは、不可能でめった。そして、インタ
ーロイキン又はウロキナーゼなどは、強い生理活性を有
するが、静脈注入の場合、血中半減期が短いため、失活
し易く、臨床的な効果を得るのに、適当なりＤＳが望ま
れている。This method also allows antibodies, interferon,
It is rarely possible to encapsulate physiologically active proteins such as interleukin, tissue-type plasminogen activator, and crokinase inside LMS. Interleukins and urokinase have strong physiological activity, but when injected intravenously, their half-life in the blood is short, so they are easily deactivated, and appropriate DS is required to obtain clinical effects. There is.
本発明者らは、これら従来のＬＭＳの欠点のない技術に
ついて検討した結果、本発明を見い出した。The present inventors discovered the present invention as a result of studying techniques that do not have the drawbacks of these conventional LMSs.
即ち、本発明は生理活性蛋白をその表面に共有結合した
マイクロリビットスフエアーに関する。That is, the present invention relates to microribit spheres having physiologically active proteins covalently bonded to their surfaces.
本発明に用いる生理活性蛋白は、生理活性を有する蛋白
であれば、どんなものでもよいが、例、ｔ バクロキナ
ーゼ、スーパーオキシド・ディスムターゼ、抗体（癌モ
ノクローナル抗体などの特異性を有する抗体）、プロテ
ィンＡ１インターロイキンやＭＡ１ｔ’などの各種リン
ホカイン、インター７二ロン又はインターフェロン誘導
物質、Ｉａ、リウトマイド因子、生理活性を有する酵素
、蛋白を置換基に有する薬物又はその他の蛋白製剤を含
む。The physiologically active protein used in the present invention may be any physiologically active protein, but examples include t-baclokinase, superoxide dismutase, antibodies (antibodies with specificity such as cancer monoclonal antibodies), protein It includes various lymphokines such as A1 interleukin and MA1t', inter7 diron or interferon inducers, Ia, rheutomide factor, physiologically active enzymes, drugs having protein as a substituent, or other protein preparations.
又、本発明に用いられるＬＭＳは、従来知られている任
意の方法により製造される。例えば、所定量の大豆油、
リン脂質、そして必要に応じて乳化補助剤、安定剤、界
面活性剤などの添加物を混合し、加熱して溶液とし、通
常のホモジナイザーにより均質化することにより製造さ
れる。Further, the LMS used in the present invention is manufactured by any conventionally known method. For example, a certain amount of soybean oil,
It is produced by mixing phospholipids and, if necessary, additives such as emulsification aids, stabilizers, and surfactants, heating the mixture to form a solution, and homogenizing it using a conventional homogenizer.
生理活性蛋白とＬＭＳとの結合は、共有結合による。The binding between the physiologically active protein and LMS is through a covalent bond.
ＬＭＳと生理活性蛋白とを共有結合させる方法としては
、種々のやり方が考えられる。例えば、ヘテロ三官能性
試薬を用いる方法があけられる。Various methods can be considered for covalently bonding LMS and physiologically active proteins. For example, a method using a heterotrifunctional reagent is possible.
例えば、蛋白架橋剤５ＰＤＰ ［Ｎ−サクシミジルー３
−（２−ピリジルジチオ）プロピオネート〕を７ミノ基
を導入したイソプレノイド（Ｃ４５或いはＣ９５）と結
合させ、この結合物を含むＬＭＳを生成し、必要な蛋白
をＬＭＳにＳ−Ｓ結合を介して共有結合させる。For example, the protein cross-linker 5PDP [N-succimidyl-3
-(2-pyridyldithio)propionate] is combined with an isoprenoid (C45 or C95) into which a 7-mino group has been introduced, an LMS containing this conjugate is produced, and the necessary protein is covalently transferred to the LMS via an S-S bond. combine.
又、架橋剤としては、ｍ−マレイミドベンゾイル−Ｎ−
（ジパルミトイルホスファチジル）エタノールアミドも
用いられる。これは、例えばジパルミトイルホス７アテ
ジルエタノールアミンとｍ−マレイミドベンゾイル−Ｎ
−ヒドロキシサクシイミドエステルとの反応により得ら
れる。In addition, as a crosslinking agent, m-maleimidobenzoyl-N-
(Dipalmitoylphosphatidyl)ethanolamide is also used. This includes, for example, dipalmitoylphos-7atezylethanolamine and m-maleimidobenzoyl-N
-obtained by reaction with hydroxysuccinimide ester.
又、本発明は薬物をその内部に含有ししかも生理活性蛋
白をその表面に共有結合したリピツドマイクロスフェア
ーに関する。The present invention also relates to lipid microspheres containing a drug therein and having a physiologically active protein covalently bonded to the surface thereof.
このリピツドマイクロスフエアーでは、前述の如くその
表面に生理活性蛋白を共有結合しているが、その内部に
薬物を含有する。このＬＭＳでは、薬物を含有している
ため、治療効果を増大てせることができる。As described above, this lipid microsphere has a physiologically active protein covalently bonded to its surface, and contains a drug inside. Since this LMS contains a drug, the therapeutic effect can be increased.
ＬＭＳに含有される薬物には特に制限がなく、その中油
溶性のものが好ましい。これら薬剤の例は、抗炎症剤（
例えば、ステロイド系、非ステロイド系の何れでもよく
、例えばデキサメサゾンパルミテート、プレドニゾロン
ノミルミテート、インドメサシンエステル、イブフロフ
ェン、フルフェナム酸など）、抗癌剤（例えば５− Ｆ
Ｕ。There are no particular restrictions on the drug contained in LMS, and oil-soluble drugs are preferred. Examples of these drugs include anti-inflammatory agents (
For example, they may be steroidal or nonsteroidal, such as dexamethasone palmitate, prednisolone nomilmitate, indomethacin ester, ibuflofen, flufenamic acid, etc., anticancer agents (such as 5-F
U.
ダウンマイシン、プレオマイシン、アントラマイシン、
マイトマイシン、６−メルカプトプリンなど）、抗生物
質（例えばエリスロマイシン、セファロスポリン類、ｋ
ニジリン類なト）、抗ウィルス剤（例えばインターフェ
ロンなど）、プロスタグランディン類を含むがこれに限
定されるものではない。downmycin, pleomycin, anthramycin,
mitomycin, 6-mercaptopurine, etc.), antibiotics (e.g. erythromycin, cephalosporins, k
These include, but are not limited to, antiviral agents (eg, interferon, etc.), and prostaglandins.
この薬物含有ＬＭＳの製造に当っては、前述のＬＭＳの
製造時に、適当量の薬物を添加混合して得ることができ
る。In producing this drug-containing LMS, it can be obtained by adding and mixing an appropriate amount of drug during the production of the LMS described above.
次に実施例を示す。 Next, examples will be shown.
実施例 １
（Ａ）１０μＭジパルミトイルホスファチジルエタノー
ルアミン（ＤＰＰＥ ）のクロロホルム・メタノール（
９対１）混合溶液に、１２μＭＮ−サクシンイミジル３
−（２−ピリジルジチオ）プロピオネート（ＳＰＤＰ）
及び２０μＭトリエタノールを加え、２時間室温で攪拌
、反応させて、５ＰＤＰとＤＰＰｇとの結合物（ＰＤＰ
−ＰＥ ）を得た。Example 1 (A) 10 μM dipalmitoylphosphatidylethanolamine (DPPE) in chloroform-methanol (
9 to 1) mixed solution, 12 μM N-succinimidyl 3
-(2-pyridyldithio)propionate (SPDP)
and 20 μM triethanol, stirred and reacted at room temperature for 2 hours to form a conjugate of 5PDP and DPPg (PDP
-PE) was obtained.
（Ｂ） （Ａ）で得たＦＤＰ−ＰＥ ０．０４％（ｗ
、、’ｖ） 、精製大豆油２０ Ｓ （Ｗ／’Ｖ’）、
精製大豆レシテｙ ２．４ ％ （Ｗ／Ｖ）、精製グリ
セリン５％（Ｗ／Ｖ）を混合し、常法に従ってマントン
・ゴーリン乳化機によシ乳化し、ＰＤＰ −ＰＥ含有Ｌ
ＭＳ （大豆油含有率２０％）を得た。(B) FDP-PE obtained in (A) 0.04% (w
,,'v), Refined soybean oil 20S (W/'V'),
2.4% (W/V) of purified soybean leishitin and 5% (W/V) of purified glycerin were mixed and emulsified using a Manton-Gorlin emulsifier according to a conventional method, and L containing PDP-PE was mixed.
MS (soybean oil content 20%) was obtained.
（Ｃ） 次に、インターフェロンを０．０１Ｍ燐酸塩
緩衝液（ｐｌ（７，５）に対しｉｓ９／ｌｎｌになるよ
うに溶解し、０、３６ ｍＭ ５ＰＤＰを反応させてＦ
ＤＰ−インターフェロンを得た。(C) Next, interferon was dissolved in 0.01M phosphate buffer (pl(7,5) to give is9/lnl, and reacted with 0.36mM 5PDP to F
DP-interferon was obtained.
インターフェロン未結合５ＰＤＰを除去した後、さらに
５０ｍＭジチオスレイトールにより還元し、還元ＰＤＰ
−インターフェロンを得た。過剰のジテオスレイトール
を除去した後、還元ＦＤＰ−インターフェロン溶液に、
（Ｂ）で得たＬＭＳを加え、室温で１晩、攪拌放置して
、インターフェロン結合ＬＭＳを得た。After removing interferon-unbound 5PDP, it was further reduced with 50mM dithiothreitol to form reduced PDP.
- Obtained interferon. After removing excess diteothreitol, the reduced FDP-interferon solution was
The LMS obtained in (B) was added and left to stir at room temperature overnight to obtain interferon-conjugated LMS.
実施例 ２
実施例１（Ｂ）において、ＤＰＰＥの代υに、Ｃ４Ｓポ
リプレノイド（インプレノイドの一種）の末端ＯＨ基を
ＮＨ７基に置換したものを用いて、ＬＭＳを製造した。Example 2 In Example 1 (B), LMS was produced using a C4S polyprenoid (a type of imprenoid) whose terminal OH group was replaced with an NH7 group instead of DPPE.
このＬＭＳを用いて、実施例１（Ｃ）と同様にして、イ
ンターフェロンの代りに、ウロキナーゼを用いて、ウロ
キナーゼ結合ＬＭＳを得た。Using this LMS, urokinase-binding LMS was obtained in the same manner as in Example 1 (C), using urokinase instead of interferon.
実施例 ６
実施例１（Ｂ）において、ＤＰＰＥの代りに、Ｃ９５ン
ラネンール（インプレノイドの−ａ［）の末端ＯＨ基を
ＮＨ２基に置換したものを用い、ＬＭＳを製造した。Example 6 In Example 1 (B), instead of DPPE, LMS was produced by using C95 nanenol (imprenoid -a[) in which the terminal OH group was replaced with an NH2 group).
このＬＭＳを用いて、実施例１（Ｃ）と同様にして、イ
ンターフェロンの代りに１イ・ンターロイキン２を用い
て、インターロイキン２結合ＬＭＳを得た。Using this LMS, interleukin 2-binding LMS was obtained in the same manner as in Example 1 (C), using 1-interleukin 2 instead of interferon.
実施例 ４
ステアリルマイトマイシンＣを２０μｇ／ａｔ を添加
する以外は、実施例１（Ｂ）の方法を行った。Example 4 The method of Example 1 (B) was carried out except that 20 μg/at of stearyl mitomycin C was added.
得られたＬＭＳにＭＭ４６マウス乳癌に対するモノクロ
ーナル抗体を実施例１（Ｃ）の方法により共有結合させ
た。A monoclonal antibody against MM46 mouse mammary cancer was covalently bonded to the obtained LMS by the method of Example 1(C).
得られたステアリルマイトマイシンＣ含有ＬＭ！３の生
体外におけるＭＭ４６細胞に対する抗腫瘍効果は次の通
りでらった。方法はＭＭ４６細胞を１０％ＦＣ３含有ハ
ンクス液で１ｘ ｊ Ｑ６ （＠／ｍに浮遊させた。９
６大のマイクロウェルプレートに細胞浮遊液１００μｌ
とＬＭＳ １０μ）を入れ、３７℃、２４時間、５％Ｃ
Ｏ２気流下にインキュベートした。The obtained stearyl mitomycin C-containing LM! The antitumor effect of No. 3 on MM46 cells in vitro was as follows. The method was to suspend MM46 cells at 1x j Q6 (@/m) in Hank's solution containing 10% FC3.9
100μl of cell suspension in 6 large microwell plate
and LMS (10μ), and incubated at 37°C, 5%C for 24 hours.
Incubated under O2 flow.
次ニ、トリパンブルー染色で細胞の生存率を測定した。Next, cell viability was measured by trypan blue staining.
生存率はｉ ｔｓｓであった。一方、ステアリルマイト
マイシンＣをＬＭＳに含有させたものは、３９５％、Ｌ
ＭＳのみでは６４．５％であった。Survival rate was itss. On the other hand, LMS containing stearyl mitomycin C was 395% LMS.
For MS alone, it was 64.5%.
この実験において、抗体の使用量は、−生存率に影響を
及ぼさない量であった。In this experiment, the amount of antibody used was - an amount that did not affect viability.
さらに、４８時間インキュベーションを行っても、同様
な結果が得られた。Furthermore, similar results were obtained even after incubation for 48 hours.
この結果から、モノクローナル抗体を結合した方が、生
存率が低く、抗腫瘍効果が増大していることが分る。These results show that monoclonal antibody binding has a lower survival rate and an increased antitumor effect.
参考例 １
蛋白として、プロティンＡ、 ＩｇＧ、ウロキナーゼを
用い、実施例１の方法に従って、これら蛋白を共有結合
により結合したＬＭＳｔ−得た。この結合率は、プロテ
ィンＡ・・で６０．０％、工ｇＧで５７、５％、ウロキ
ナーゼ４０．０％であった。Reference Example 1 Protein A, IgG, and urokinase were used as proteins, and according to the method of Example 1, LMSt-covalently bound these proteins was obtained. The binding rate was 60.0% for protein A, 57.5% for protein G, and 40.0% for urokinase.
一方、これら蛋白を吸着法（ＬＭＳとこれら蛋白とを単
に混合することにより両者を結合させる方法）によりＬ
ＭＳに結合させた場合には、その結合率は、プロティン
Ａで４．０％、塊Ｇで３０，０係、ウロキナーゼで２０
％であった。On the other hand, these proteins were absorbed into LMS by an adsorption method (a method of binding LMS and these proteins by simply mixing them).
When bound to MS, the binding rate was 4.0% for protein A, 30.0% for block G, and 20% for urokinase.
%Met.
又、吸着法で得られた蛋白吸着ＬＭＳは、血清とともに
インキュベートすると、吸着しｆｃ蛋白は剥ｍ Ｌ、て
しまう。ＬＭＳは、静脈中に投与されるので、目的部位
に到達する間に、蛋白が保持されず、目的を達すること
ができない。これに対し、本発明で得られたＬＭＳには
、このような欠点がない。Furthermore, when the protein-adsorbed LMS obtained by the adsorption method is incubated with serum, it adsorbs and the fc protein is stripped off. Since LMS is administered intravenously, the protein is not retained during the delivery to the target site and the target cannot be achieved. In contrast, the LMS obtained according to the present invention does not have such drawbacks.
Claims (1)
イクロスフェアー。 ２）薬物をその内部に含有ししかも生理活性蛋白をその
表面に共有結合したリピッドマイクロスフェアー。[Claims] 1) Lipid microspheres having physiologically active proteins covalently bonded to their surfaces. 2) Lipid microspheres that contain a drug inside and have a physiologically active protein covalently bonded to their surface.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61126697A JPS62284000A (en) | 1986-05-31 | 1986-05-31 | Physiologically active protein bonded lipid microsphere |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61126697A JPS62284000A (en) | 1986-05-31 | 1986-05-31 | Physiologically active protein bonded lipid microsphere |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62284000A true JPS62284000A (en) | 1987-12-09 |
Family
ID=14941608
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61126697A Pending JPS62284000A (en) | 1986-05-31 | 1986-05-31 | Physiologically active protein bonded lipid microsphere |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62284000A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5137725A (en) * | 1986-04-22 | 1992-08-11 | L'oreal | Dispersion of lipidic spherules |
| EP0706798A1 (en) | 1994-07-22 | 1996-04-17 | Sanwa Kagaku Kenkyusho Co., Ltd. | Pharmaceutical composition containing biologically active peptide or protein |
-
1986
- 1986-05-31 JP JP61126697A patent/JPS62284000A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5137725A (en) * | 1986-04-22 | 1992-08-11 | L'oreal | Dispersion of lipidic spherules |
| EP0706798A1 (en) | 1994-07-22 | 1996-04-17 | Sanwa Kagaku Kenkyusho Co., Ltd. | Pharmaceutical composition containing biologically active peptide or protein |
| US5733877A (en) * | 1994-07-22 | 1998-03-31 | Sanwa Kagaku Kenkyusho Co., Ltd. | Pharmaceutical composition containing biologically active peptide or protein |
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